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Studies on the Riboflavin, Niacin, Pantothenic Acid and Choline Requirements of Young Bobwhite Quail
Studies on the Riboflavin, Niacin, Pantothenic Acid and Choline Requirements of Young Bobwhite Quail J. A . SERAFIN
United States Fish & Wildlife Service, Patuxent Wildlife Research Center, Laurel, Maryland 20810 (Received for publication November 23, 1973)
ABSTRACT Four experiments were conducted to examine the riboflavin, niacin, pantothenic acid and choline requirements of young Bobwhite quail. Quail fed purified diets deficient in either riboflavin, niacin, pantothenic acid or choline grew poorly and high mortality occurred by 5 weeks of age. Under the conditions of these experiments, it was found that: (1) young quail require approximately 3.8 mg. riboflavin/kg. diet for satisfactory growth and survival; (2) no more than 31 mg. niacin/kg. diet are required for normal growth and survival of young quail; (3) the requirement for pantothenic acid is higher than has previously been reported, quail in these studies requiring 12.6 mg. pantothenic acid/kg. feed for growth and survival; and (4) the requirement for choline for reducing mortality is approximately 1000 mg./kg., while the amount necessary for normal growth of young quail is no greater than 1500 mg./kg. when the diet contains ample amounts of methionine. Quail fed a niacin-deficient diet developed stiff, shortened feathers and an erythema about the head; those receiving a riboflavin-deficient ration developed enlarged hocks and bowed legs, as did quail fed diets low or devoid of choline. Aside from slow growth, poor feathering was the only other indication that a deficient diet was being fed when quail were placed on a basal ration without pantothenic acid for five weeks. POULTRY SCIENCE 53: 1522-1532, 1974
nutrients for Bobwhite quail. Very few reports exist describing vitamin deficiency NCREASING numbers of Bobwhite quail states in quail. In view of the limited informa(Colinus virginianus) are being raised by tion available on nutrient requirements and the game bird industry for hunting preserves considering the increased interest in Boband for table food. Use of Bobwhite quail white quail, a study was begun to evaluate to study the influence of pesticides and other nutrient needs of this species. This report pollutants in the environment has also in- describes the results of a series of expericreased recently. For the past several years ments with purified diets designed to investia program has been in progress to propagate gate the riboflavin, niacin, pantothenic acid masked Bobwhite quail (Colinus virginianus and choline requirements of Bobwhite quail. ridgwayi) in an effort to prevent possible Characteristics of these vitamin deficiencies extinction of the species (Erickson, 1968). are also compared with classical descriptions The requirements of Bobwhite quail for most for the chick and other domestic species. nutrients have not been specifically identiEXPERIMENTAL PROCEDURES fied. A review of the nutrition of pheasants (Phasianus colchicus), Bobwhite and CoturTrials of five week duration were conductnix quail (Coturnix coturnix japonica) by ed during all seasons of the year with quail Howes and Beane (1966) indicated that few of mixed sex. Hatching eggs were obtained studies have been conducted to determine from a breeding flock raised and maintained specific vitamin requirements of quail. The to provide production during all months of National Research Council, which recently the year. Seasonal variation in production reviewed nutritional needs for poultry, Bob- resulted in fewer chicks for some trials than white quail and pheasants (N.R.C., 1971), for others. Quail were housed in electrically lists specific requirement values of only six heated, thermostatically controlled battery INTRODUCTION
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QUAIL VITAMIN REQUIREMENTS
TABLE 1.—Composition of basal diet used in studies of vitamin requirements of young Bobwhite quail Amount (percent) Glucose 58.69 Isolated soybean protein1 27.00 Soybean oil 3.00 Cellulose 3.00 Vitamin mixture2 1.00 Mineral mixture3 6.31 Glycine 0.30 DL-methionine 0.70 Protein (N x 6.25) 25.50 1 RP-100, Ralston-Purina Company, St. Louis, Missouri. 2 The composition of the vitamin mixtures used in these studies is shown in Table 2. 3 Supplied the following per kilogram of diet: (in grams) CaHPCy2H 2 0, 21.6; CaC03, 18.3; KH2P04, 13.9; NaCl, 6.0; MgS04, 2.5; (in milligrams) FeS0 4 -7H 2 0, 333.0; MnSCyH 2 0, 333.0; KI, 2.6; CuSCy5H 2 0, 16.7; CoCl2-6H20, 1.6; Na2Mo04-2H20, 8.3; ZnC03, 115; and Na2Se03, 0.22. Ingredient
brooders with raised wire floors located in a room with continuous forced draft ventilation held at a temperature of approximately 70° F. The brooding temperature was maintained at 105° F. for the first week of each experiment. The average weight of quail at the beginning of an experiment ranged between 6.9 and 7.1 grams. All birds were debeaked at 5 days of age to prevent picking. Feed and water were supplied ad libitum. The composition of the basal purfied diet and vitamin mixtures used in these studies is shown in Tables 1 and 2. In the few instances where purified diets have been fed to Bobwhite quail poor growth responses and high mortality were observed (Scott et al., 1964). For this reason a practical quail starter ration was fed groups of quail to evaluate responses of quail fed purified diets. The composition of this ration appears in Table 3. The pantothenic acid content of the pantothenic acid-deficient basal diet was deter-
TABLE 2.— Vitamin levels used in vitamin mixes for studies of riboflavin, niacin, pantothenic acid and choline requirements of young Bobwhite quail Vitamin1 Thiamine-HC1, mg. Riboflavin, mg. Niacin, mg. Pantothenic acid, mg. Choline, mg. Inositol, mg. Pyridoxine • HC1, mg. Folic acid, mg. Biotin, mg. Vitamin B, 2 (micrograms) Menadione sodium bisulfite, mg. Vitamin A (I.U.) Vitamin D 3 (I.U.) a-tocopheryl acetate (I.U.) Ethoxyquin, mg.
Amount/kg diet 15.0 15.02 50.03 20.04 1336.05 250.0 6.0 6.0 1.6 30.0 1.52 5000.0 4500.0 110.0 125.0
1 Water soluble vitamins were obtained from Nutritional Biochemicals Corp., Cleveland, Ohio. 2 The vitamin mixture used for the studies concerning riboflavin contained no riboflavin. 3 The vitamin mixture used for the studies concerning niacin contained no niacin. 4 The vitamin mixture used for the studies concerning pantothenic acid contained no pantothenic acid. 5 The vitamin mixture used for the studies concerning choline contained no choline. mined by a method described in the U.S. Pharmacopeia (1965) employing Lactobacillus plantarum ATCC 8014 (Difco Laboratories, Detroit, Michigan).1 Liberation of pantothenic acid from coenzyme A, which permitted 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 described in the A.O.A.C. (1960) using L. plantarum ATCC 8014. The choline content of the choline-def icient basal diet was determined by the method described by Entenman et al. (1944). The culture media for the pan-
1. Use of trade names does not imply endorsement of commercial products by the Federal Government.
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J. A. SERAFIN
TABLE 3.—Composition of practical Bobwhite quail starter ration Ingredient Yellow corn meal Dehulled soybean meal (50%) Fish meal (60%) Alfalfa meal (17%) Soybean oil Brewers dried yeast Distillers dried solubles Dried whey Dicalcium phosphate Limestone Vitamin mixture 1 Iodized salt Trace mineral mixture 2 DL-methionine Protein (N x 6.25)
Amount (percent) 44.43 35.0 6.0 3.0 3.0 2.5 2.0 1.5 1.0 0.5 0.5 0.25 0.25 0.07 29.2
1 Supplies inmg. /kg. diet: Niacin, 44.0; Calcium pantothenate, 8.8; Riboflavin, 3.3; Choline chloride, 125.0; Menadione sodium bisulfite, 2.2; Zinc bacitracin, 220.0; Ethoxyquin, 125.0; Vitamin B l2 , 6.6 meg; Vitamin A, 22068; 22068 I.U.; Vitamin D3, 1653 I.U.; a-tocopheryl acetate, 110 I.U. premixed in carrier. 2 Supplies in mg./kg. diet: MnS0 4 H 2 0, 375.1; ZnO, 124.8; FeSCy7H 2 0, 99.0; CuS0 4 -5H 2 0, 22.0; NaMoCy5H 2 0, 8.2; CoCl2-5H20, 1.7; Na2Se03, 0.22 premixed in carrier. tothenic acid assays was obtained from Difco Laboratories, and media for the niacin assays was prepared using ingredients supplied by Difco Laboratories and Nutritional Biochemicals Corp., Cleveland, Ohio. U.S.P. reference standards were obtained from United States Pharmacopeia Convention, Inc., Bethesda, Md. Data were tested for significance using the analysis of variance (Steel and Torrie, 1960). Differences between treatment means were tested using Duncan's multiple range test (Duncan, 1955). Percentages were transformed to arc-sin values before statistical treatment. RESULTS AND DISCUSSION Riboflavin. To study the riboflavin requirement, the basal diet was fed alone and sup-
plemented with riboflavin at 0.75, 1.5, 2.5, 3.5 and 5.0 mg./kg. These levels of addition were selected because the requirements by other species of birds which have been studied are known to be less than 5.0 mg./kg. Each diet was supplied to two pens of 25 quail each. The results are presented in Table 4. Growth was significantly improved by additions of riboflavin up to 3.5 mg./kg. in the purified diets. The response of quail fed the purified diet supplemented with 3.5 and 5.0 mg. of added riboflavin/kg. compared well with the results from those supplied a practical ration. Mortality in these groups was not excessively high and feed utilization was not significantly different from that for quail fed a starter diet. Feed utilization and mortality was not significantly influenced by additions to the purified diet of riboflavin beyond 2.5 mg./kg. A level of 5.0 mg. of added riboflavin per kg. of feed did not improve growth rate or decrease mortality beyond that observed with 3.5 mg. of added riboflavin/kg. in the diet. TABLE 4.—Experiment 1: Effect of riboflavin supplementation of a basal purified diet for young Bobwhite quail'2 Treatment Dietary riboflavin Added
Total 4
mg./kg. of diet 0 0.75 1.50 2.50 3.50 5.00 Practical
0.34 1.09 1.84 2.84 3.84 5.34 starter
Body ' weight 3 (35 days)
Feed/ gain
Mortality Percent
10.80 4.59 s 3.32b 2.13 c 2.15° 2.17° 2.48c
98 86" 59" 27= 27c 28c llc
Grams 165 24 s 33 b 66c 79" 80" 86e
1 Values represent the average of two groups of 25 quail per treatment. 2 Values not followed by the same letter superscript differ significantly (P < 0.01). 3 The error mean square for the body weight data (6 d.f.) was 2.5. 4 Determined by calculation. 5 Only one bird remained alive at the completion of the experiment in the basal treatment and the group was not included in statistical analysis.
