- Email: [email protected]
Methionine, Choline, and Sulfate Supplementation of Practical-Type Diets for Young Turkeys
Methionine, Choline, and Sulfate Supplementation of Practical-Type Diets for Young Turkeys M. E. BLAIR,1 L. M. POTTER, B. A. BLISS, and J. R. SHELTON Department of Poultry Science, Virginia Polytechnic Institute and State University Blacksburg, Virginia 24061 (Received for publication April 17, 1985)
1986 Poultry Science 65:130-137
INTRODUCTION
Practical-type diets for poultry composed primarily of ground yellow corn and dehulled soybean meal are first limiting in sulfur amino acids and thus require methionine supplementation. In addition to other functions, methionine serve*; a« a dnnnr of methvl srouDS and as a source of sulfur. Whether choline, acting as a supplier of methyl groups, can spare methionine has been a subject of several studies which have been reviewed by Fidler (1981). Results from recent experiments have provided renewed interest on this topic. Pesti et al. (1979, 1980) reported significant increases (ca. 12%) in body weight gain from the additions of .04 to .39% choline to practical-type diets for poults and chicks from 0 to 2 and 3 weeks of age, respectively. The addition of .23% methionine to the same diets produced
greater body weight gains. These authors suggested that the methionine requirement may be reduced .025 to .05% in such diets by the supplementation of .07 to .08% choline. Miles et al. (1983) also reported large increases (ca. 28%) in body weights of poults between 0 and 3 weeks of age from the simultaneous addition of , 1 % potassium sulfate and .066% choline to a 25% protein corn-soybean meal diet but much larger increases (ca. 51%) from the addition of .25% methionine, indicating that both sulfate and choline may be involved in sparing methionine. The objective of this investigation was to determine whether choline or inorganic sulfate can reduce the requirement for methionine in low-protein, practical-type diets for young turkeys between 3 and 8 weeks of age. MATERIALS AND METHODS
1
Portion of a thesis presented by the senior author as partial fulfillment of the requirements for the degree of Master of Science at Virginia Polytechnic Institute and State University.
Three similar experiments were conducted utilizing a total of 2,448 poults. For the first experiment, medium-type turkeys at 4 weeks of age were randomized into 96 battery pens (48 pens of each sex) until each contained 9 poults.
130
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 2, 2015
ABSTRACT Three experiments were conducted using a total of 2,448 poults to study the interrelationships of methionine, choline, and sulfate in practical-type diets. A 6 X 2 X 2 factorial was used involving DL-methionine at increments of .06% from 0 to .30%, choline chloride at 0 and .20%, and potassium sulfate at 0 and .10%. The variables were added to a 21% protein basal diet containing 61% ground yellow corn and 32% dehulled soybean meal but devoid of supplemental choline or sulfate. Each of the 24 diets was fed to four pens of poults in each experiment. Mediumtype turkeys between 4 and 8 weeks of age were used in the first experiment and Large White turkeys between 3 and 7 weeks of age in each of the next two experiments. With diets containing 0, .06, .12, .18, .24, and .30% added methionine, body weight gains were 1340, 1457, 1520, 1559, 1538, and 1558 g, respectively, indicating that at least .18% added methionine (.86% total dietary sulfur amino acids) was required in a 21% protein diet for maximum growth. Average increases in body weight gain, feed consumption, and feed efficiency from the three highest levels of added methionine over the unsupplemented diets were 15.8, 4.8, and 10.6%, respectively. These parameters failed to be affected by adding choline to diets already containing 1250 mg choline/kg. Added sulfate significantly increased average body weight and feed efficiency only in methionine-deficient diets containing 0 and .06% added methionine, and these increases amounted to 2.8 and 3.5% in body weight and to 1.7 and 2.7% in feed efficiency. (Key words: methionine, choline, sulfate, turkeys)
METHIONINE, CHOLINE, AND SULFATE
RESULTS Mean body weight gains, feed consumptions, and feed efficiencies of the turkeys fed each diet from 3 to 7 or 4 to 8 weeks of age are presented in Table 2. Analyses of variance of combined results from the three experiments are presented in Table 3. Means of these parameters from the three experiments are shown in Table 4. From additions of .06, .12, .18, .24, and .30% DL-methionine, body weight gains were significantly improved (P<.001) over the basal diet by 8.7, 13.4, 16.3, 14.8, and 16.3%, respectively (Table 4). Average increases in body weight gain, feed consumption, and feed
efficiency for the three highest levels of added methionine over the unsupplemented diet were 15.8, 4.8, and 10.6%, respectively (Table 4). The addition of choline to the diet failed to affect body weight gain, feed consumption, and feed efficiency (Table 4). Perosis did not occur in the turkeys fed diets without added choline. By calculation (National Research Council, 1984), the basal diet contained .125% choline (1250 mg/kg), which appeared to be sufficient to obtain maximum body weight gain and feed efficiency.
TABLE 1. Composition of basal diet Ingredients
Amount (g/kg)
Ground yellow corn Glucose monohydrate, cerelose Stabilized fat Dehulled soybean meal Defluorinated phosphate Iodized salt Trace mineral mix 1 ' 2 Vitamin and feed additive mix 1 ' 3 L-Lysine HC1 Total Calculated analyses4 Protein, % Calcium, % Phosphorus, total, % Phosphorus, available, % Lysine, % Methionine, % Cystine, % Sulfur, % Choline, mg/kg Metabolizable energy, kcal/kg
608.97 11 10 320 38 3 2 1.03 6 1,000.00 21.4 1.32 1.05 .80 1.64 .35 .33 .11 1,250 2,952
' Designed to be sulfate- and choline-free. 2 Supplied the following amounts of trace minerals in mg/kg complete diet: 150 manganese, 101 zinc, 70 iron, 10 copper, and 2.6 iodine from manganese carbonate, zinc oxide, ferric citrate, cupric oxide, and potassium iodate, respectively. 3 Supplied the following amounts of vitamins and feed additives in mg/kg complete diet unless otherwise stated: 13,202 IU vitamin A, 3,312 ICU vitamin D 3 , 11 IU vitamin E, 4.4 menadione sodium bisulfite complex, 1.1 thiamine HC1, 5.5 riboflavin, 16.5 calcium pantothenate (D), 66.1 niacin, .015 vitamin B 1 2 , 1.1 folic acid, .11 biotin, 2.2 pyridoxine HC1, 125 ethoxyquin, .2 selenium, and 26.4 penicillin. 4 Based on composition values obtained from the National Research Council (1984) publication, Nutrient Requirements of Poultry.
