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The Effect of Dietary Methionine on the Methionine and Cystine Content of Poultry Meat1
The Effect of Dietary Methionine on the Methionine and Cystine Content of Poultry Meat1 JACK L. FRY 2 AND W. J. STADELMAN Purdue University, Lafayette, Indiana (Received for publication August 28, 1959)
TABLE 1.—Basal ration for dietary methionine study
M
OST present-day rations for growing chicks now contain DL-methionine as a supplement to the amino acids in the feed ingredients. This additional amino acid is used because growing rations are usually high in soybean oil meal with the ration subsequently being low in methionine. Although it is added for its stimulation of growth the authors felt that the dietary methionine might increase the methionine and cystine in the carcass thus providing a more nutritious food product. This study was designed to study the effects of age and dietary methionine on the methionine and cystine in the poultry meat.
Ground yellow corn Fish-n-fifty (50%) Di-calcium phosphate (18%) Ground limestone Iodized salt Manganese sulfate (70% tech. grade) Antibiotic—B12 supplement 1 Vitamin mixture 4-8-18 2 Dry choline (25% choline CI) Vit. E (20,000 I.U. Vit E/lb.) Vit. D3 (3,000 I.C.U. D 3 /gm.) Vit. A (5,000 I.U. vit A/gm.) Vit. K supplement 3
% 65.354 30.0 2.2 1.3 0.45 0.05 0.1 0.04 0.3 0.013 0.03 0.15 0.013
1 Contains 2 g. penicillin and 3 mg. vitamin B12 per pound of supplement. 2 Each pound supplies 4 g. riboflavin, 8 g. calcium pantothenate, and 18 g. niacin. 3 Contains 2.5 g. of menadione sodium bisulfite per pound of supplement.
MATERIALS AND METHODS Day-old broiler-type male chicks were assigned to battery pens at random, banded, and vaccinated for Newcastle disease immediately and for bronchitis after three days. To the basal ration (Table 1), 0%, .05% and . 5 % DL-methionine were added. Each ration was replicated twice by pens and each pen contained 50 chicks. Chicks were grown in large starting batteries until 6 weeks of age and were then transferred to holding batteries. At the end of 6, 9, and 12 weeks one-third of the birds were removed, weighed, slaughtered, and eviscerated. Dressed carcasses were cooled in crushed ice rather than ice water to prevent pos1 Journal Paper No. 1474 of the Purdue Agricultural Experiment Station, Lafayette, Indiana. 2 Present address: Poultry Department, Kansas State University, Manhattan, Kansas.
sible leaching of amino acids. Polyethylene bags were used for packaging and the birds were frozen at —6°F. ( —21°C.) and held until assayed. Birds to be assayed were selected at random from each lot at each age and methionine level combination. A composite was used, and, due to variation in size of birds of different ages, six 6-week old birds, four 9-week old birds, and three 12-week old birds were used from each group. The pectoralis major muscles from a given lot were combined, ground in a food chopper, dried by lyophilization and fat extracted with petroleum ether. In addition assays were made on the hearts and livers of the 12-week old birds. Details of analysis are reported (Fry and Stadelman, 1960 a, b). Briefly one gram samples of the moisture-free, fatfree samples were hydrolyzed with 3 N
614
615
DIETARY METHIONINE AND SULPHUR AMINO ACIDS IN MEAT TABLE 2.—Average live weights* of birds fed different levels of VL-methionine
TABLE 3.—Effect
of dietary DL-•methionine on muscle methionine *
Age Replicate
Age Replicate
9 12 6 weeks weeks weeks
Methionine Level
6 9 12 weeks weeks weeks
Methionine Level
0%
1 2 Av.
1.82 1.92 1.86
3.37 3.23 3.30
4.92 5.06 4.98
0%
1 2 Av.
2.82 2.77 2.79
2.83 2.93 2.88
2.79 2.83 2.81
.05%
1 2 Av.
1.86 2.00 1.93
3.18 3.26 3.22
5.12 5.21 5.17
.05%
1 2 Av.