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Growth was extremely slow among quail fed the basal diet alone and the basal supplemented with 0.75 mg./kg. of added riboflavin. Quail were emaciated, weak, and unthrifty. Mortality ranged from 83% to 100% among these groups. Most of the deaths occurred between the 14th and 21st days of age. Only one chick survived the 35 day experimental period among the groups fed the basal diet. Quail fed this diet and the basal supplemented with 0.75 mg. of riboflavin/kg. developed enlarged hocks and bowed legs. However, no incidence of paralysis of the legs, also called "curled toe paralysis," a symptom normally associated with a deficiency of riboflavin in chicks, was observed among young quail. This observation is consistent with recent findings by Wyatt et al. (1973) who studied symptoms of riboflavin deficiency in three strains of broiler chicks and found a low incidence of curled toe paralysis. It has been generally believed that curled toe paralysis is the most obvious symptom among chicks suffering a lack of riboflavin (N.R.C., 1971). Wyatt et al. (1973) concluded that while riboflavin deficiency can cause curled toe paralysis, such a deficiency does not cause it obligatorily and that curled toe paralysis is a minor aspect of
riboflavin deficiency in modern broiler chickens. This appears to be true also in the case of Bobwhite quail. No descriptions of a riboflavin deficiency in Bobwhite quail seem to have been previously reported. The findings in this study indicate that curled toe paralysis is not associated with a lack of riboflavin in diets for yong quail. Based upon the response to additions of riboflavin to a purified diet and a comparison with the results obtained from feeding a practical starter diet to quail it was possible to estimate the approximate riboflavin requirement. No improvement was found in growth, appearance, feed utilization or reduced mortality by adding more than 3.5 mg. of riboflavin/kg. to the diet. Since the basal diet contained approximately 0.34 mg. of riboflavin/kg., it appears that under the conditions of this experiment, the total riboflavin requirement of young Bobwhite quail is no greater than 3.8 mg./kg. The results of this study are similar to those of Scott et al. (1959) with young pheasants. These investigators found the riboflavin requirement for young pheasant chicks to be approximately 3.4 mg./kg. Quail and pheasant chicks appear to have a nearly identical requirement which is only slightly higher than
TABLE 5.—Experiment 2: Effect of niacin supplementation of a purified basal diet for young Bobwhite quail12 Treatment Dietary niacin Added
Total
mg./kg. of diet 0 1.2 15 16.2 30 31.2 60 61.2 75 76.2 Practical starter
Body weight3 (35 days) Trial 1 Trial 2 Grams 37a 81" 96c 95c 96c 96c
13a 68b 78 b 78" 79" 85"
Feed/gain Trial 1 Trial 2 4.30" 2.41" 2.25" 2.30" 2.32 b 2.54"
4.58 a 2.48" 2.24b 2.40 b 2.42b 2.60"
Mortality Trial 1 Trial 24 Percent 88a 10b 4»
4b 4" 8b
90" 36b 21b 13b 18b 7
b
1 Values represent the average of two groups of 13 quail per treatment in Trial 1 and 20 quail per treatment in Trial 2. 2 Values not having the same letter superscript differ significantly (P < 0.01). 3 The error mean squares for the body weight data (6 d.f.) were 12 (Trial 1) and 81 (Trial 2). 4 As in footnote 2, but P < 0.05.
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J. A. SERAFIN
the amount of riboflavin needed by the chick and poult. Mitchell (1964) in a review of the riboflavin requirement for a variety of animals including the mouse, rat, dog, piglet, poultry and man indicated that variation in the requirement for riboflavin generally ranged within the limits of 1-3 mg./kg. of food. There appeared to be no correlation between the amount of riboflavin required and species size. The observed requirement of various species for this vitamin seems to fall within a narrow range compared with requirements for other vitamins such as choline or vitamin A which are known to differ considerably. The results from the experiment reported here suggest that the riboflavin requirement of Bobwhite quail is only slightly higher than accepted values for most other species studied. Niacin. Two trials were conducted to observe the response by Bobwhite quail to additions of niacin to a purified, deficient diet. Treatments consisted of duplicate groups of 13 Bobwhite quail supplied the basal diet alone or supplemented with levels of niacin ranging from 15 to 75 mg./kg. of diet in Trial 1 (Table 5). Trial 2 was identical to Trial 1 except that duplicate groups of 20 quail were used for each treatment. Results showed that growth was improved by additions of 15 and 30 mg. of niacin/kg. of diet but was not influenced by additions beyond 30 mg. /kg. In Trial 1, quail supplied the basal diet supplemented with 15 mg. of niacin/kg. grew significantly less than those treatments receiving greater amounts of niacin. Feed utilization was significantly improved in both trials by addition of niacin to the basal ration. Growth, feed utilization and mortality of quail supplied diets containing additions of 30 mg. of niacin/kg. or more were essentially the same as responses observed among quail fed the practical ration which contained approximately 80 mg. of niacin/kg. by calculation. Growth was severely retarded among quail
fed the basal diet and reduced among quail supplied the basal supplemented with 15 mg. of niacin/kg. Most of the mortality that was observed in these groups occurred between 7 and 21 days of age. Surviving quail on these diets developed stiff, shortened body feathers, while no feathers were present on their heads. Many quail developed an erythema, principally in the region of the head. No evidence of an enlargement of the tibiotarsal joint or of leg bowing was observed among quail fed niacin deficient diets. An estimate of the niacin requirement for Bobwhite quail was made with data from these trials. Since quail 5 weeks of age can be expected to obtain a weight of 95-100 grams when supplied a complete ration, it is apparent that satisfactory growth was not achieved in Trial 2. The reason for this is not known. Although quail grew at a slower rate in Trial 2 than was expected, the responses to the addition of niacin were essentially the same in both trials. A statistically significant growth response was obtained by supplementing the diet with 30 mg. of niacin/kg. compared to 15 mg./kg. in Trial 1, but not in Trial 2, although it appeared that 15 mg./kg. was insufficient to permit a high rate of growth in both studies. Analysis of the basal purified diet indicated a niacin content of approximately 1.2 mg./kg. Based upon these results it would appear that Bobwhite quail require no more than 31.2 mg. of niacin/kg. for maximum growth. This amount is similar to the requirement for the young chick (N.R.C., 1971), but is considerably lower than the amount required by the young poult and pheasant chick (N.R.C., 1971) to provide for growth and survival. Pantothenic Acid. The pantothenic acid requirement of Bobwhite quail was examined in two trials in which graded levels of calcium pantothenate were added to the basal diet to supply pantothenic acid in amounts up to 30 mg./kg. In Trial 1 duplicate groups of
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TABLE 6.—Experiment 3, Trial 1: Effect of pantothenic acid supplementation of a purified diet for young Bobwhite quail' Treatment Dietary pantothenic acid Added
Total
mg./kg. of diet
0 5 7.5 10.0 15.0
20.0 25.0 30.0 Practical
Body weight2 Feed/ (35 days) gain' Mortality Grams
0.12 5.12 7.62 10.12 15.12
Part l 4 05 39 s 72" 80 b 90 c
4.13 6 3.74a 2.04" 2.16b 2.21 b
20.12 25.12 30.12 starter
Part 2 7 90° 90 c 91 c 91<=
2.23 b 2.21" 2.19b 2.60"
1 Values not having the same letter superscript differ significantly (P < 0.05). 2 The error mean square for the body weight data (13 d.f.) was 4. 3 As in footnote 1, but P < 0.01. 4 Values represent the average of two groups of 25 quail per treatment. 5 Since 100% mortality occurred before completion of the experiment, this group was not included in statistical analysis. 6 Efficiency of feed utilization, 0-14 days of age, for basal treatment only. 7 Values represent the average of three groups of 50 quail per treatment. 25 quail per treatment were fed the basal diet alone and supplemented with 5, 7.5, 10 and 15 mg. of pantothenic acid/kg. Triplicate groups of 50 quail per treatment were supplied the basal diet supplemented with 20.0, 25.0 and 30.0 mg. of pantothenic acid/kg. Levels above 15 mg./kg. were used when preliminary observations suggested the requirement for pantothenic acid by Bobwhite quail might be considerably higher than was supposed. The results of this study (Table 6) showed that the basal diet was deficient in pantothenic acid since all quail in this treatment died. Each increment in the pantothenic acid content of the diet resulted in a marked increase i n growth up to 15 mg. / kg. of added pantothen-
ic acid. Further additions had no significant effect upon growth. Feed utilization was improved by the addition of 5 and 7.5 mg. of pantothenic acid/kg. compared to the basal ration. No statistically significant change in feed utilization was observed from additions of pantothenic acid in excess of 7.5 mg./kg. Mortality was markedly reduced by adding pantothenic acid to the basal ration. Among those treatments supplied purified diets there was no significant influence on mortality by additions of pantothenic acid greater than 10.0 mg./kg. No quail fed the basal diet survived longer than 21 days. Quail supplied the unsupplemented diet grew slowly and exhibited poor feathering. This diet was found to contain 0.12 mg. of pantothenic acid/kg. by microbiological assay. Although perosis, skin lesions, and crusty scabs about the beak and eyes have been observed in chicks and young pheasants suffering from a pantothenic acid deficiency, none of these conditions was observed in this study among quail fed deficient diets. Nearly all mortality among quail fed the basal diet and that containing 5.0 mg./kg. of added pantothenic acid occurred
TABLE 7.—Experiment 3, Trial 2: Effect of pantothenic acid, supplementation of1 a2 purified diet for young Bobwhite quail ' Treatment Dietary pantothenic acid Added Total mg./kg. of diet
Body weight' Feed/4 (35 days) gain Grams
Mortality Percent
10.0 10.12 84a 2.25a 14" 12.5 12.62 88" 2.26a 15a 15.0 15.12 89b 2.20" 9ab Practical starter 90b 2.60b 5b 1 Values not having the same letter superscript differ significantly (P < 0.05). 2 Values represent the average of six groups of 25 quail per treatment. 3 The error mean square for the body weight data (20 d.f.) was 4.8. 4 As in footnote 1, but P < 0.01.