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 2, 2015
For the second and third experiments, a similar procedure was followed using Large White turkeys at 3 weeks of age with 8 females or 8 (9 in the second experiment) males randomized into each pen. Diets were assigned randomly to pens of poults within a sex. Water and food were provided ad libitum to 8 weeks of age in the first experiment and to 7 weeks of age in the latter two experiments. The composition of the basal diet used in the three experiments is presented in Table 1. The vitamin and trace mineral premixes were choline- and sulfate-free, respectively. The variables in the 24 diets of the 6 x 2 x 2 factorial were DL-methionine at 0, .06, .12, .18, .24, and .30%, choline chloride at 0 and .20%, and potassium sulfate at 0 and .10%, assuming 99, 60, and 99% purity, respectively. For each experiment, three identical mixes containing all constant ingredients were prepared. The variables with glucose monohydrate were added to equal parts of each mix to form the 24 diets. Each diet was fed to two pens of male and two pens of female poults in each experiment. Body weights of the poults by pens were determined at weekly intervals. Average body weight gain, feed consumption, and feed efficiency for each pen were calculated. All data for each period in each experiment were subjected to analysis of variance. Because the effects of the variables under study from the three experiments were similar, the results from the 288 observations were combined for analysis. Also, the interaction effects of diet with all combinations of experiment, sex, and replicate were combined for the error term to test for significance among diets.
131
132
BLAIR ET AL. TABLE 2. Average body weight gain, feed consumption, and feed efficiency of turkeys fed each diet (average of three experiments)* Potassium sulfate
Level of added DLmethionine
10%
0%
0% Calcium chloride
.20% Calcium chloride
.00 .06 .12 .18 .24 .30
1296 : 1419 1523 1559 1542 1555
1347 1445 1521 1571 1533 1550
.00 .06 .12 .18 .24 .30
2767 2906 2977 2988 2923 2932
2826 2905 2963 2966 2934 2930
0% Choline chloride
.20% Choline chloride
Body
(%)
1344 1494 1525 1532 1544 1567
2873 2890 2920 2978 2936 2952
2785 2962 2961 2957 2925 2963
Feed coi
.00 .06 .12 .18 .24 .30
4644 4854 5084 5193 5240 5272
—~ Feed 4739 4945 5108 5 264 5198 5252
.4748 .5052 .5133 .5253 .5184 .5266
.4791 .5008 .5116 .5154 .5242 .5244
'Combined data for Large White turkeys from 3 to 7 weeks of age in two experiments and Medium White turkeys from 4 to 8 weeks in one experiment. 2 Each value is an average of twelve values representing the turkeys in 6 pens of males and 6 pens of females with 8 or 9 poults/pen.
The plots of average body weight gain and feed efficiency of turkeys on level of added methionine in diets with 0 or .20% added choline chloride are shown in Figure 1. The increases in body weight gain from the three highest levels of added methionine to diets, with or without choline, were essentially the same (15.2 and 16.5%, respectively). Also, the observed increases in body weight gain and feed efficiency from added choline to diets containing varying amounts of added methionine were not significant except for the .0069 (1.5%) increase in feed efficiency when added to the diets without methionine supplementation. The addition of .10% potassium sulfate resulted in significantly higher average feed efficiency during the 4-week experiments (Table 4). Body weight gain was 14 g more for those turkeys fed diets with, than without, added sulfate, a difference which approached significance (.10»P>.05; Table 3).
Plots of average body weight gain and feed efficiency on level of added methionine in diets with 0 or .10% added potassium sulfate are shown in Figure 2. When diets contained 0 and .06% supplemental methionine, potassium sulfate additions increased body weight gain 2.8 and 3.5% and feed efficiency 1.7 and 2.7%, respectively (each significant, except for the 2.8% which approached significance; Table 5). In diets containing higher levels of added methionine, added potassium sulfate failed to influence either body weight gain or feed efficiency. DISCUSSION
For the 4-week experiments of this study, body weight gain and feed efficiency of the poults increased with each increment of added methionine to the .18% level. The basal diet contained .35% methionine and .33% cystine by calculation (National Research Council,
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 2, 2015
1373 1470 1511 1575 1534 1561
133
METHIONINE, CHOLINE, AND SULFATE
TABLE 3. Analyses of variance of body weight gains, feed consumptions, and feed efficiencies Mean squares Source
23 5 1 1 5 5 1 5 2 1 1 2 2 1 2 253 46 2 23 23 46 46 23 46
Body wt. gain
Feed consumption
Feed efficiency
X 102
X 102
X 10"6
792 * * * 3,459*** 15 139 21 71 41 53 64,05 3 * * * 59,291*** 732*** 1,055*** 181* 325* 144 49 82 206 42 41 40 36 60 42
406*** 1,553*** 6 37 51 69 3 183 102,892*** 123,897*** 561* 7,567*** 1,297*** 633* 696*** 140 202 444 147 131 133 84 108 127
452,279*** 19,698*** 95 801* 127 432* 447 280 106,160*** 49,746*** 2,445*** 4,252*** 464 265 213 145 213 125 188 99 131 130 81 140
'Standard deviations calculated from the error mean squares were 70.3 g, 118.3 g, and .0120 for body weight gain, feed consumption, and feed efficiency, respectively. *P<.05. ***P<.001.
1984). Thus, the total sulfur amino acid requirement of Large White turkeys between 3 and 7 weeks of age and Medium White turkeys between 4 and 8 weeks of age is about .86% in a 21% protein diet based on the results of this study. This value may be compared to .90% stated by the National Research Council (1984) and 1.00% reported by Potter and Shelton (1979) for turkeys between 4 and 8 weeks of age. In the latter report, a 24 or 27% protein diet produced optimum body weight gain, and the stated total sulfur amino acid requirement was based on these accompanying levels of protein. When diets contained 18 or 21% protein in that study, the total sulfur amino acid requirement appeared to be about .70 and .85%, respectively. However, body weight gains were only about 70 and 85%, respectively, that from diets containing 1.00% total sulfur amino acids and 24 or 27% protein.