2.84 2.79 2.81
2.87 2.83 2.85
2.85 2.83 2.84
•5%
1 2 Av.
1.93 1.90 1.92
3.44 3.44 3.44
5.02 5.23 5.12
•5%
1 2 Av.
2.90 2.81 2.85
2.96 2.90 2.93
2.87 2.87 2.87
* Live weights in pounds of all remaining birds in each group.
* Grams methionine per 16.0 grams of nitrogen.
HC1 in sealed tubes at 15 pounds autoclave pressure for 6 hours. The amino acid content was determined by microbiological assay using Leuconostoc mesenteroides P-60 ATCC No. 8042 as the test organism and measuring acid production after 72 hours incubation at 30°C.
The corresponding cystine values are presented in Table 5 followed by the analysis of the data in Table 6. In the case of cystine the effect of age has a highly significant effect on the cystine content of the pectoralis major with the birds at six weeks of age having lower levels than those at 9 and 12 weeks of age. This is not too surprising when one considers the fact that the major part of the feathering of the bird is accomplished during the first six weeks of its life. The protein of feathers is 6.7% cystine (Ward, Binkley and Snell, 1955), and it might be assumed that some of the cystine from the diet of birds less than six weeks of age is being utilized in the production of feathers rather than meat. As feather growth
RESULTS AND DISCUSSION The average live weights of the birds prior to slaughter are presented in Table 2. The added dietary DL-methionine was not statistically significant in its effect on live weight. The muscle methionine values of the birds on the different levels of methionine are presented in Table 3 and followed by the analysis of variance in Table 4. It will be observed that neither methionine level nor age had significant effects on the methionine content of the pectoralis major muscle. An interaction of treatment X age might have been expected but such was not the case. Though not significant there appears to be a slight trend toward higher methionine levels with increasing DL-methionine and increasing age, particularly from 6 weeks to 9 weeks.
TABLE 4.-—Anal-vsis
of variance of muscle methionine data
Source
d.f.
M.S.
Fobs
Treatment Replicate Age TXR AXR TXA RXTXA Error Total
2 1 2 2 2 4 4 54 71
.0217 .0070 .0273 .0124 .0083 .0020 .0035 .0090
2.41 .78 3.03 1.38 .92 .22 .38
616
J. L. FRY AND W. J. STADELMAN TABLE 5.-—Effect of
dietary DL-•methionine on muscle cystine*
TABLE 7.—Effect of dietary x>L-methionine on liver methionine and cystine*
Age cate
12 9 6 weeks weeks weeks
Methionine Level 0%
.05%
•5%
Replicate
Methionine level 0%
• 05%
.5%
Methionine
1 2 Av.
2.32 2.29 2.30
2.28 2.29 2.28
2.32 2.25 2.28
Cystine
1 2 Av.
1.69 1.53 1.61
1.50 1.67 1.59
1.56 1.63 1.60
1 2 Av.
1.18 1.22 1.20
1.23 1.28 1.25
1.29 1.28 1.29
1 2 Av.
1.23 1.21 1.22
1.26 1.23 1.25
1.28 1.26 1.27
* Grams amino acid per 16.0 grams nitrogen.
1 2 Av.
1.20 1.25 1.22
1.27 1.27 1.27
1.30 1.28 1.29
TABLE 8.—Effect of dietary DL-methionine on heart methionine and cystine*
* Grams cystine per 16.0 grams nitrogen. Replicate
slows down, more cystine may be channeled toward the formation of protein of muscle. Tables 7 and 8 present the cystine and methionine values of the liver and heart of 12 week old birds on different levels of dietary methionine. Little or no change was observed in the methionine and cystine content of liver, and only slight increases were noted in the case of heart. It may be of interest, however, that both liver and heart are considerably higher in cystine and lower in methionine than is the pectoralis major muscle. In general added DL-methionine can be said to have little effect on the methionine or cystine content of birds consuming TABLE 6.—An•alysis of variance
muscle cystine data Source
d.f.