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among birds between 14 and 21 days of age; all chicks in these groups were emaciated and weakened. A second trial containing six groups of 25 quail per treatment was conducted to evaluate more precisely the requirement for pantothenic acid. In this trial 10.0, 12.5, and 15.0 mg. of pantothenic acid/kg. were included in the diets. The data from this trial appears in Table 7 and shows a similar response to that observed in Trial 1. Quail fed levels of 10 and 15 mg./kg. of added pantothenic acid grew at essentially the same rate in each trial. Although the addition of 12.5mg. of pantothenic acid/kg. resulted in a significant increase in growth compared with quail fed the ration containig 10.0 mg. of added pantothenic acid/kg. ,theadditionof 15.0mg. of pantothenic acid/kg. produced no better growth than was observed when 12.5 mg./kg. was included in the diet. Growth on the purified diets was equal to that obtained when quail were supplied a practical starter ration. There was no statistically significant influence on feed utilization by additonal supplementation of the purified diet with pantothenic acid compared to the response obtained when 10.0 mg. /kg. of added pantothenic acid was present. No statistically significant changes in mortality occurred from supplementing the purified diet with 10.0, 12.5 or 15.0 mg. of pantothenic acid/kg. From the results of these trials it was possible to determine a pantothenic acid requirement for young Bobwhite quail. Since the addition of 15 mg. of pantothenic acid/kg. failed to improve growth or feed utilization and had no influence on mortality compared to the response obtained when a diet containing 12.62 mg. of pantothenic acid/kg. was supplied quail, it appears that the quantitative requirement for pantothenic acid to insure satisfactory growth, feed utUization and low mortality is approximately 12.6 mg./kg. of diet. This value is considerably higher than others which have been reported. Scott et
al. (1964) indicated the minimum pantothenic acid requirement for growth of quail was 10 mg./kg. The requirement established for young pheasants and quail by the National Research Council (1971) is also 10 mg./kg. This value is the same as that recommended for the chick and slightly lower than that established for the poult. From the results of this study it would appear that a value of 10 mg./kg. is somewhat low since an improvement in growth was observed when quail were fed diets containing amounts of pantothenic acid greater than 10 mg./kg. Based upon these results it would appear that rations for young quail should contain approximately 15 mg. of pantothenic acid/kg. to insure meeting minimum needs. Most unsupplemented practical quail rations, however, contain less than this amount, indicating that the vitamin may be present in inadequate or marginal levels. This would suggest that addition of pantothenic acid in amounts as high as 10 mg./kg. to practical rations may be necessary to provide a sufficient quantity to satisfy the minimum requirement and provide an adequate margin for safety. Choline. Experiment 4 consisted of two trials conducted to study the choline requirement of Bobwhite quail. In the first trial the basal diet was fed alone and supplemented with choline at levels of 500, 1000, 1500 and 2000 mg./kg. The levels above 1000 mg./kg. were chosen since the requirements for the chick, young pheasant and poult are approximately 1300, 1500 and 1900 mg./kg., respectively, (N.R.C., 1971; Howes and Beane, 1966) and it was postulated that the requirement for quail would be in this range. Two additional treatments were included in these trials in which choline was supplied in the diet as a dry, commercial premix consisting of 25% choline chloride according to the manufacturer's analysis. Diets were formulated to contain 500 and 1000 mg. of cholin-
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TABLE 8.—Experiment 4: Effect of choline supplementation of a purified diet for young Bobwhite quail'-2 Treatment Dietary choline Added Total mg./kg. 0 0.1 500 500.1 1000 1000.1 1500 1500.1 2000.1 2000 2500 2500.1 5006 10006 Practical starter
Body weight3 (35 days) Trial 1 Trial 2 Grams 51" 76a 88"° 97cd
98d
— 80* 94cd 92cd
—
72a 88 b 94 bc 94bc
96 c 72a 91bc
—
Feed/ gain Trial 1 Trial 2
2.57 2.34bc 2.28 ab 2.33 bc 2.20 s
—
2.41 c 2.30 abc 2.53 d
—
2.345 2.24 2.27 2.33 2.26 2.44 2.32
—
Mortality Trial 1 Trial 2 Percent 39 85 12 10 10
— 18 12 11
—
245 15 10 9 12 30 10
—
1 Values represent the average of two groups of 25 quail per treatment in each trial, except for the basal treatment. 2 Values not having the same letter superscript differ significantly (P < 0.05). 3 The error mean squares for the body weight data (7 d.f.) were 13 (Trial 1) and 9.6 (Trial 2). 4 Average of 25 quail. This group was not included in statistical analysis. 5 None of the treatment differences were statistically significant (P < 0.05). 6 Supplied as a dry, commercial premix, 25% choline chloride. e/kg. with this preparation for comparison with similar treatments receiving choline supplied as a 70% choline chloride solution. Trial 2 was similar to the first except that an additional treatment was included in which the diet contained 2500 mg. of choline/kg. The data from these trials are shown in Table 8. The response to additions of choline to the basal diet was virtually identical in both trials. The results obtained from supplying choline in the form of a dry, commercial premix consisting of 25% choline chloride were not significantly different from the response observed when choline was added as a 70% choline chloride solution, indicating that the potency of the premix was consistent with the stated value. Growth was satisfactory at levels of 1500 mg. of added choline/ kg. or more compared to results obtained with a practical starter diet. It is interesting to note that modest growth occurred when quail were supplied the basal ration. This contrasts with results from similar groups in Experiments 1, 2 and 3, when quail supplied basal diets essentially devoid of riboflavin,
niacin and pantothenic acid, respectively, grew poorly and failed to live beyond 14-21 days of age. The basal diet used in this study contained no more than 100 mg. of choline/ kg. by analysis. The response by quail supplied this diet raises a question concerning the ability of quail to survive on a diet containing an extremely small amount of choline. It is well known that chicks can synthesize choline from monomethylethanolamine or dimethylethanolamine, and that with adequate dietary methionine, supplemental dimethylethanolamine can overcome a choline deficiency. Although it has been reported that de novo biosynthesis of choline occurs in animals (Bremer et ah, 1960) there appears to be no evidence suggesting this plays a significant role in satisfying the requirement for choline in birds. Whether quail can grow with such small amounts of dietary choline as was present in the basal ration or whether minimal needs are furnished by synthesis from precursors or by other means remains unclear at this time. Quail supplied the basal ration developed
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J. A. SERAFIN
enlarged hocks and slipped tendons by 12 days of age. These changes are also associated with a choline deficiency in the chick. The leg bones of quail supplied the basal diet were also relatively short and stocky, resembling a condition observed in chicks suffering from an inadequacy of zinc (Young et al, 1958; N.R.C., 1971). Quail so affected walked with an awkward gait resulting from enlarged and stiffened joints as well as movement on their hocks instead of their toes and heels. Addition of 500 mg. of choline/kg. reduced the incidence of leg shortening but did not prevent enlarged hocks and slipped tendons in virtually all birds in this treatment. Addition of 1000 mg. of choline/kg. of diet prevented both of these conditions but did not seem to be sufficient for normal growth. Examination of the body weight data indicated that growth was significantly less when 500 mg. of choline/kg. was added to the diet compared to treatments receiving additions of 1000 mg. of choline/kg. or more. There was considerable variation among average weights of groups of quail within treatments in this experiment (as well as in Experiment 2, Trial 2). This variation was greater than occurred in other studies and precluded significant differences from being demonstrated between dietary treatments where data suggested an improvement in growth had taken place. The results obtained from supplying quail the basal diet supplemented with choline inamounts ranging from 1000 to 2500 mg. /kg. demonstrates this effect. Although the response to 1000 mg. of added choline/kg. to the diet was not significantly different than that obtained with some higher levels, maximum growth was not achieved with this level of supplementation. Including choline in diets at levels of 2000 and 2500 mg./kg. failed to improve growth significantly compared to results obtained with 1500 mg. of choline/kg. These responses introduced some uncertainty in determining the choline requirement. However, the data do suggest that Bobwhite
quail require no more than 1500 mg. of choline/kg. diet for normal growth under the conditions of this study. It should be noted that the diet used in this experiment contained 25.5% protein and 0.7% added DL-methionine giving a total sulfur amino acid content of approximately 1.31% (5.14% of protein). In a study of the protein and methionine requirements for Bobwhite quail it was found that they require between 0.95 and 1.0% total sulfur amino acids for maximum growth. 2 It is believed that the level of methionine necessary for normal growth is related to the need for choline since choline can spare methionine used for transmethylation purposes. In studies where DL-methionine was omitted from the basal ration (Table 1) choline did not support normal growth even at levels in excess of 2500 mg./kg. On the other hand, with much lower levels of choline in the diet, quail grew well, weighing approximately 95 grams at 5 weeks of age when 0.24% DL-methionine was added to the basal diet. 2 This information suggests that the studies presented in Experiment 4 were conducted using diets containing an ample amount of methionine, eliminating a sparing action by choline. GENERAL DISCUSSION Results of these experiments demonstrate that Bobwhite quail fed a purified diet adequately supplemented with nutrients grow well. Purified diets of the kind used in these studies support excellent growth in White Plymouth Rock and other domestic chicks (Scott et al, 1969). Since very little research has been conducted with Bobwhite quail using purified diets, there was some uncertainty whether such a diet would support satisfactory growth and survival. Consequently a preliminary effort was made to determine whether the diet selected for these studies