The poorer growth from the 18 or 21% protein diets was believed to be due to a deficiency of other amino acids. Thus, the basal diet in the current study was supplemented with .6% L-lysine HCl in an effort to correct a possible deficiency of this amino acid. Based on the results of other experiments from this laboratory (Stas and Potter, 1982), amino acids other than lysine may have been deficient in the 21% protein diet of the present study. Thus, it is postulated that the total sulfur amino acid requirement would be greater than .86% if the basal diet had contained additional protein as in those reported by Potter and Shelton (1979) or had contained supplemental amino acids such as threonine, valine, and isoleucine to produce optimum growth. The addition of choline to the diets of this study failed to increase body weight but did increase feed efficiency in the absence of added
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 2, 2015
Diet (D) M e t h i o n i n e (M) Choline (C) Sulfate (S) M X C MX S C XS MX C X S E x p e r i m e n t (E) Sex Replicate (R) E X Sex EX R Sex X R E X Sex X R Error 1 D X E S X E D X Sex D X R D X E X Sex D X E X R D X Sex X R D X E X Sex X R
Degrees of freedom
BLAIR ETAL.
134
TABLE 4. Effects of dietary variables on measured responses during the 4-week
Variable
Body wt. gain1
DL-Methionine, % .00 .06 .12 .18 .24 .30
13402 1457 1520 1559 1538 1558
Feed consumption 1 I
63*** 39** -21 20
Choline chloride, % .00 .20
1493 1498
1 -t 7 * * *
. . .—
2813 2916 2955 2972 2930 2944
103*** 39 17 -42 14
2920 2923
Difference Potassium sulfate, % .00 .10 Difference
Feed efficiency1
.4730 .4965 .5110 .5216 .5216 .5259
.0235*** .0145*** .0106*** .0000 .0043
.5077 .5088 .0011
1488 1502 14
2918 2925
.5066 .5099 .0033*
1 Data collected in two experiments with Large White turkeys from 3 to 7 weeks of age and in one experiment with Medium White turkeys from 4 to 8 weeks of age. 2 Each value represents 48 pens of turkeys fed diets containing each level of methionine and 144 pens containing each level of choline or sulfate. 3 Differences for significance between average body weight gains, feed consumptions, or feed efficiencies associated with DL-methionine must be greater than 28 g, 48 g, or 0048 and with choline chloride or with potassium sulfate 16 g, 28 g, and .0028, respectively.
*Significant at the 5% level. "Significant at the 1% level. **'Significant at the .1% level.
methionine. Thus, the choline requirement of turkeys 4 to 8 weeks of age appears to be not greater than .125% or 1250 mg/kg diet, the amount in the unsupplemented diet. This value may be compared to the established requirement of 1900 mg/kg diet for turkeys 0 to 4 weeks of age and an estimated requirement of 1600 mg/kg diet for turkeys 4 to 8 weeks of age as stated by the National Research Council (1984). The latter values are believed to be based primarily on the findings of Evans (1943) and supported by those of Gartley et al. (1951) and Gogus and Griminger (1957). Harms and Miles (1984) also obtained no increase in body weights of poults to 3 weeks of age from added choline over that from a 25.4% protein basal diet containing .25% added methionine and a calculated 1490 mg choline/kg. Thus, the
choline requirement of turkeys between 4 and 8 weeks of age may be less than 1600 mg/kg diet when practical-type diets are fed as in the present study. Practical-type diets for turkeys and chickens on occasion have been shown to be deficient in choline (Marvel et al, 1944; Gerry et al, 1948; Sherwood and Sloan, 1954; Quillin et al, 1961; Slinger et al, 1962;Pesti et al, 1980; Baker et al, 1983; Harms and Miles, 1984). In these studies, large increases in body weights occurred from added dietary choline at an early age, especially in the absence of supplemental methionine; but also in these and other studies, increases were more difficult to demonstrate at the older ages. It has been hypothesized that synthesis of choline increases with age of turkeys and chickens, thus possibly explaining
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 2, 2015
Difference .06 vs. .00 .12 vs. .06 .18 vs. .12 .24 os. .18 .30 os. .24
experiments
METHIONINE, CHOLINE, AND SULFATE 1600
1500
|
1450
* a i4oo 1350
1300
1600 .53 1550
.52 .51
| 1500 u>
S -50
l-
.49
1 "SO *
u_ .48
§ 1400
w o
.47 .46
1350 06 .12 .18 .24 ADDED METHIONINE, %
.30
FIG. 1. Increases in body weight gains and feed efficiencies during the 4-week experiments for turkeys fed diets containing various levels of added choline, chloride, and methionine. A significant increase (P<.05) in feed efficiency occurred when choline chloride was added at the 0% level of added methionine.
the lack of response from choline in the present experiment. The failure of choline to significantly affect body weight gain, feed consumption, and feed efficiency in the presence and absence of added methionine, except in the unsupplemented diet, indicates that choline had little or no effect in sparing or decreasing the requirement for methionine in practical-type diets after 3 or 4 weeks of age. These observations indicate that the 21% corn-soybean meal basal diet is deficient in sulfur amino acids or methionine per se rather than in labile methyl groups. The results of the current study with turkeys also support
1300
0
06 .12 .18 .24 ADDED METHIONINE, %
FIG. 2. Increases in body weight gains and feed efficiencies during the 4-week experiments for turkeys fed diets containing various levels of added potassium, sulfate, and methionine. Added potassium sulfate increased body weight gain (P<.05) at .06% added methionine and feed efficiency at 0 (P<.05) and .06% (P<.001) added methionine.