M.S.
Treatment Replicate Age TXR AXR TXA RXTXA Error Total
2 1 2 2 2 4 4 54 71
.0015 .0004 .0264 .0051 .0026 .0008 .0010 .0021
Significant at .01 level.
of •fobs
.71 .19 12.57** 2.43 1.24 .38 .48
Methionine level 0%
.05%
.5%
Methionine
1 2 Av.
2.65 2.62 2.64
2.69 2.71 2.70
2.64 2.74 2.69
Cystine
1 2 Av.
1.63 1.63 1.63
1.67 1.72 1.69
1.64 1.73 1.68
* Grams amino acid per 16.0 grams nitrogen.
it in levels up to . 5 % of the diet. Additions of methionine to the diet should be made for growth response and certainly not for increased amino acids in the meat. SUMMARY The supplemental levels of 0%, .05%, and . 5 % dietary DL-methionine were found to have little effect on the methionine or cystine content of poultry meat. Average values for the amino acid content of the pectoralis major per 16.0 grams of nitrogen were 2.85 grams of methionine and 1.25 grams of cystine. Age, though not significant for methionine, was shown to be an important factor affecting the cystine content of muscle. Probably tied in with feather formation, the cystine content of poultry meat is higher at 9 and 12 weeks than at 6 weeks of age.
DIETARY METHIONINE AND SULPHUR AMINO ACIDS IN MEAT REFERENCES Fry, J. L., and W. J. Stadelman, 1960a. Optimum conditions of hydrolysis for microbiological assay of amino acids in poultry meat. 1. Methionine and cystine. J. Ag. Food Chem. (in press). Fry, J. L., and W. J. Stadelman, 1960b. The effect
617
of cooking and carcass part on the methionine and cystine content of poultry meat. Food Research (in press). Ward, W. H., G. H. Binkley and N. S. Snell, 1955. Amino acid composition of normal wool, wool fractions, mohair, feather and feather fractions. Textile Research J. 25: 314-325.
The Influence of Orally and Parenterally Administered Salts of Terephthalic Acid on Oxytetracycline Serum Levels in Chickens K. E. PRICE, 1 Z. ZOLLI, JR. AND G. A. DONOVAN Chas. Pfizer &" Co., Inc., Terre Haute, Ind. (Received for publication August 28, 1959)
I
T HAS been demonstrated previously that concurrent supplementation of poultry rations with terephthalic acid and oxytetracycline2 results in higher antibiotic serum levels than those obtained with oxytetracycline alone (Price et ah, 1959b; Price and Zolli, 1959a). A possible mode of action by which terephthalic acid potentiates the tetracycline antibiotics has been the subject of a separate report (Price and Zolli, 1959b). In this, it was shown that the relatively soluble sodium salt of terephthalic acid increased oxytetracycline serum levels, whether administered via the gastrointestinal tract or parenterally. Gastrointestinal tract administration was accomplished by injection into a ligated segment of the small intestine, while parenteral administration was by various routes. Although the latter studies were limited, it appeared that the antibiotic serum level response was inversely proportional to the rate of release of the terephthalic 1
Present address: Bristol Laboratories, Syracuse, N.Y. 2 Terramycin, trademark of Chas. Pfizer & Co., Inc.
acid salt from the site of injection, as less potentiation was obtained from administration of terephthalic acid intravenously than intramuscularly. The greatest potentiating effect was with subcutaneous administration. The purpose of the present investigation was to determine the influence of several terephthalic acid salts and the dimethyl ester of terephthalic acid on oxytetracycline serum levels when the antibiotic and these substances were concurrently administered in the feed, in the drinking water, or parenterally. Efforts were made also to establish optimum concentrations of the terephthalate derivatives for maximum potentiation of oxytetracycline. EXPERIMENTAL PROCEDURE Experiment 1. Eighty-five five-weekold male Nichols White Cross chicks were allotted into 17 groups of 5 birds each, groups being approximately equal in weight. Birds were placed in finishing batteries and given the appropriate supplement in a diet which was basically corn-soy and fairly typical of commercial rations. Calcium and phosphorus, which
TABLE 1.—Basal ration for dietary methionine study
M
OST present-day rations for growing chicks now contain DL-methionine as a supplement to the amino acids in the feed ingredients. This additional amino acid is used because growing rations are usually high in soybean oil meal with the ration subsequently being low in methionine. Although it is added for its stimulation of growth the authors felt that the dietary methionine might increase the methionine and cystine in the carcass thus providing a more nutritious food product. This study was designed to study the effects of age and dietary methionine on the methionine and cystine in the poultry meat.