2. Unpublished data, J. A. Serafin.
QUAIL VITAMIN REQUIREMENTS
was low or deficient in nutrients other than the vitamins studied. No deficiencies were found, and since survival and growth appeared normal among control groups receiving the purified diets, it was assumed that adequate amounts of other nutrients were incorporated in the rations. The protein level selected for this investigation was 25.5%, an amount slightly lower than is sometimes recommended for practical starting rations (Howes and Beane, 1966). DL-methionine was included to reduce the possibility of this amino acid limiting growth. Results from Experiments 2, 3 and 4 showed that when the basal purified diets were adequately supplemented with vitamins, growth rates were identical with those from groups fed a practical ration (Table 3) indicating that the purified diet used in this investigation was adequate in protein and other nutrients. Mortality during the five week experiments generally was less than 10% when diets and environmental factors were optimal. By maintaining the brooding temperature at 105° F. during the first 5-7 days of age of quail, it has been possible to prevent mortality during this period from exceeding 1-2%. Virtually no mortality occurred in groups of quail maintained beyond five weeks of age on purified diets, and these birds appeared no different from comparable groups raised on a practical ration. Scott et al. (1964) reported 90-100% mortality among Bobwhite quail fed a purified diet, and suggested they may have a requirement for an unknown factor. In their studies, a diet containing corn starch and isolated soybean protein was used, while a diet containing glucose and isolated soybean protein was used in the studies reported here. The vitamin mixtures used in these two investigations differed somewhat both quantitatively and qualitatively, however, which may account for the differences in survival. Interestingly, characteristic signs of vitamin deficiencies in chicks were not always
1531
observed when the corresponding deficiency was produced in young quail. None of the quail fed diets deficient in riboflavin developed curled toes, a common indication of the absence of the vitamin from diets fed young chicks. Aside from reduced growth and poor feathering, none of the other conditions observed when chicks and pheasants are supplied diets containing deficient amounts of pantothenic acid were observed when quail were supplied a similarly deficient diet in Experiment 3. Spivey-Fox et al. (1966) found no dermatitis or feather depigmentation among Coturnix quail fed a pantothenic aciddeficient diet, indicating that this species of quail may also differ from the chick in its response to inadequate levels of dietary vitamins. Quail fed diets containing insufficient niacin for normal growth in Experiment 2 failed to develop enlarged tibiotarsal joints, bowed legs or a dermatitis, conditions often found in the domestic chick. Young quail supplied these diets, however, developed a severe erythema about the head. This condition is one of the early signs of a nicotinic acid deficiency in man, but does not appear to have been observed before in birds. From a review of the nutrition of Coturnix quail (Vohra, 1971), it is apparent that although knowledge of this species has increased considerably since its introduction as a laboratory animal in 1957, precise requirements for most of the water soluble vitamins remain unreported for it as well as for Bobwhite quail. A comparison of values which have been determined shows that some differences exist between these two closely related species. Spivey-Fox et al. (1966) reported that Coturnix quail required as much as 30 mg. of pantothenic acid/kg. of diet early in life to support normal growth and feathering. The results in Experiment 3 showed that 15 mg. of pantothenic acid/kg. of diet appeared to support normal growth and development with no indication of poor feathering, skin lesions or other abnormal-
1532
J. A. SERAFIN
ities. Shellenberger and Lee (1966) have pointed out that the vitamin A requirement of young Coturnix quail and Bobwhite quail differ considerably. Further comparisons cannot be made now since precise requirements of both species for other vitamins are lacking. Studies of additional vitamin requirements of Coturnix and Bobwhite quail are needed before it will be possible to determine whether significant differences exist between the two species. ACKNOWLEDGMENTS The capable technical assistance of Kathleen Kennedy and Mrs. Bertha O. Preston is gratefully acknowledged. REFERENCES Association of Official Agricultural Chemists, 1960. Official Methods of Analysis, 9th Ed. Assoc, of Official Agr. Chemists, Washington, D.C. Bremer, J., P. H. Figard and D. M. Greenberg, 1960. The biosynthesis of choline and its relation to phospholipid metabolism. Biochim. Biophys. Acta, 43: 477. Duncan, D. R., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Entenman, C , A. Taurog and I. L. Chaikoff, 1944. The determination of choline in phospholipids. J. Biol. Chem. 155: 13-18. Erickson, R. C , 1968. A federal research program for endangered species. Transactions of the Thirtythird North American Wildlife and Natural Resources Conference, March 11-13, 1968. Wildlife Management Institute, Washington, D.C. 20005. Howes, J. R., and W. L. Beane, 1966. The nutrition of pheasants, Bobwhite and Coturnix quail. Feedstuffs, 38: 18-21.
Mitchell, H. H., 1964. Comparative Nutrition of Man and Domestic Animals. Vol. II, Academic Press, New York, pgs. 95-107. National Research Council, 1971. Nutrient Requirements of Poultry. Publication 01861-7, 2101 Constitution Avenue, Washington, D.C. 20418. 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, 1965. U.S. Pharmocopeial Convention, Inc., 17th Ed., Washington, D . C , Calcium Pantothenate Assay, 840-842. Scott, M. L., E. R. Holm and R. E. Reynolds, 1959. Studies on the niacin, riboflavin, choline, manganese and zinc requirements of young Ringnecked pheasants for growth, feathering and prevention of leg disorders. Poultry Sci. 38: 1344-1350. Scott, M. L., E. R. Holm and R. E. Reynolds, 1964. Studies on the pantothenic acid and unidentified factor requirements of young Ringnecked pheasants and Bobwhite quail. Poultry Sci. 43: 1534-1539. Scott, M. L., M. C. Nesheim and R. J. Young, 1969. Nutrition of the Chicken. M. L. Scott and Associates, Ithaca, New York. Shellenberger, T. E., and J. M. Lee, 1966. Effect of vitamin A on growth, egg production and reproduction of Japanese quail. Poultry Sci. 45: 708-713. Spivey-Fox, M. R., G. A. Hudson and M. E. Hintz, 1966. Pantothenic acid requirement of young Japanese quail. Fed. Proc. 25: 721. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics, McGraw-Hill, New York. Vohra,P., 1971. A review of the nutrition of Japanese quail. World's Poultry Sci. J. 27: 26-34. Wyatt, R. D., H. T. Tung, W. E. Donaldson and P. B. Hamilton, 1973. A new description of riboflavin deficiency syndrome in chickens. Poultry Sci. 52: 237-244. Young, R. J., H. M. Edwards, Jr. and M. B. Gillis, 1958. Studies on zinc in poultry nutrition. Poultry Sci. 37: 1100-1107.
AUGUST 4-8. ANNUAL MEETING OF AGRICULTURAL INSTITUTE OF CANADA AND AFFILIATED SOCIETIES. LAVAL UNIVERSITY, QUEBEC, QUEBEC. AUGUST 5-8. 63RD ANNUAL MEETING OF THE POULTRY SCIENCE ASSOCIATION, INC. WEST VIRGINIA STATE UNIVERSITY, MORGANTOWN, WEST VIRGINIA. AUGUST 11-17. FIFTEENTH WORLD'S POULTRY CONGRESS, NEW ORLEANS, LOUISIANA. AUGUST 15-17. ANNUAL MEETING OF THE INTERNATIONAL EGG COMMISSION, NEW ORLEANS, LOUISIANA.