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 2, 2015
those with chicks by Baker et al. (1983) who concluded that excessive choline had minimal capacity to reduce the dietary requirement for methionine. The addition of .10% potassium sulfate to diets deficient in methionine, or sulfur amino acids in the present experiments, significantly increased body weight and feed efficiency only about 3 and 2%, respectively, in contrast to 16 and 10% from .18% or more added DLmethionine. However, sulfate addition to diets with sufficient methionine failed to increase body weights or feed efficiencies. In previous studies (Potter et al, 1983 ; Potter and Shelton,
1550 |
135
136
B L A I R E T AL. T A B L E 5. Increase in body weight gain, feed consumption, and feed efficiency sulfate to diets containing graded levels of added methionine by
from adding experiments
potassium
Experiment1
Added methionine
Average
(%)
Increase in b o d y weight gain, g 6 -17 -30 33 -37 -38
59
56
.00 .06 .12 .18 .24 .30
58 3 -50 -18 30 47
46 30 -56 65 -18 25
LSD
103
104
LSD2
68 138*** 51 -41 25 28
36 50* -4 -12 1 12
(2.8%) (3.5%)
40
77 Increase in feed c o n s u m p t i o n , g
33 62 -30 -9 1 27
35 130* 54 -51 34 84
68
129 Increase in feed efficiency
.00 .06 .12 .18 .24 .30
563 105 -34 68 16 84
LSD
124
12 66 60 35 -16 -37 98
167** 220*** 62 -41 15 -67
78* (1.7%) 1 3 0 * * * (2.7%) 29 -25 -6 -7
118
68
1 Medium White t u r k e y s 3 t o 7 weeks of age used in E x p e r i m e n t 1 and Large White t u r k e y s 4 t o 8 weeks of age used in E x p e r i m e n t s 2 a n d 3. 2
LSD = Least significant difference.
' M u l t i p l y each b y 10~ 4 . 'Significant at t h e 5% level. ' • S i g n i f i c a n t at t h e 1% level. * * ' S i g n i f i c a n t at t h e . 1 % level.
1984), added sulfate to methionine-deficient diets for turkeys failed to increase body weights at any age except for increases of 3 and 2% at 2 and 4 weeks of age, respectively, in the second study. In contrast, methionine additions increased body weights about 9 and 7% at 2 weeks of age and 10 and 4% at 7 or 8 weeks of age, respectively, in these same studies. Similarly, Sloan and Harms (1972) have also demonstrated that sulfate and methionine increased body weights of turkeys to 3 weeks of age 4 and 11%, respectively, when added to diets deficient in methionine. Using diets with essentially the same formulation, Miles et al. (1983) obtained even larger increases (10 and 43%) in 3-week body weights of turkeys from added sulfate or added methionine, but sulfate failed
to increase body weights when added to the methionine-supplemented diet. Results of the present study support those in previous reports that sulfate has the capacity to increase body weights of young turkeys when added to methionine deficient diets but not when added to diets sufficient in methionine. Therefore, neither inorganic sulfate nor choline reduced the requirement for methionine in practicaltype diets for young turkeys in this study.
ACKNOWLEDGMENT The poults used in the last two experiments of this study were supplied by Wampler Hatchery, Harrisonburg, VA.
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 2, 2015
36 28 -33 -27 14 44
.00 .06 .12 .18 .24 .30
METHIONINE, CHOLINE, AND SULFATE
Pesti, G. M., A. E. Harper, and M. L. Sunde, 1979. Sulfur amino acid and methyl donor status of corn-soy diets fed to starting broiler chicks and turkey poults. Poultry Sci. 58:1541-1547. Pesti, G. M., A. E. Harper, and M. L. Sunde, 1980. Choline/methionine nutrition of starting broiler chicks. Three models for estimating the choline requirement with economic considerations. Poultry Sci. 59:1073-1081. Potter, L. M., and J. R. Shelton, 1979. Methionine and protein requirements of young turkeys. Poultry Sci. 58:609-615. Potter, L. M., and J. R. Shelton, 1984. Methionine, cystine, sodium sulfate, and Fermacto-500 supplementation of practical-type diets for young turkeys. Poultry Sci. 63:987-992. Potter, L. M., J. R. Shelton, and D. J. Castaldo, 1983. Supplementary inorganic sulfate and methionine for young turkeys. Poultry Sci. 62:2398-2402. Quillin, E. C , G. F. Combs, R. D. Creek, and G. L. Romoser, 1961. Effect of choline on the methionine requirements of broiler chickens. Poultry Sci. 40:639-645. Sherwood, D. H., and H. J. Sloan, 1954. Vitamin B I 2 and choline in corn-soy rations for starting poults. Poultry Sci. 33:1015-1021. Slinger, S. J., W. F. Pepper, and I. R. Sibbald, 1962. Interrelationships between methionine, choline, sodium chloride and reserpine in growing turkeys. Poultry Sci. 41:974-982. Sloan, D. R., and R. H. Harms, 1972. Utilization of inorganic sulfate by turkey poults. Poultry Sci. 51:1673-1675. Stas, R. J., and L. M. Potter, 1982. Amino acid supplementation of a 21% protein corn-soybean meal diet for young turkeys. Nutr. Rep. Int. 26: 487-494.
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 2, 2015
REFERENCES Baker, D. H., K. M. Halpin, G. L. Czarnecki, and C. M. Parsons, 1983. The choline-methionine interrelationship for growth of the chick. Poultry Sci. 62:133-137. Evans, R. J., 1943. The choline requirements of turkey poults. Poultry Sci. 22:266-267. Fidler, J. W., 1981. Sparing action between methionine and choline in poultry diets. Pages 1 8 - 2 0 in Proc. 41st Annu. Meet. Am. Feed Mfr. Assoc. Gartley, K. M., S. J. Slinger, A. M. Morphet, and C. R. Cameron, 1951. The choline requirements of Broadbreasted Bronze Turkeys in the presence of APF concentrates. Poultry Sci. 30:913. (Abstr.) Gerry, R. W., C. W. Carrick, and S. M. Hauge, 1948. Methionine and choline in a simplified chick ration. Poultry Sci. 27:161-168. Gogus, K., and P. Griminger, 1957. On the vitamin requirements of poults. 2. Choline. Poultry Sci. 36:1121. (Abstr.) Harms, R. H., and R. D. Miles, 1984. Effects of supplemental methionine and potassium sulfate on the choline requirement of the turkey poult. Poultry Sci. 63:1464-1466. Marvel, J. A., C. W. Carrick, R. E. Roberts, and S. M. Hauge, 1944. The supplementary value of choline and methionine in a corn and soybean oil meal chick ration. Poultry Sci. 23:294-297. Miles, R. D., N. Ruiz, and R. H. Harms, 1983. The interrelationships between methionine, choline, and sulfate in turkey diets. Proc. Soc. Exp. Biol. Med. 173:32-34. National Research Council, 1984. Nutrient Requirements of Domestic Animals. 1. Nutrient Requirements of Poultry. 8th ed. Natl. Acad. Sci., Washington, DC.