Ground yellow corn Fish-n-fifty (50%) Di-calcium phosphate (18%) Ground limestone Iodized salt Manganese sulfate (70% tech. grade) Antibiotic—B12 supplement 1 Vitamin mixture 4-8-18 2 Dry choline (25% choline CI) Vit. E (20,000 I.U. Vit E/lb.) Vit. D3 (3,000 I.C.U. D 3 /gm.) Vit. A (5,000 I.U. vit A/gm.) Vit. K supplement 3
% 65.354 30.0 2.2 1.3 0.45 0.05 0.1 0.04 0.3 0.013 0.03 0.15 0.013
1 Contains 2 g. penicillin and 3 mg. vitamin B12 per pound of supplement. 2 Each pound supplies 4 g. riboflavin, 8 g. calcium pantothenate, and 18 g. niacin. 3 Contains 2.5 g. of menadione sodium bisulfite per pound of supplement.
MATERIALS AND METHODS Day-old broiler-type male chicks were assigned to battery pens at random, banded, and vaccinated for Newcastle disease immediately and for bronchitis after three days. To the basal ration (Table 1), 0%, .05% and . 5 % DL-methionine were added. Each ration was replicated twice by pens and each pen contained 50 chicks. Chicks were grown in large starting batteries until 6 weeks of age and were then transferred to holding batteries. At the end of 6, 9, and 12 weeks one-third of the birds were removed, weighed, slaughtered, and eviscerated. Dressed carcasses were cooled in crushed ice rather than ice water to prevent pos1 Journal Paper No. 1474 of the Purdue Agricultural Experiment Station, Lafayette, Indiana. 2 Present address: Poultry Department, Kansas State University, Manhattan, Kansas.
sible leaching of amino acids. Polyethylene bags were used for packaging and the birds were frozen at —6°F. ( —21°C.) and held until assayed. Birds to be assayed were selected at random from each lot at each age and methionine level combination. A composite was used, and, due to variation in size of birds of different ages, six 6-week old birds, four 9-week old birds, and three 12-week old birds were used from each group. The pectoralis major muscles from a given lot were combined, ground in a food chopper, dried by lyophilization and fat extracted with petroleum ether. In addition assays were made on the hearts and livers of the 12-week old birds. Details of analysis are reported (Fry and Stadelman, 1960 a, b). Briefly one gram samples of the moisture-free, fatfree samples were hydrolyzed with 3 N
614
615
DIETARY METHIONINE AND SULPHUR AMINO ACIDS IN MEAT TABLE 2.—Average live weights* of birds fed different levels of VL-methionine
TABLE 3.—Effect
of dietary DL-•methionine on muscle methionine *
Age Replicate
Age Replicate
9 12 6 weeks weeks weeks
Methionine Level
6 9 12 weeks weeks weeks
Methionine Level
0%
1 2 Av.
1.82 1.92 1.86
3.37 3.23 3.30
4.92 5.06 4.98
0%
1 2 Av.
2.82 2.77 2.79
2.83 2.93 2.88
2.79 2.83 2.81
.05%
1 2 Av.
1.86 2.00 1.93
3.18 3.26 3.22
5.12 5.21 5.17
.05%
1 2 Av.
2.84 2.79 2.81
2.87 2.83 2.85
2.85 2.83 2.84
•5%
1 2 Av.