United States Fish & Wildlife Service, Patuxent Wildlife Research Center, Laurel, Maryland 20810 (Received for publication November 23, 1973)
ABSTRACT Four experiments were conducted to examine the riboflavin, niacin, pantothenic acid and choline requirements of young Bobwhite quail. Quail fed purified diets deficient in either riboflavin, niacin, pantothenic acid or choline grew poorly and high mortality occurred by 5 weeks of age. Under the conditions of these experiments, it was found that: (1) young quail require approximately 3.8 mg. riboflavin/kg. diet for satisfactory growth and survival; (2) no more than 31 mg. niacin/kg. diet are required for normal growth and survival of young quail; (3) the requirement for pantothenic acid is higher than has previously been reported, quail in these studies requiring 12.6 mg. pantothenic acid/kg. feed for growth and survival; and (4) the requirement for choline for reducing mortality is approximately 1000 mg./kg., while the amount necessary for normal growth of young quail is no greater than 1500 mg./kg. when the diet contains ample amounts of methionine. Quail fed a niacin-deficient diet developed stiff, shortened feathers and an erythema about the head; those receiving a riboflavin-deficient ration developed enlarged hocks and bowed legs, as did quail fed diets low or devoid of choline. Aside from slow growth, poor feathering was the only other indication that a deficient diet was being fed when quail were placed on a basal ration without pantothenic acid for five weeks. POULTRY SCIENCE 53: 1522-1532, 1974
nutrients for Bobwhite quail. Very few reports exist describing vitamin deficiency NCREASING numbers of Bobwhite quail states in quail. In view of the limited informa(Colinus virginianus) are being raised by tion available on nutrient requirements and the game bird industry for hunting preserves considering the increased interest in Boband for table food. Use of Bobwhite quail white quail, a study was begun to evaluate to study the influence of pesticides and other nutrient needs of this species. This report pollutants in the environment has also in- describes the results of a series of expericreased recently. For the past several years ments with purified diets designed to investia program has been in progress to propagate gate the riboflavin, niacin, pantothenic acid masked Bobwhite quail (Colinus virginianus and choline requirements of Bobwhite quail. ridgwayi) in an effort to prevent possible Characteristics of these vitamin deficiencies extinction of the species (Erickson, 1968). are also compared with classical descriptions The requirements of Bobwhite quail for most for the chick and other domestic species. nutrients have not been specifically identiEXPERIMENTAL PROCEDURES fied. A review of the nutrition of pheasants (Phasianus colchicus), Bobwhite and CoturTrials of five week duration were conductnix quail (Coturnix coturnix japonica) by ed during all seasons of the year with quail Howes and Beane (1966) indicated that few of mixed sex. Hatching eggs were obtained studies have been conducted to determine from a breeding flock raised and maintained specific vitamin requirements of quail. The to provide production during all months of National Research Council, which recently the year. Seasonal variation in production reviewed nutritional needs for poultry, Bob- resulted in fewer chicks for some trials than white quail and pheasants (N.R.C., 1971), for others. Quail were housed in electrically lists specific requirement values of only six heated, thermostatically controlled battery INTRODUCTION
I
1522
1523
QUAIL VITAMIN REQUIREMENTS
TABLE 1.—Composition of basal diet used in studies of vitamin requirements of young Bobwhite quail Amount (percent) Glucose 58.69 Isolated soybean protein1 27.00 Soybean oil 3.00 Cellulose 3.00 Vitamin mixture2 1.00 Mineral mixture3 6.31 Glycine 0.30 DL-methionine 0.70 Protein (N x 6.25) 25.50 1 RP-100, Ralston-Purina Company, St. Louis, Missouri. 2 The composition of the vitamin mixtures used in these studies is shown in Table 2. 3 Supplied the following per kilogram of diet: (in grams) CaHPCy2H 2 0, 21.6; CaC03, 18.3; KH2P04, 13.9; NaCl, 6.0; MgS04, 2.5; (in milligrams) FeS0 4 -7H 2 0, 333.0; MnSCyH 2 0, 333.0; KI, 2.6; CuSCy5H 2 0, 16.7; CoCl2-6H20, 1.6; Na2Mo04-2H20, 8.3; ZnC03, 115; and Na2Se03, 0.22. Ingredient
brooders with raised wire floors located in a room with continuous forced draft ventilation held at a temperature of approximately 70° F. The brooding temperature was maintained at 105° F. for the first week of each experiment. The average weight of quail at the beginning of an experiment ranged between 6.9 and 7.1 grams. All birds were debeaked at 5 days of age to prevent picking. Feed and water were supplied ad libitum. The composition of the basal purfied diet and vitamin mixtures used in these studies is shown in Tables 1 and 2. In the few instances where purified diets have been fed to Bobwhite quail poor growth responses and high mortality were observed (Scott et al., 1964). For this reason a practical quail starter ration was fed groups of quail to evaluate responses of quail fed purified diets. The composition of this ration appears in Table 3. The pantothenic acid content of the pantothenic acid-deficient basal diet was deter-
TABLE 2.— Vitamin levels used in vitamin mixes for studies of riboflavin, niacin, pantothenic acid and choline requirements of young Bobwhite quail Vitamin1 Thiamine-HC1, mg. Riboflavin, mg. Niacin, mg. Pantothenic acid, mg. Choline, mg. Inositol, mg. Pyridoxine • HC1, mg. Folic acid, mg. Biotin, mg. Vitamin B, 2 (micrograms) Menadione sodium bisulfite, mg. Vitamin A (I.U.) Vitamin D 3 (I.U.) a-tocopheryl acetate (I.U.) Ethoxyquin, mg.
Amount/kg diet 15.0 15.02 50.03 20.04 1336.05 250.0 6.0 6.0 1.6 30.0 1.52 5000.0 4500.0 110.0 125.0
1 Water soluble vitamins were obtained from Nutritional Biochemicals Corp., Cleveland, Ohio. 2 The vitamin mixture used for the studies concerning riboflavin contained no riboflavin. 3 The vitamin mixture used for the studies concerning niacin contained no niacin. 4 The vitamin mixture used for the studies concerning pantothenic acid contained no pantothenic acid. 5 The vitamin mixture used for the studies concerning choline contained no choline. mined by a method described in the U.S. Pharmacopeia (1965) employing Lactobacillus plantarum ATCC 8014 (Difco Laboratories, Detroit, Michigan).1 Liberation of pantothenic acid from coenzyme A, which permitted 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 described in the A.O.A.C. (1960) using L. plantarum ATCC 8014. The choline content of the choline-def icient basal diet was determined by the method described by Entenman et al. (1944). The culture media for the pan-
1. Use of trade names does not imply endorsement of commercial products by the Federal Government.
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J. A. SERAFIN
TABLE 3.—Composition of practical Bobwhite quail starter ration Ingredient Yellow corn meal Dehulled soybean meal (50%) Fish meal (60%) Alfalfa meal (17%) Soybean oil Brewers dried yeast Distillers dried solubles Dried whey Dicalcium phosphate Limestone Vitamin mixture 1 Iodized salt Trace mineral mixture 2 DL-methionine Protein (N x 6.25)
Amount (percent) 44.43 35.0 6.0 3.0 3.0 2.5 2.0 1.5 1.0 0.5 0.5 0.25 0.25 0.07 29.2
1 Supplies inmg. /kg. diet: Niacin, 44.0; Calcium pantothenate, 8.8; Riboflavin, 3.3; Choline chloride, 125.0; Menadione sodium bisulfite, 2.2; Zinc bacitracin, 220.0; Ethoxyquin, 125.0; Vitamin B l2 , 6.6 meg; Vitamin A, 22068; 22068 I.U.; Vitamin D3, 1653 I.U.; a-tocopheryl acetate, 110 I.U. premixed in carrier. 2 Supplies in mg./kg. diet: MnS0 4 H 2 0, 375.1; ZnO, 124.8; FeSCy7H 2 0, 99.0; CuS0 4 -5H 2 0, 22.0; NaMoCy5H 2 0, 8.2; CoCl2-5H20, 1.7; Na2Se03, 0.22 premixed in carrier. tothenic acid assays was obtained from Difco Laboratories, and media for the niacin assays was prepared using ingredients supplied by Difco Laboratories and Nutritional Biochemicals Corp., Cleveland, Ohio. U.S.P. reference standards were obtained from United States Pharmacopeia Convention, Inc., Bethesda, Md. Data were tested for significance using the analysis of variance (Steel and Torrie, 1960). Differences between treatment means were tested using Duncan's multiple range test (Duncan, 1955). Percentages were transformed to arc-sin values before statistical treatment. RESULTS AND DISCUSSION Riboflavin. To study the riboflavin requirement, the basal diet was fed alone and sup-
plemented with riboflavin at 0.75, 1.5, 2.5, 3.5 and 5.0 mg./kg. These levels of addition were selected because the requirements by other species of birds which have been studied are known to be less than 5.0 mg./kg. Each diet was supplied to two pens of 25 quail each. The results are presented in Table 4. Growth was significantly improved by additions of riboflavin up to 3.5 mg./kg. in the purified diets. The response of quail fed the purified diet supplemented with 3.5 and 5.0 mg. of added riboflavin/kg. compared well with the results from those supplied a practical ration. Mortality in these groups was not excessively high and feed utilization was not significantly different from that for quail fed a starter diet. Feed utilization and mortality was not significantly influenced by additions to the purified diet of riboflavin beyond 2.5 mg./kg. A level of 5.0 mg. of added riboflavin per kg. of feed did not improve growth rate or decrease mortality beyond that observed with 3.5 mg. of added riboflavin/kg. in the diet. TABLE 4.—Experiment 1: Effect of riboflavin supplementation of a basal purified diet for young Bobwhite quail'2 Treatment Dietary riboflavin Added
Total 4
mg./kg. of diet 0 0.75 1.50 2.50 3.50 5.00 Practical
0.34 1.09 1.84 2.84 3.84 5.34 starter
Body ' weight 3 (35 days)
Feed/ gain
Mortality Percent
10.80 4.59 s 3.32b 2.13 c 2.15° 2.17° 2.48c
98 86" 59" 27= 27c 28c llc
Grams 165 24 s 33 b 66c 79" 80" 86e
1 Values represent the average of two groups of 25 quail per treatment. 2 Values not followed by the same letter superscript differ significantly (P < 0.01). 3 The error mean square for the body weight data (6 d.f.) was 2.5. 4 Determined by calculation. 5 Only one bird remained alive at the completion of the experiment in the basal treatment and the group was not included in statistical analysis.