137
1986 Poultry Science 65:130-137
INTRODUCTION
Practical-type diets for poultry composed primarily of ground yellow corn and dehulled soybean meal are first limiting in sulfur amino acids and thus require methionine supplementation. In addition to other functions, methionine serve*; a« a dnnnr of methvl srouDS and as a source of sulfur. Whether choline, acting as a supplier of methyl groups, can spare methionine has been a subject of several studies which have been reviewed by Fidler (1981). Results from recent experiments have provided renewed interest on this topic. Pesti et al. (1979, 1980) reported significant increases (ca. 12%) in body weight gain from the additions of .04 to .39% choline to practical-type diets for poults and chicks from 0 to 2 and 3 weeks of age, respectively. The addition of .23% methionine to the same diets produced
greater body weight gains. These authors suggested that the methionine requirement may be reduced .025 to .05% in such diets by the supplementation of .07 to .08% choline. Miles et al. (1983) also reported large increases (ca. 28%) in body weights of poults between 0 and 3 weeks of age from the simultaneous addition of , 1 % potassium sulfate and .066% choline to a 25% protein corn-soybean meal diet but much larger increases (ca. 51%) from the addition of .25% methionine, indicating that both sulfate and choline may be involved in sparing methionine. The objective of this investigation was to determine whether choline or inorganic sulfate can reduce the requirement for methionine in low-protein, practical-type diets for young turkeys between 3 and 8 weeks of age. MATERIALS AND METHODS
1
Portion of a thesis presented by the senior author as partial fulfillment of the requirements for the degree of Master of Science at Virginia Polytechnic Institute and State University.
Three similar experiments were conducted utilizing a total of 2,448 poults. For the first experiment, medium-type turkeys at 4 weeks of age were randomized into 96 battery pens (48 pens of each sex) until each contained 9 poults.
130
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 2, 2015
ABSTRACT Three experiments were conducted using a total of 2,448 poults to study the interrelationships of methionine, choline, and sulfate in practical-type diets. A 6 X 2 X 2 factorial was used involving DL-methionine at increments of .06% from 0 to .30%, choline chloride at 0 and .20%, and potassium sulfate at 0 and .10%. The variables were added to a 21% protein basal diet containing 61% ground yellow corn and 32% dehulled soybean meal but devoid of supplemental choline or sulfate. Each of the 24 diets was fed to four pens of poults in each experiment. Mediumtype turkeys between 4 and 8 weeks of age were used in the first experiment and Large White turkeys between 3 and 7 weeks of age in each of the next two experiments. With diets containing 0, .06, .12, .18, .24, and .30% added methionine, body weight gains were 1340, 1457, 1520, 1559, 1538, and 1558 g, respectively, indicating that at least .18% added methionine (.86% total dietary sulfur amino acids) was required in a 21% protein diet for maximum growth. Average increases in body weight gain, feed consumption, and feed efficiency from the three highest levels of added methionine over the unsupplemented diets were 15.8, 4.8, and 10.6%, respectively. These parameters failed to be affected by adding choline to diets already containing 1250 mg choline/kg. Added sulfate significantly increased average body weight and feed efficiency only in methionine-deficient diets containing 0 and .06% added methionine, and these increases amounted to 2.8 and 3.5% in body weight and to 1.7 and 2.7% in feed efficiency. (Key words: methionine, choline, sulfate, turkeys)
METHIONINE, CHOLINE, AND SULFATE
RESULTS Mean body weight gains, feed consumptions, and feed efficiencies of the turkeys fed each diet from 3 to 7 or 4 to 8 weeks of age are presented in Table 2. Analyses of variance of combined results from the three experiments are presented in Table 3. Means of these parameters from the three experiments are shown in Table 4. From additions of .06, .12, .18, .24, and .30% DL-methionine, body weight gains were significantly improved (P<.001) over the basal diet by 8.7, 13.4, 16.3, 14.8, and 16.3%, respectively (Table 4). Average increases in body weight gain, feed consumption, and feed
efficiency for the three highest levels of added methionine over the unsupplemented diet were 15.8, 4.8, and 10.6%, respectively (Table 4). The addition of choline to the diet failed to affect body weight gain, feed consumption, and feed efficiency (Table 4). Perosis did not occur in the turkeys fed diets without added choline. By calculation (National Research Council, 1984), the basal diet contained .125% choline (1250 mg/kg), which appeared to be sufficient to obtain maximum body weight gain and feed efficiency.
TABLE 1. Composition of basal diet Ingredients
Amount (g/kg)
Ground yellow corn Glucose monohydrate, cerelose Stabilized fat Dehulled soybean meal Defluorinated phosphate Iodized salt Trace mineral mix 1 ' 2 Vitamin and feed additive mix 1 ' 3 L-Lysine HC1 Total Calculated analyses4 Protein, % Calcium, % Phosphorus, total, % Phosphorus, available, % Lysine, % Methionine, % Cystine, % Sulfur, % Choline, mg/kg Metabolizable energy, kcal/kg
608.97 11 10 320 38 3 2 1.03 6 1,000.00 21.4 1.32 1.05 .80 1.64 .35 .33 .11 1,250 2,952
' Designed to be sulfate- and choline-free. 2 Supplied the following amounts of trace minerals in mg/kg complete diet: 150 manganese, 101 zinc, 70 iron, 10 copper, and 2.6 iodine from manganese carbonate, zinc oxide, ferric citrate, cupric oxide, and potassium iodate, respectively. 3 Supplied the following amounts of vitamins and feed additives in mg/kg complete diet unless otherwise stated: 13,202 IU vitamin A, 3,312 ICU vitamin D 3 , 11 IU vitamin E, 4.4 menadione sodium bisulfite complex, 1.1 thiamine HC1, 5.5 riboflavin, 16.5 calcium pantothenate (D), 66.1 niacin, .015 vitamin B 1 2 , 1.1 folic acid, .11 biotin, 2.2 pyridoxine HC1, 125 ethoxyquin, .2 selenium, and 26.4 penicillin. 4 Based on composition values obtained from the National Research Council (1984) publication, Nutrient Requirements of Poultry.