1.93 1.90 1.92
3.44 3.44 3.44
5.02 5.23 5.12
•5%
1 2 Av.
2.90 2.81 2.85
2.96 2.90 2.93
2.87 2.87 2.87
* Live weights in pounds of all remaining birds in each group.
* Grams methionine per 16.0 grams of nitrogen.
HC1 in sealed tubes at 15 pounds autoclave pressure for 6 hours. The amino acid content was determined by microbiological assay using Leuconostoc mesenteroides P-60 ATCC No. 8042 as the test organism and measuring acid production after 72 hours incubation at 30°C.
The corresponding cystine values are presented in Table 5 followed by the analysis of the data in Table 6. In the case of cystine the effect of age has a highly significant effect on the cystine content of the pectoralis major with the birds at six weeks of age having lower levels than those at 9 and 12 weeks of age. This is not too surprising when one considers the fact that the major part of the feathering of the bird is accomplished during the first six weeks of its life. The protein of feathers is 6.7% cystine (Ward, Binkley and Snell, 1955), and it might be assumed that some of the cystine from the diet of birds less than six weeks of age is being utilized in the production of feathers rather than meat. As feather growth
RESULTS AND DISCUSSION The average live weights of the birds prior to slaughter are presented in Table 2. The added dietary DL-methionine was not statistically significant in its effect on live weight. The muscle methionine values of the birds on the different levels of methionine are presented in Table 3 and followed by the analysis of variance in Table 4. It will be observed that neither methionine level nor age had significant effects on the methionine content of the pectoralis major muscle. An interaction of treatment X age might have been expected but such was not the case. Though not significant there appears to be a slight trend toward higher methionine levels with increasing DL-methionine and increasing age, particularly from 6 weeks to 9 weeks.
TABLE 4.-—Anal-vsis
of variance of muscle methionine data
Source
d.f.
M.S.
Fobs
Treatment Replicate Age TXR AXR TXA RXTXA Error Total
2 1 2 2 2 4 4 54 71
.0217 .0070 .0273 .0124 .0083 .0020 .0035 .0090
2.41 .78 3.03 1.38 .92 .22 .38
616
J. L. FRY AND W. J. STADELMAN TABLE 5.-—Effect of
dietary DL-•methionine on muscle cystine*
TABLE 7.—Effect of dietary x>L-methionine on liver methionine and cystine*
Age cate
12 9 6 weeks weeks weeks
Methionine Level 0%
.05%
•5%
Replicate
Methionine level 0%
• 05%
.5%
Methionine
1 2 Av.
2.32 2.29 2.30
2.28 2.29 2.28
2.32 2.25 2.28
Cystine
1 2 Av.
1.69 1.53 1.61
1.50 1.67 1.59
1.56 1.63 1.60
1 2 Av.
1.18 1.22 1.20
1.23 1.28 1.25
1.29 1.28 1.29
1 2 Av.
1.23 1.21 1.22
1.26 1.23 1.25
1.28 1.26 1.27
* Grams amino acid per 16.0 grams nitrogen.
1 2 Av.
1.20 1.25 1.22
1.27 1.27 1.27
1.30 1.28 1.29
TABLE 8.—Effect of dietary DL-methionine on heart methionine and cystine*
* Grams cystine per 16.0 grams nitrogen. Replicate
slows down, more cystine may be channeled toward the formation of protein of muscle. Tables 7 and 8 present the cystine and methionine values of the liver and heart of 12 week old birds on different levels of dietary methionine. Little or no change was observed in the methionine and cystine content of liver, and only slight increases were noted in the case of heart. It may be of interest, however, that both liver and heart are considerably higher in cystine and lower in methionine than is the pectoralis major muscle. In general added DL-methionine can be said to have little effect on the methionine or cystine content of birds consuming TABLE 6.—An•alysis of variance
muscle cystine data Source
d.f.
M.S.