1525
QUAIL VITAMIN REQUIREMENTS
Growth was extremely slow among quail fed the basal diet alone and the basal supplemented with 0.75 mg./kg. of added riboflavin. Quail were emaciated, weak, and unthrifty. Mortality ranged from 83% to 100% among these groups. Most of the deaths occurred between the 14th and 21st days of age. Only one chick survived the 35 day experimental period among the groups fed the basal diet. Quail fed this diet and the basal supplemented with 0.75 mg. of riboflavin/kg. developed enlarged hocks and bowed legs. However, no incidence of paralysis of the legs, also called "curled toe paralysis," a symptom normally associated with a deficiency of riboflavin in chicks, was observed among young quail. This observation is consistent with recent findings by Wyatt et al. (1973) who studied symptoms of riboflavin deficiency in three strains of broiler chicks and found a low incidence of curled toe paralysis. It has been generally believed that curled toe paralysis is the most obvious symptom among chicks suffering a lack of riboflavin (N.R.C., 1971). Wyatt et al. (1973) concluded that while riboflavin deficiency can cause curled toe paralysis, such a deficiency does not cause it obligatorily and that curled toe paralysis is a minor aspect of
riboflavin deficiency in modern broiler chickens. This appears to be true also in the case of Bobwhite quail. No descriptions of a riboflavin deficiency in Bobwhite quail seem to have been previously reported. The findings in this study indicate that curled toe paralysis is not associated with a lack of riboflavin in diets for yong quail. Based upon the response to additions of riboflavin to a purified diet and a comparison with the results obtained from feeding a practical starter diet to quail it was possible to estimate the approximate riboflavin requirement. No improvement was found in growth, appearance, feed utilization or reduced mortality by adding more than 3.5 mg. of riboflavin/kg. to the diet. Since the basal diet contained approximately 0.34 mg. of riboflavin/kg., it appears that under the conditions of this experiment, the total riboflavin requirement of young Bobwhite quail is no greater than 3.8 mg./kg. The results of this study are similar to those of Scott et al. (1959) with young pheasants. These investigators found the riboflavin requirement for young pheasant chicks to be approximately 3.4 mg./kg. Quail and pheasant chicks appear to have a nearly identical requirement which is only slightly higher than
TABLE 5.—Experiment 2: Effect of niacin supplementation of a purified basal diet for young Bobwhite quail12 Treatment Dietary niacin Added
Total
mg./kg. of diet 0 1.2 15 16.2 30 31.2 60 61.2 75 76.2 Practical starter
Body weight3 (35 days) Trial 1 Trial 2 Grams 37a 81" 96c 95c 96c 96c
13a 68b 78 b 78" 79" 85"
Feed/gain Trial 1 Trial 2 4.30" 2.41" 2.25" 2.30" 2.32 b 2.54"
4.58 a 2.48" 2.24b 2.40 b 2.42b 2.60"
Mortality Trial 1 Trial 24 Percent 88a 10b 4»
4b 4" 8b
90" 36b 21b 13b 18b 7
b
1 Values represent the average of two groups of 13 quail per treatment in Trial 1 and 20 quail per treatment in Trial 2. 2 Values not having the same letter superscript differ significantly (P < 0.01). 3 The error mean squares for the body weight data (6 d.f.) were 12 (Trial 1) and 81 (Trial 2). 4 As in footnote 2, but P < 0.05.
1526
J. A. SERAFIN
the amount of riboflavin needed by the chick and poult. Mitchell (1964) in a review of the riboflavin requirement for a variety of animals including the mouse, rat, dog, piglet, poultry and man indicated that variation in the requirement for riboflavin generally ranged within the limits of 1-3 mg./kg. of food. There appeared to be no correlation between the amount of riboflavin required and species size. The observed requirement of various species for this vitamin seems to fall within a narrow range compared with requirements for other vitamins such as choline or vitamin A which are known to differ considerably. The results from the experiment reported here suggest that the riboflavin requirement of Bobwhite quail is only slightly higher than accepted values for most other species studied. Niacin. Two trials were conducted to observe the response by Bobwhite quail to additions of niacin to a purified, deficient diet. Treatments consisted of duplicate groups of 13 Bobwhite quail supplied the basal diet alone or supplemented with levels of niacin ranging from 15 to 75 mg./kg. of diet in Trial 1 (Table 5). Trial 2 was identical to Trial 1 except that duplicate groups of 20 quail were used for each treatment. Results showed that growth was improved by additions of 15 and 30 mg. of niacin/kg. of diet but was not influenced by additions beyond 30 mg. /kg. In Trial 1, quail supplied the basal diet supplemented with 15 mg. of niacin/kg. grew significantly less than those treatments receiving greater amounts of niacin. Feed utilization was significantly improved in both trials by addition of niacin to the basal ration. Growth, feed utilization and mortality of quail supplied diets containing additions of 30 mg. of niacin/kg. or more were essentially the same as responses observed among quail fed the practical ration which contained approximately 80 mg. of niacin/kg. by calculation. Growth was severely retarded among quail
fed the basal diet and reduced among quail supplied the basal supplemented with 15 mg. of niacin/kg. Most of the mortality that was observed in these groups occurred between 7 and 21 days of age. Surviving quail on these diets developed stiff, shortened body feathers, while no feathers were present on their heads. Many quail developed an erythema, principally in the region of the head. No evidence of an enlargement of the tibiotarsal joint or of leg bowing was observed among quail fed niacin deficient diets. An estimate of the niacin requirement for Bobwhite quail was made with data from these trials. Since quail 5 weeks of age can be expected to obtain a weight of 95-100 grams when supplied a complete ration, it is apparent that satisfactory growth was not achieved in Trial 2. The reason for this is not known. Although quail grew at a slower rate in Trial 2 than was expected, the responses to the addition of niacin were essentially the same in both trials. A statistically significant growth response was obtained by supplementing the diet with 30 mg. of niacin/kg. compared to 15 mg./kg. in Trial 1, but not in Trial 2, although it appeared that 15 mg./kg. was insufficient to permit a high rate of growth in both studies. Analysis of the basal purified diet indicated a niacin content of approximately 1.2 mg./kg. Based upon these results it would appear that Bobwhite quail require no more than 31.2 mg. of niacin/kg. for maximum growth. This amount is similar to the requirement for the young chick (N.R.C., 1971), but is considerably lower than the amount required by the young poult and pheasant chick (N.R.C., 1971) to provide for growth and survival. Pantothenic Acid. The pantothenic acid requirement of Bobwhite quail was examined in two trials in which graded levels of calcium pantothenate were added to the basal diet to supply pantothenic acid in amounts up to 30 mg./kg. In Trial 1 duplicate groups of
1527
QUAIL VITAMIN REQUIREMENTS
TABLE 6.—Experiment 3, Trial 1: Effect of pantothenic acid supplementation of a purified diet for young Bobwhite quail' Treatment Dietary pantothenic acid Added
Total
mg./kg. of diet
0 5 7.5 10.0 15.0
20.0 25.0 30.0 Practical
Body weight2 Feed/ (35 days) gain' Mortality Grams
0.12 5.12 7.62 10.12 15.12
Part l 4 05 39 s 72" 80 b 90 c
4.13 6 3.74a 2.04" 2.16b 2.21 b
20.12 25.12 30.12 starter
Part 2 7 90° 90 c 91 c 91<=
2.23 b 2.21" 2.19b 2.60"
1 Values not having the same letter superscript differ significantly (P < 0.05). 2 The error mean square for the body weight data (13 d.f.) was 4. 3 As in footnote 1, but P < 0.01. 4 Values represent the average of two groups of 25 quail per treatment. 5 Since 100% mortality occurred before completion of the experiment, this group was not included in statistical analysis. 6 Efficiency of feed utilization, 0-14 days of age, for basal treatment only. 7 Values represent the average of three groups of 50 quail per treatment. 25 quail per treatment were fed the basal diet alone and supplemented with 5, 7.5, 10 and 15 mg. of pantothenic acid/kg. Triplicate groups of 50 quail per treatment were supplied the basal diet supplemented with 20.0, 25.0 and 30.0 mg. of pantothenic acid/kg. Levels above 15 mg./kg. were used when preliminary observations suggested the requirement for pantothenic acid by Bobwhite quail might be considerably higher than was supposed. The results of this study (Table 6) showed that the basal diet was deficient in pantothenic acid since all quail in this treatment died. Each increment in the pantothenic acid content of the diet resulted in a marked increase i n growth up to 15 mg. / kg. of added pantothen-
ic acid. Further additions had no significant effect upon growth. Feed utilization was improved by the addition of 5 and 7.5 mg. of pantothenic acid/kg. compared to the basal ration. No statistically significant change in feed utilization was observed from additions of pantothenic acid in excess of 7.5 mg./kg. Mortality was markedly reduced by adding pantothenic acid to the basal ration. Among those treatments supplied purified diets there was no significant influence on mortality by additions of pantothenic acid greater than 10.0 mg./kg. No quail fed the basal diet survived longer than 21 days. Quail supplied the unsupplemented diet grew slowly and exhibited poor feathering. This diet was found to contain 0.12 mg. of pantothenic acid/kg. by microbiological assay. Although perosis, skin lesions, and crusty scabs about the beak and eyes have been observed in chicks and young pheasants suffering from a pantothenic acid deficiency, none of these conditions was observed in this study among quail fed deficient diets. Nearly all mortality among quail fed the basal diet and that containing 5.0 mg./kg. of added pantothenic acid occurred
TABLE 7.—Experiment 3, Trial 2: Effect of pantothenic acid, supplementation of1 a2 purified diet for young Bobwhite quail ' Treatment Dietary pantothenic acid Added Total mg./kg. of diet
Body weight' Feed/4 (35 days) gain Grams
Mortality Percent
10.0 10.12 84a 2.25a 14" 12.5 12.62 88" 2.26a 15a 15.0 15.12 89b 2.20" 9ab Practical starter 90b 2.60b 5b 1 Values not having the same letter superscript differ significantly (P < 0.05). 2 Values represent the average of six groups of 25 quail per treatment. 3 The error mean square for the body weight data (20 d.f.) was 4.8. 4 As in footnote 1, but P < 0.01.