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 2, 2015
For the second and third experiments, a similar procedure was followed using Large White turkeys at 3 weeks of age with 8 females or 8 (9 in the second experiment) males randomized into each pen. Diets were assigned randomly to pens of poults within a sex. Water and food were provided ad libitum to 8 weeks of age in the first experiment and to 7 weeks of age in the latter two experiments. The composition of the basal diet used in the three experiments is presented in Table 1. The vitamin and trace mineral premixes were choline- and sulfate-free, respectively. The variables in the 24 diets of the 6 x 2 x 2 factorial were DL-methionine at 0, .06, .12, .18, .24, and .30%, choline chloride at 0 and .20%, and potassium sulfate at 0 and .10%, assuming 99, 60, and 99% purity, respectively. For each experiment, three identical mixes containing all constant ingredients were prepared. The variables with glucose monohydrate were added to equal parts of each mix to form the 24 diets. Each diet was fed to two pens of male and two pens of female poults in each experiment. Body weights of the poults by pens were determined at weekly intervals. Average body weight gain, feed consumption, and feed efficiency for each pen were calculated. All data for each period in each experiment were subjected to analysis of variance. Because the effects of the variables under study from the three experiments were similar, the results from the 288 observations were combined for analysis. Also, the interaction effects of diet with all combinations of experiment, sex, and replicate were combined for the error term to test for significance among diets.
131
132
BLAIR ET AL. TABLE 2. Average body weight gain, feed consumption, and feed efficiency of turkeys fed each diet (average of three experiments)* Potassium sulfate
Level of added DLmethionine
10%
0%
0% Calcium chloride
.20% Calcium chloride
.00 .06 .12 .18 .24 .30
1296 : 1419 1523 1559 1542 1555
1347 1445 1521 1571 1533 1550
.00 .06 .12 .18 .24 .30
2767 2906 2977 2988 2923 2932
2826 2905 2963 2966 2934 2930
0% Choline chloride
.20% Choline chloride
Body
(%)
1344 1494 1525 1532 1544 1567
2873 2890 2920 2978 2936 2952
2785 2962 2961 2957 2925 2963
Feed coi
.00 .06 .12 .18 .24 .30
4644 4854 5084 5193 5240 5272
—~ Feed 4739 4945 5108 5 264 5198 5252
.4748 .5052 .5133 .5253 .5184 .5266
.4791 .5008 .5116 .5154 .5242 .5244
'Combined data for Large White turkeys from 3 to 7 weeks of age in two experiments and Medium White turkeys from 4 to 8 weeks in one experiment. 2 Each value is an average of twelve values representing the turkeys in 6 pens of males and 6 pens of females with 8 or 9 poults/pen.
The plots of average body weight gain and feed efficiency of turkeys on level of added methionine in diets with 0 or .20% added choline chloride are shown in Figure 1. The increases in body weight gain from the three highest levels of added methionine to diets, with or without choline, were essentially the same (15.2 and 16.5%, respectively). Also, the observed increases in body weight gain and feed efficiency from added choline to diets containing varying amounts of added methionine were not significant except for the .0069 (1.5%) increase in feed efficiency when added to the diets without methionine supplementation. The addition of .10% potassium sulfate resulted in significantly higher average feed efficiency during the 4-week experiments (Table 4). Body weight gain was 14 g more for those turkeys fed diets with, than without, added sulfate, a difference which approached significance (.10»P>.05; Table 3).
Plots of average body weight gain and feed efficiency on level of added methionine in diets with 0 or .10% added potassium sulfate are shown in Figure 2. When diets contained 0 and .06% supplemental methionine, potassium sulfate additions increased body weight gain 2.8 and 3.5% and feed efficiency 1.7 and 2.7%, respectively (each significant, except for the 2.8% which approached significance; Table 5). In diets containing higher levels of added methionine, added potassium sulfate failed to influence either body weight gain or feed efficiency. DISCUSSION
For the 4-week experiments of this study, body weight gain and feed efficiency of the poults increased with each increment of added methionine to the .18% level. The basal diet contained .35% methionine and .33% cystine by calculation (National Research Council,
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 2, 2015
1373 1470 1511 1575 1534 1561
133
METHIONINE, CHOLINE, AND SULFATE
TABLE 3. Analyses of variance of body weight gains, feed consumptions, and feed efficiencies Mean squares Source
23 5 1 1 5 5 1 5 2 1 1 2 2 1 2 253 46 2 23 23 46 46 23 46
Body wt. gain
Feed consumption
Feed efficiency
X 102
X 102
X 10"6
792 * * * 3,459*** 15 139 21 71 41 53 64,05 3 * * * 59,291*** 732*** 1,055*** 181* 325* 144 49 82 206 42 41 40 36 60 42
406*** 1,553*** 6 37 51 69 3 183 102,892*** 123,897*** 561* 7,567*** 1,297*** 633* 696*** 140 202 444 147 131 133 84 108 127
452,279*** 19,698*** 95 801* 127 432* 447 280 106,160*** 49,746*** 2,445*** 4,252*** 464 265 213 145 213 125 188 99 131 130 81 140
'Standard deviations calculated from the error mean squares were 70.3 g, 118.3 g, and .0120 for body weight gain, feed consumption, and feed efficiency, respectively. *P<.05. ***P<.001.
1984). Thus, the total sulfur amino acid requirement of Large White turkeys between 3 and 7 weeks of age and Medium White turkeys between 4 and 8 weeks of age is about .86% in a 21% protein diet based on the results of this study. This value may be compared to .90% stated by the National Research Council (1984) and 1.00% reported by Potter and Shelton (1979) for turkeys between 4 and 8 weeks of age. In the latter report, a 24 or 27% protein diet produced optimum body weight gain, and the stated total sulfur amino acid requirement was based on these accompanying levels of protein. When diets contained 18 or 21% protein in that study, the total sulfur amino acid requirement appeared to be about .70 and .85%, respectively. However, body weight gains were only about 70 and 85%, respectively, that from diets containing 1.00% total sulfur amino acids and 24 or 27% protein.