Treatment Replicate Age TXR AXR TXA RXTXA Error Total
2 1 2 2 2 4 4 54 71
.0015 .0004 .0264 .0051 .0026 .0008 .0010 .0021
Significant at .01 level.
of •fobs
.71 .19 12.57** 2.43 1.24 .38 .48
Methionine level 0%
.05%
.5%
Methionine
1 2 Av.
2.65 2.62 2.64
2.69 2.71 2.70
2.64 2.74 2.69
Cystine
1 2 Av.
1.63 1.63 1.63
1.67 1.72 1.69
1.64 1.73 1.68
* Grams amino acid per 16.0 grams nitrogen.
it in levels up to . 5 % of the diet. Additions of methionine to the diet should be made for growth response and certainly not for increased amino acids in the meat. SUMMARY The supplemental levels of 0%, .05%, and . 5 % dietary DL-methionine were found to have little effect on the methionine or cystine content of poultry meat. Average values for the amino acid content of the pectoralis major per 16.0 grams of nitrogen were 2.85 grams of methionine and 1.25 grams of cystine. Age, though not significant for methionine, was shown to be an important factor affecting the cystine content of muscle. Probably tied in with feather formation, the cystine content of poultry meat is higher at 9 and 12 weeks than at 6 weeks of age.
DIETARY METHIONINE AND SULPHUR AMINO ACIDS IN MEAT REFERENCES Fry, J. L., and W. J. Stadelman, 1960a. Optimum conditions of hydrolysis for microbiological assay of amino acids in poultry meat. 1. Methionine and cystine. J. Ag. Food Chem. (in press). Fry, J. L., and W. J. Stadelman, 1960b. The effect
617
of cooking and carcass part on the methionine and cystine content of poultry meat. Food Research (in press). Ward, W. H., G. H. Binkley and N. S. Snell, 1955. Amino acid composition of normal wool, wool fractions, mohair, feather and feather fractions. Textile Research J. 25: 314-325.
The Influence of Orally and Parenterally Administered Salts of Terephthalic Acid on Oxytetracycline Serum Levels in Chickens K. E. PRICE, 1 Z. ZOLLI, JR. AND G. A. DONOVAN Chas. Pfizer &" Co., Inc., Terre Haute, Ind. (Received for publication August 28, 1959)
I
T HAS been demonstrated previously that concurrent supplementation of poultry rations with terephthalic acid and oxytetracycline2 results in higher antibiotic serum levels than those obtained with oxytetracycline alone (Price et ah, 1959b; Price and Zolli, 1959a). A possible mode of action by which terephthalic acid potentiates the tetracycline antibiotics has been the subject of a separate report (Price and Zolli, 1959b). In this, it was shown that the relatively soluble sodium salt of terephthalic acid increased oxytetracycline serum levels, whether administered via the gastrointestinal tract or parenterally. Gastrointestinal tract administration was accomplished by injection into a ligated segment of the small intestine, while parenteral administration was by various routes. Although the latter studies were limited, it appeared that the antibiotic serum level response was inversely proportional to the rate of release of the terephthalic 1
Present address: Bristol Laboratories, Syracuse, N.Y. 2 Terramycin, trademark of Chas. Pfizer & Co., Inc.
acid salt from the site of injection, as less potentiation was obtained from administration of terephthalic acid intravenously than intramuscularly. The greatest potentiating effect was with subcutaneous administration. The purpose of the present investigation was to determine the influence of several terephthalic acid salts and the dimethyl ester of terephthalic acid on oxytetracycline serum levels when the antibiotic and these substances were concurrently administered in the feed, in the drinking water, or parenterally. Efforts were made also to establish optimum concentrations of the terephthalate derivatives for maximum potentiation of oxytetracycline. EXPERIMENTAL PROCEDURE Experiment 1. Eighty-five five-weekold male Nichols White Cross chicks were allotted into 17 groups of 5 birds each, groups being approximately equal in weight. Birds were placed in finishing batteries and given the appropriate supplement in a diet which was basically corn-soy and fairly typical of commercial rations. Calcium and phosphorus, which