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J. A. SERAFIN
among birds between 14 and 21 days of age; all chicks in these groups were emaciated and weakened. A second trial containing six groups of 25 quail per treatment was conducted to evaluate more precisely the requirement for pantothenic acid. In this trial 10.0, 12.5, and 15.0 mg. of pantothenic acid/kg. were included in the diets. The data from this trial appears in Table 7 and shows a similar response to that observed in Trial 1. Quail fed levels of 10 and 15 mg./kg. of added pantothenic acid grew at essentially the same rate in each trial. Although the addition of 12.5mg. of pantothenic acid/kg. resulted in a significant increase in growth compared with quail fed the ration containig 10.0 mg. of added pantothenic acid/kg. ,theadditionof 15.0mg. of pantothenic acid/kg. produced no better growth than was observed when 12.5 mg./kg. was included in the diet. Growth on the purified diets was equal to that obtained when quail were supplied a practical starter ration. There was no statistically significant influence on feed utilization by additonal supplementation of the purified diet with pantothenic acid compared to the response obtained when 10.0 mg. /kg. of added pantothenic acid was present. No statistically significant changes in mortality occurred from supplementing the purified diet with 10.0, 12.5 or 15.0 mg. of pantothenic acid/kg. From the results of these trials it was possible to determine a pantothenic acid requirement for young Bobwhite quail. Since the addition of 15 mg. of pantothenic acid/kg. failed to improve growth or feed utilization and had no influence on mortality compared to the response obtained when a diet containing 12.62 mg. of pantothenic acid/kg. was supplied quail, it appears that the quantitative requirement for pantothenic acid to insure satisfactory growth, feed utUization and low mortality is approximately 12.6 mg./kg. of diet. This value is considerably higher than others which have been reported. Scott et
al. (1964) indicated the minimum pantothenic acid requirement for growth of quail was 10 mg./kg. The requirement established for young pheasants and quail by the National Research Council (1971) is also 10 mg./kg. This value is the same as that recommended for the chick and slightly lower than that established for the poult. From the results of this study it would appear that a value of 10 mg./kg. is somewhat low since an improvement in growth was observed when quail were fed diets containing amounts of pantothenic acid greater than 10 mg./kg. Based upon these results it would appear that rations for young quail should contain approximately 15 mg. of pantothenic acid/kg. to insure meeting minimum needs. Most unsupplemented practical quail rations, however, contain less than this amount, indicating that the vitamin may be present in inadequate or marginal levels. This would suggest that addition of pantothenic acid in amounts as high as 10 mg./kg. to practical rations may be necessary to provide a sufficient quantity to satisfy the minimum requirement and provide an adequate margin for safety. Choline. Experiment 4 consisted of two trials conducted to study the choline requirement of Bobwhite quail. In the first trial the basal diet was fed alone and supplemented with choline at levels of 500, 1000, 1500 and 2000 mg./kg. The levels above 1000 mg./kg. were chosen since the requirements for the chick, young pheasant and poult are approximately 1300, 1500 and 1900 mg./kg., respectively, (N.R.C., 1971; Howes and Beane, 1966) and it was postulated that the requirement for quail would be in this range. Two additional treatments were included in these trials in which choline was supplied in the diet as a dry, commercial premix consisting of 25% choline chloride according to the manufacturer's analysis. Diets were formulated to contain 500 and 1000 mg. of cholin-
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QUAIL VITAMIN REQUIREMENTS
TABLE 8.—Experiment 4: Effect of choline supplementation of a purified diet for young Bobwhite quail'-2 Treatment Dietary choline Added Total mg./kg. 0 0.1 500 500.1 1000 1000.1 1500 1500.1 2000.1 2000 2500 2500.1 5006 10006 Practical starter
Body weight3 (35 days) Trial 1 Trial 2 Grams 51" 76a 88"° 97cd
98d
— 80* 94cd 92cd
—
72a 88 b 94 bc 94bc
96 c 72a 91bc
—
Feed/ gain Trial 1 Trial 2
2.57 2.34bc 2.28 ab 2.33 bc 2.20 s
—
2.41 c 2.30 abc 2.53 d
—
2.345 2.24 2.27 2.33 2.26 2.44 2.32
—
Mortality Trial 1 Trial 2 Percent 39 85 12 10 10
— 18 12 11
—
245 15 10 9 12 30 10
—
1 Values represent the average of two groups of 25 quail per treatment in each trial, except for the basal treatment. 2 Values not having the same letter superscript differ significantly (P < 0.05). 3 The error mean squares for the body weight data (7 d.f.) were 13 (Trial 1) and 9.6 (Trial 2). 4 Average of 25 quail. This group was not included in statistical analysis. 5 None of the treatment differences were statistically significant (P < 0.05). 6 Supplied as a dry, commercial premix, 25% choline chloride. e/kg. with this preparation for comparison with similar treatments receiving choline supplied as a 70% choline chloride solution. Trial 2 was similar to the first except that an additional treatment was included in which the diet contained 2500 mg. of choline/kg. The data from these trials are shown in Table 8. The response to additions of choline to the basal diet was virtually identical in both trials. The results obtained from supplying choline in the form of a dry, commercial premix consisting of 25% choline chloride were not significantly different from the response observed when choline was added as a 70% choline chloride solution, indicating that the potency of the premix was consistent with the stated value. Growth was satisfactory at levels of 1500 mg. of added choline/ kg. or more compared to results obtained with a practical starter diet. It is interesting to note that modest growth occurred when quail were supplied the basal ration. This contrasts with results from similar groups in Experiments 1, 2 and 3, when quail supplied basal diets essentially devoid of riboflavin,
niacin and pantothenic acid, respectively, grew poorly and failed to live beyond 14-21 days of age. The basal diet used in this study contained no more than 100 mg. of choline/ kg. by analysis. The response by quail supplied this diet raises a question concerning the ability of quail to survive on a diet containing an extremely small amount of choline. It is well known that chicks can synthesize choline from monomethylethanolamine or dimethylethanolamine, and that with adequate dietary methionine, supplemental dimethylethanolamine can overcome a choline deficiency. Although it has been reported that de novo biosynthesis of choline occurs in animals (Bremer et ah, 1960) there appears to be no evidence suggesting this plays a significant role in satisfying the requirement for choline in birds. Whether quail can grow with such small amounts of dietary choline as was present in the basal ration or whether minimal needs are furnished by synthesis from precursors or by other means remains unclear at this time. Quail supplied the basal ration developed
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J. A. SERAFIN
enlarged hocks and slipped tendons by 12 days of age. These changes are also associated with a choline deficiency in the chick. The leg bones of quail supplied the basal diet were also relatively short and stocky, resembling a condition observed in chicks suffering from an inadequacy of zinc (Young et al, 1958; N.R.C., 1971). Quail so affected walked with an awkward gait resulting from enlarged and stiffened joints as well as movement on their hocks instead of their toes and heels. Addition of 500 mg. of choline/kg. reduced the incidence of leg shortening but did not prevent enlarged hocks and slipped tendons in virtually all birds in this treatment. Addition of 1000 mg. of choline/kg. of diet prevented both of these conditions but did not seem to be sufficient for normal growth. Examination of the body weight data indicated that growth was significantly less when 500 mg. of choline/kg. was added to the diet compared to treatments receiving additions of 1000 mg. of choline/kg. or more. There was considerable variation among average weights of groups of quail within treatments in this experiment (as well as in Experiment 2, Trial 2). This variation was greater than occurred in other studies and precluded significant differences from being demonstrated between dietary treatments where data suggested an improvement in growth had taken place. The results obtained from supplying quail the basal diet supplemented with choline inamounts ranging from 1000 to 2500 mg. /kg. demonstrates this effect. Although the response to 1000 mg. of added choline/kg. to the diet was not significantly different than that obtained with some higher levels, maximum growth was not achieved with this level of supplementation. Including choline in diets at levels of 2000 and 2500 mg./kg. failed to improve growth significantly compared to results obtained with 1500 mg. of choline/kg. These responses introduced some uncertainty in determining the choline requirement. However, the data do suggest that Bobwhite
quail require no more than 1500 mg. of choline/kg. diet for normal growth under the conditions of this study. It should be noted that the diet used in this experiment contained 25.5% protein and 0.7% added DL-methionine giving a total sulfur amino acid content of approximately 1.31% (5.14% of protein). In a study of the protein and methionine requirements for Bobwhite quail it was found that they require between 0.95 and 1.0% total sulfur amino acids for maximum growth. 2 It is believed that the level of methionine necessary for normal growth is related to the need for choline since choline can spare methionine used for transmethylation purposes. In studies where DL-methionine was omitted from the basal ration (Table 1) choline did not support normal growth even at levels in excess of 2500 mg./kg. On the other hand, with much lower levels of choline in the diet, quail grew well, weighing approximately 95 grams at 5 weeks of age when 0.24% DL-methionine was added to the basal diet. 2 This information suggests that the studies presented in Experiment 4 were conducted using diets containing an ample amount of methionine, eliminating a sparing action by choline. GENERAL DISCUSSION Results of these experiments demonstrate that Bobwhite quail fed a purified diet adequately supplemented with nutrients grow well. Purified diets of the kind used in these studies support excellent growth in White Plymouth Rock and other domestic chicks (Scott et al, 1969). Since very little research has been conducted with Bobwhite quail using purified diets, there was some uncertainty whether such a diet would support satisfactory growth and survival. Consequently a preliminary effort was made to determine whether the diet selected for these studies