The poorer growth from the 18 or 21% protein diets was believed to be due to a deficiency of other amino acids. Thus, the basal diet in the current study was supplemented with .6% L-lysine HCl in an effort to correct a possible deficiency of this amino acid. Based on the results of other experiments from this laboratory (Stas and Potter, 1982), amino acids other than lysine may have been deficient in the 21% protein diet of the present study. Thus, it is postulated that the total sulfur amino acid requirement would be greater than .86% if the basal diet had contained additional protein as in those reported by Potter and Shelton (1979) or had contained supplemental amino acids such as threonine, valine, and isoleucine to produce optimum growth. The addition of choline to the diets of this study failed to increase body weight but did increase feed efficiency in the absence of added
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 2, 2015
Diet (D) M e t h i o n i n e (M) Choline (C) Sulfate (S) M X C MX S C XS MX C X S E x p e r i m e n t (E) Sex Replicate (R) E X Sex EX R Sex X R E X Sex X R Error 1 D X E S X E D X Sex D X R D X E X Sex D X E X R D X Sex X R D X E X Sex X R
Degrees of freedom
BLAIR ETAL.
134
TABLE 4. Effects of dietary variables on measured responses during the 4-week
Variable
Body wt. gain1
DL-Methionine, % .00 .06 .12 .18 .24 .30
13402 1457 1520 1559 1538 1558
Feed consumption 1 I
63*** 39** -21 20
Choline chloride, % .00 .20
1493 1498
1 -t 7 * * *
. . .—
2813 2916 2955 2972 2930 2944
103*** 39 17 -42 14
2920 2923
Difference Potassium sulfate, % .00 .10 Difference
Feed efficiency1
.4730 .4965 .5110 .5216 .5216 .5259
.0235*** .0145*** .0106*** .0000 .0043
.5077 .5088 .0011
1488 1502 14
2918 2925
.5066 .5099 .0033*
1 Data collected in two experiments with Large White turkeys from 3 to 7 weeks of age and in one experiment with Medium White turkeys from 4 to 8 weeks of age. 2 Each value represents 48 pens of turkeys fed diets containing each level of methionine and 144 pens containing each level of choline or sulfate. 3 Differences for significance between average body weight gains, feed consumptions, or feed efficiencies associated with DL-methionine must be greater than 28 g, 48 g, or 0048 and with choline chloride or with potassium sulfate 16 g, 28 g, and .0028, respectively.
*Significant at the 5% level. "Significant at the 1% level. **'Significant at the .1% level.
methionine. Thus, the choline requirement of turkeys 4 to 8 weeks of age appears to be not greater than .125% or 1250 mg/kg diet, the amount in the unsupplemented diet. This value may be compared to the established requirement of 1900 mg/kg diet for turkeys 0 to 4 weeks of age and an estimated requirement of 1600 mg/kg diet for turkeys 4 to 8 weeks of age as stated by the National Research Council (1984). The latter values are believed to be based primarily on the findings of Evans (1943) and supported by those of Gartley et al. (1951) and Gogus and Griminger (1957). Harms and Miles (1984) also obtained no increase in body weights of poults to 3 weeks of age from added choline over that from a 25.4% protein basal diet containing .25% added methionine and a calculated 1490 mg choline/kg. Thus, the
choline requirement of turkeys between 4 and 8 weeks of age may be less than 1600 mg/kg diet when practical-type diets are fed as in the present study. Practical-type diets for turkeys and chickens on occasion have been shown to be deficient in choline (Marvel et al, 1944; Gerry et al, 1948; Sherwood and Sloan, 1954; Quillin et al, 1961; Slinger et al, 1962;Pesti et al, 1980; Baker et al, 1983; Harms and Miles, 1984). In these studies, large increases in body weights occurred from added dietary choline at an early age, especially in the absence of supplemental methionine; but also in these and other studies, increases were more difficult to demonstrate at the older ages. It has been hypothesized that synthesis of choline increases with age of turkeys and chickens, thus possibly explaining
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 2, 2015
Difference .06 vs. .00 .12 vs. .06 .18 vs. .12 .24 os. .18 .30 os. .24
experiments
METHIONINE, CHOLINE, AND SULFATE 1600
1500
|
1450
* a i4oo 1350
1300
1600 .53 1550
.52 .51
| 1500 u>
S -50
l-
.49
1 "SO *
u_ .48
§ 1400
w o
.47 .46
1350 06 .12 .18 .24 ADDED METHIONINE, %
.30
FIG. 1. Increases in body weight gains and feed efficiencies during the 4-week experiments for turkeys fed diets containing various levels of added choline, chloride, and methionine. A significant increase (P<.05) in feed efficiency occurred when choline chloride was added at the 0% level of added methionine.
the lack of response from choline in the present experiment. The failure of choline to significantly affect body weight gain, feed consumption, and feed efficiency in the presence and absence of added methionine, except in the unsupplemented diet, indicates that choline had little or no effect in sparing or decreasing the requirement for methionine in practical-type diets after 3 or 4 weeks of age. These observations indicate that the 21% corn-soybean meal basal diet is deficient in sulfur amino acids or methionine per se rather than in labile methyl groups. The results of the current study with turkeys also support
1300
0
06 .12 .18 .24 ADDED METHIONINE, %
FIG. 2. Increases in body weight gains and feed efficiencies during the 4-week experiments for turkeys fed diets containing various levels of added potassium, sulfate, and methionine. Added potassium sulfate increased body weight gain (P<.05) at .06% added methionine and feed efficiency at 0 (P<.05) and .06% (P<.001) added methionine.
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 2, 2015
those with chicks by Baker et al. (1983) who concluded that excessive choline had minimal capacity to reduce the dietary requirement for methionine. The addition of .10% potassium sulfate to diets deficient in methionine, or sulfur amino acids in the present experiments, significantly increased body weight and feed efficiency only about 3 and 2%, respectively, in contrast to 16 and 10% from .18% or more added DLmethionine. However, sulfate addition to diets with sufficient methionine failed to increase body weights or feed efficiencies. In previous studies (Potter et al, 1983 ; Potter and Shelton,
1550 |
135
136
B L A I R E T AL. T A B L E 5. Increase in body weight gain, feed consumption, and feed efficiency sulfate to diets containing graded levels of added methionine by
from adding experiments
potassium
Experiment1
Added methionine
Average
(%)
Increase in b o d y weight gain, g 6 -17 -30 33 -37 -38
59
56
.00 .06 .12 .18 .24 .30
58 3 -50 -18 30 47
46 30 -56 65 -18 25
LSD
103
104
LSD2
68 138*** 51 -41 25 28
36 50* -4 -12 1 12
(2.8%) (3.5%)
40
77 Increase in feed c o n s u m p t i o n , g
33 62 -30 -9 1 27
35 130* 54 -51 34 84
68
129 Increase in feed efficiency
.00 .06 .12 .18 .24 .30
563 105 -34 68 16 84
LSD
124
12 66 60 35 -16 -37 98
167** 220*** 62 -41 15 -67
78* (1.7%) 1 3 0 * * * (2.7%) 29 -25 -6 -7
118
68
1 Medium White t u r k e y s 3 t o 7 weeks of age used in E x p e r i m e n t 1 and Large White t u r k e y s 4 t o 8 weeks of age used in E x p e r i m e n t s 2 a n d 3. 2
LSD = Least significant difference.
' M u l t i p l y each b y 10~ 4 . 'Significant at t h e 5% level. ' • S i g n i f i c a n t at t h e 1% level. * * ' S i g n i f i c a n t at t h e . 1 % level.
1984), added sulfate to methionine-deficient diets for turkeys failed to increase body weights at any age except for increases of 3 and 2% at 2 and 4 weeks of age, respectively, in the second study. In contrast, methionine additions increased body weights about 9 and 7% at 2 weeks of age and 10 and 4% at 7 or 8 weeks of age, respectively, in these same studies. Similarly, Sloan and Harms (1972) have also demonstrated that sulfate and methionine increased body weights of turkeys to 3 weeks of age 4 and 11%, respectively, when added to diets deficient in methionine. Using diets with essentially the same formulation, Miles et al. (1983) obtained even larger increases (10 and 43%) in 3-week body weights of turkeys from added sulfate or added methionine, but sulfate failed
to increase body weights when added to the methionine-supplemented diet. Results of the present study support those in previous reports that sulfate has the capacity to increase body weights of young turkeys when added to methionine deficient diets but not when added to diets sufficient in methionine. Therefore, neither inorganic sulfate nor choline reduced the requirement for methionine in practicaltype diets for young turkeys in this study.
ACKNOWLEDGMENT The poults used in the last two experiments of this study were supplied by Wampler Hatchery, Harrisonburg, VA.
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 2, 2015
36 28 -33 -27 14 44
.00 .06 .12 .18 .24 .30
METHIONINE, CHOLINE, AND SULFATE
Pesti, G. M., A. E. Harper, and M. L. Sunde, 1979. Sulfur amino acid and methyl donor status of corn-soy diets fed to starting broiler chicks and turkey poults. Poultry Sci. 58:1541-1547. Pesti, G. M., A. E. Harper, and M. L. Sunde, 1980. Choline/methionine nutrition of starting broiler chicks. Three models for estimating the choline requirement with economic considerations. Poultry Sci. 59:1073-1081. Potter, L. M., and J. R. Shelton, 1979. Methionine and protein requirements of young turkeys. Poultry Sci. 58:609-615. Potter, L. M., and J. R. Shelton, 1984. Methionine, cystine, sodium sulfate, and Fermacto-500 supplementation of practical-type diets for young turkeys. Poultry Sci. 63:987-992. Potter, L. M., J. R. Shelton, and D. J. Castaldo, 1983. Supplementary inorganic sulfate and methionine for young turkeys. Poultry Sci. 62:2398-2402. Quillin, E. C , G. F. Combs, R. D. Creek, and G. L. Romoser, 1961. Effect of choline on the methionine requirements of broiler chickens. Poultry Sci. 40:639-645. Sherwood, D. H., and H. J. Sloan, 1954. Vitamin B I 2 and choline in corn-soy rations for starting poults. Poultry Sci. 33:1015-1021. Slinger, S. J., W. F. Pepper, and I. R. Sibbald, 1962. Interrelationships between methionine, choline, sodium chloride and reserpine in growing turkeys. Poultry Sci. 41:974-982. Sloan, D. R., and R. H. Harms, 1972. Utilization of inorganic sulfate by turkey poults. Poultry Sci. 51:1673-1675. Stas, R. J., and L. M. Potter, 1982. Amino acid supplementation of a 21% protein corn-soybean meal diet for young turkeys. Nutr. Rep. Int. 26: 487-494.
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 2, 2015
REFERENCES Baker, D. H., K. M. Halpin, G. L. Czarnecki, and C. M. Parsons, 1983. The choline-methionine interrelationship for growth of the chick. Poultry Sci. 62:133-137. Evans, R. J., 1943. The choline requirements of turkey poults. Poultry Sci. 22:266-267. Fidler, J. W., 1981. Sparing action between methionine and choline in poultry diets. Pages 1 8 - 2 0 in Proc. 41st Annu. Meet. Am. Feed Mfr. Assoc. Gartley, K. M., S. J. Slinger, A. M. Morphet, and C. R. Cameron, 1951. The choline requirements of Broadbreasted Bronze Turkeys in the presence of APF concentrates. Poultry Sci. 30:913. (Abstr.) Gerry, R. W., C. W. Carrick, and S. M. Hauge, 1948. Methionine and choline in a simplified chick ration. Poultry Sci. 27:161-168. Gogus, K., and P. Griminger, 1957. On the vitamin requirements of poults. 2. Choline. Poultry Sci. 36:1121. (Abstr.) Harms, R. H., and R. D. Miles, 1984. Effects of supplemental methionine and potassium sulfate on the choline requirement of the turkey poult. Poultry Sci. 63:1464-1466. Marvel, J. A., C. W. Carrick, R. E. Roberts, and S. M. Hauge, 1944. The supplementary value of choline and methionine in a corn and soybean oil meal chick ration. Poultry Sci. 23:294-297. Miles, R. D., N. Ruiz, and R. H. Harms, 1983. The interrelationships between methionine, choline, and sulfate in turkey diets. Proc. Soc. Exp. Biol. Med. 173:32-34. National Research Council, 1984. Nutrient Requirements of Domestic Animals. 1. Nutrient Requirements of Poultry. 8th ed. Natl. Acad. Sci., Washington, DC.
137