2. Unpublished data, J. A. Serafin.
QUAIL VITAMIN REQUIREMENTS
was low or deficient in nutrients other than the vitamins studied. No deficiencies were found, and since survival and growth appeared normal among control groups receiving the purified diets, it was assumed that adequate amounts of other nutrients were incorporated in the rations. The protein level selected for this investigation was 25.5%, an amount slightly lower than is sometimes recommended for practical starting rations (Howes and Beane, 1966). DL-methionine was included to reduce the possibility of this amino acid limiting growth. Results from Experiments 2, 3 and 4 showed that when the basal purified diets were adequately supplemented with vitamins, growth rates were identical with those from groups fed a practical ration (Table 3) indicating that the purified diet used in this investigation was adequate in protein and other nutrients. Mortality during the five week experiments generally was less than 10% when diets and environmental factors were optimal. By maintaining the brooding temperature at 105° F. during the first 5-7 days of age of quail, it has been possible to prevent mortality during this period from exceeding 1-2%. Virtually no mortality occurred in groups of quail maintained beyond five weeks of age on purified diets, and these birds appeared no different from comparable groups raised on a practical ration. Scott et al. (1964) reported 90-100% mortality among Bobwhite quail fed a purified diet, and suggested they may have a requirement for an unknown factor. In their studies, a diet containing corn starch and isolated soybean protein was used, while a diet containing glucose and isolated soybean protein was used in the studies reported here. The vitamin mixtures used in these two investigations differed somewhat both quantitatively and qualitatively, however, which may account for the differences in survival. Interestingly, characteristic signs of vitamin deficiencies in chicks were not always
1531
observed when the corresponding deficiency was produced in young quail. None of the quail fed diets deficient in riboflavin developed curled toes, a common indication of the absence of the vitamin from diets fed young chicks. Aside from reduced growth and poor feathering, none of the other conditions observed when chicks and pheasants are supplied diets containing deficient amounts of pantothenic acid were observed when quail were supplied a similarly deficient diet in Experiment 3. Spivey-Fox et al. (1966) found no dermatitis or feather depigmentation among Coturnix quail fed a pantothenic aciddeficient diet, indicating that this species of quail may also differ from the chick in its response to inadequate levels of dietary vitamins. Quail fed diets containing insufficient niacin for normal growth in Experiment 2 failed to develop enlarged tibiotarsal joints, bowed legs or a dermatitis, conditions often found in the domestic chick. Young quail supplied these diets, however, developed a severe erythema about the head. This condition is one of the early signs of a nicotinic acid deficiency in man, but does not appear to have been observed before in birds. From a review of the nutrition of Coturnix quail (Vohra, 1971), it is apparent that although knowledge of this species has increased considerably since its introduction as a laboratory animal in 1957, precise requirements for most of the water soluble vitamins remain unreported for it as well as for Bobwhite quail. A comparison of values which have been determined shows that some differences exist between these two closely related species. Spivey-Fox et al. (1966) reported that Coturnix quail required as much as 30 mg. of pantothenic acid/kg. of diet early in life to support normal growth and feathering. The results in Experiment 3 showed that 15 mg. of pantothenic acid/kg. of diet appeared to support normal growth and development with no indication of poor feathering, skin lesions or other abnormal-
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J. A. SERAFIN
ities. Shellenberger and Lee (1966) have pointed out that the vitamin A requirement of young Coturnix quail and Bobwhite quail differ considerably. Further comparisons cannot be made now since precise requirements of both species for other vitamins are lacking. Studies of additional vitamin requirements of Coturnix and Bobwhite quail are needed before it will be possible to determine whether significant differences exist between the two species. ACKNOWLEDGMENTS The capable technical assistance of Kathleen Kennedy and Mrs. Bertha O. Preston is gratefully acknowledged. REFERENCES Association of Official Agricultural Chemists, 1960. Official Methods of Analysis, 9th Ed. Assoc, of Official Agr. Chemists, Washington, D.C. Bremer, J., P. H. Figard and D. M. Greenberg, 1960. The biosynthesis of choline and its relation to phospholipid metabolism. Biochim. Biophys. Acta, 43: 477. Duncan, D. R., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Entenman, C , A. Taurog and I. L. Chaikoff, 1944. The determination of choline in phospholipids. J. Biol. Chem. 155: 13-18. Erickson, R. C , 1968. A federal research program for endangered species. Transactions of the Thirtythird North American Wildlife and Natural Resources Conference, March 11-13, 1968. Wildlife Management Institute, Washington, D.C. 20005. Howes, J. R., and W. L. Beane, 1966. The nutrition of pheasants, Bobwhite and Coturnix quail. Feedstuffs, 38: 18-21.
Mitchell, H. H., 1964. Comparative Nutrition of Man and Domestic Animals. Vol. II, Academic Press, New York, pgs. 95-107. National Research Council, 1971. Nutrient Requirements of Poultry. Publication 01861-7, 2101 Constitution Avenue, Washington, D.C. 20418. 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, 1965. U.S. Pharmocopeial Convention, Inc., 17th Ed., Washington, D . C , Calcium Pantothenate Assay, 840-842. Scott, M. L., E. R. Holm and R. E. Reynolds, 1959. Studies on the niacin, riboflavin, choline, manganese and zinc requirements of young Ringnecked pheasants for growth, feathering and prevention of leg disorders. Poultry Sci. 38: 1344-1350. Scott, M. L., E. R. Holm and R. E. Reynolds, 1964. Studies on the pantothenic acid and unidentified factor requirements of young Ringnecked pheasants and Bobwhite quail. Poultry Sci. 43: 1534-1539. Scott, M. L., M. C. Nesheim and R. J. Young, 1969. Nutrition of the Chicken. M. L. Scott and Associates, Ithaca, New York. Shellenberger, T. E., and J. M. Lee, 1966. Effect of vitamin A on growth, egg production and reproduction of Japanese quail. Poultry Sci. 45: 708-713. Spivey-Fox, M. R., G. A. Hudson and M. E. Hintz, 1966. Pantothenic acid requirement of young Japanese quail. Fed. Proc. 25: 721. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics, McGraw-Hill, New York. Vohra,P., 1971. A review of the nutrition of Japanese quail. World's Poultry Sci. J. 27: 26-34. Wyatt, R. D., H. T. Tung, W. E. Donaldson and P. B. Hamilton, 1973. A new description of riboflavin deficiency syndrome in chickens. Poultry Sci. 52: 237-244. Young, R. J., H. M. Edwards, Jr. and M. B. Gillis, 1958. Studies on zinc in poultry nutrition. Poultry Sci. 37: 1100-1107.
AUGUST 4-8. ANNUAL MEETING OF AGRICULTURAL INSTITUTE OF CANADA AND AFFILIATED SOCIETIES. LAVAL UNIVERSITY, QUEBEC, QUEBEC. AUGUST 5-8. 63RD ANNUAL MEETING OF THE POULTRY SCIENCE ASSOCIATION, INC. WEST VIRGINIA STATE UNIVERSITY, MORGANTOWN, WEST VIRGINIA. AUGUST 11-17. FIFTEENTH WORLD'S POULTRY CONGRESS, NEW ORLEANS, LOUISIANA. AUGUST 15-17. ANNUAL MEETING OF THE INTERNATIONAL EGG COMMISSION, NEW ORLEANS, LOUISIANA.