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Influence of Choline and Sulfate on Copper and Toxicity and Substitition of and Antagonism Between Methionine and Copper Supplements to Chick Diets1
Influence of Choline and Sulfate on Copper and Toxicity and Substitition of and Antagonism Between Methionine and Copper Supplements to Chick Diets 1 JEUN-SHYH WANG, STEPHEN R. ROGERS, and GENE M. PESTI2 Department of Poultry Science, Livestock-Poultry Building, University of Georgia, Athens, Georgia 30602 (Received for publication March 10, 1986) ABSTRACT Studies were conducted to determine if the deleterious affects on chick growth of the primary antagonism between methionine and copper involves the homocysteine moiety or labile methyl group of methionine. A . 1 % choline supplement resulted in performance response similar to that of a .2% L-methionine supplement in the absence but not in the presence of 500 mg/kg copper from cupric sulfate. Similar results were observed when the levels of methionine and choline were doubled. Sulfate, with or without choline, had little effect in the presence of cupric sulfate. When cupric acetate was used instead of cupric sulfate, a small but nearly significant (P = .08) response to poiassium sulfate was observed. Maximum performance with .29% supplemental methionine and 188 mg/kg Cu was predicted from a response surface analysis. The methionine requirement was increased by feeding copper. However, the increase in methionine requirement was accompanied by an improvement in growth rate and feed efficiency. This may explain why levels used of methionine and total sulfur-containing amino acids appear to be higher under field conditions (with pharmacological levels of copper) than in laboratory conditions (without pharmacological levels of copper). The primary antagonism between methionine and copper involves the homocysteine moiety, not the labile methyl groups. (Key words: methionine, copper, choline, sulfate) 1987 Poultry Science 66:1500-1507 INTRODUCTION
The chick's nutritional requirement for copper is approximately 8 mg/kg (National Research Council, 1984). Copper is often fed at much higher pharmacological levels (100 to 300 mg/kg Cu) because of its growth-promoting properties (Fisher et al., 1970; Fisher, 1973). Although copper and methionine have been demonstrated to be antagonists (Jensen and Maurice, 1976), quantification of the relationship between them at levels common in feeds has not been performed. Baker and Robbins (1979) and Robbins and Baker (1980) reported that the addition of 125 mg Cu per kilogram diet to a crystalline amino acid diet significantly influenced the total sulphur-containing amino acid requirement of chicks. However, Waldroup et al. (1979) did not observe this with chicks fed a corn and soybean meal basal diet. The question of how much (if any) increase in methionine requirement results from the addi-
' Supported by State and Hatch funds allocated to the Georgia Agricultural Experiment Stations of the University of Georgia. 2 To whom correspondence should be addressed.
tion of pharmacological levels of cupric sulfate (400 to 1,000 mg/kg) to a diet has not been answered. Choline has also been shown to influence, by donating labile methyl groups, the methionine requirement of chicks fed practical diets (Quillin et al, 1961; Pesti et al, 1979, 1980). The first experiments reported herein were designed to determine if the primary antagonism is between cupric sulfate and methionine or between cupric sulfate and labile methyl groups. The influence of sulfate and methionine supplements was then determined in the presence of 500 mg/kg Cu. Finally a response surface depicting the response to various levels of methionine and cupric sulfate supplements was determined. MATERIALS AND METHODS
Day-old male broiler chicks (Peterson x Arbor Acres) from a commercial hatchery were used in each experiment and grown to 3 wk of age in temperature-controlled Petersime battery brooders with raised wire floors and constant illumination. Feed and water were supplied ad libitum. A corn soybean meal poultry oil-based
1500
METHIONINE AND COPPER IN CHICK DIETS
diet was fed in each experiment. Mortality was recorded daily. Feed consumption data were adjusted to account for any deaths on a chick day basis. Analyses of variance and orthogonal comparisons were calculated using the general linear models procedures of the Statistical Analysis System (SAS) Institute, Inc. (1982). The ingredient and nutrient composition of the basal diets are given in Table 1. In Experiment 1, two levels of L-methionine (basal and basal plus .2%) and two levels of choline (basal and basal plus .1%) were fed in the presence of 0 or 500 mg/kg Cu (from cupric sulfate). Each diet was fed to five randomly assigned pens of 10 male chicks each. In Experiment 2, two levels of methionine (basal and basal plus .4%), two levels of choline (basal and basal plus .2%) and two levels of sulfate (basal and basal plus .15% from potassium sulfate) in addition to two levels of copper were fed to five randomly assigned pens of 10 male chicks each. Experiment 3 had a 3 x 2 factorial design with three levels of L-methionine (basal and basal plus .2 or .4%) and two levels of potassium sulfate (basal and basal plus 758 mg/kg) in the presence of 500 mg/kg Cu in the form of cupric acetate. A negative control diet without copper, methionine, or sulfate was also fed. Each diet was fed to three pens of 10 chicks each. In Experiment 4, four levels of methionine (basal and basal plus . 1 , .2, or .4%) and four levels of copper as cupric sulfate (basal and basal plus 125, 250, or 500 mg/kg) were fed in each of two trials. Data were pooled to fit the response surface. Each diet was fed to two or three randomly assigned pens of 10 male chicks each in each trial, for a total of five replicates for each diet. The basal diet was supplemented to contain 2,000 mg/kg choline. The following response model was fitted: Y = b 0 + T + b. Met + b 2 Met2 + b 3 Cu + b4XSuB cu2 + b 5 Cu • Met where Y represents the weight gain, feed efficiency, or feed consumption, T = trial (0 or 1), Met = methionine concentration (%), and Cu = copper concentration in milligrams per kilogram. Terms where coefficients were not significantly different from zero (P>.10) were removed from the models. Models with all coefficients significantly different from zero were then calculated. The general linear models procedure (SAS Institute Inc., 1982) was used for the analyses.
1501
Choline was determined by the Reineckate technique described by Lim and Schall (1963). Methionine and cystine analyses were conducted by another laboratory using a modification of the method of Moore et al. (1958). RESULTS
Experiment 1. In the absence of supplemental copper, gain and feed efficiency responses to . 1 % supplemental choline were equivalent to responses from the .2% L-methionine supplement (Table 2). However, in the presence of 500 mg/kg Cu, response to L-methionine was much greater than response to choline. This resulted in the significant methionine x copper and methionine x choline interactions, and lack of a copper x choline interaction (P>.05). Experiment 2. The supplementation of .4% L-methionine completely overcame the adverse effects of feeding 500 mg/kg Cu from cupric sulfate (Table 3). Supplementation of .2% choline resulted in a significant response, as in Experiment 1. Supplemental sulfate, either alone or in addition to choline, was without effect. Experiment 3. In the presence of 500 mg/kg Cu from cupric acetate there was a large and significant response in gain and feed efficiency to L-methionine (Table 4). A much smaller, nearly significant (P = .08) response in gain to sulfate supplementation was observed. Experiment 4. At low copper levels, chick gains were higher but were lower with increased supplemental methionine (Table 5, Figure 1). At high copper levels, gains were higher with higher methionine levels. Conversely, at low methionine levels gains were lower with increased copper; but at high methionine levels gains were higher and then lower with higher copper levels. Feed efficiency trends were similar although efficiency gains were smaller (Table 6, Figure 2). Response surface equations accounted for 86% of the variation in weight gain and 68% of the variation in feed efficiency (Table 7). DISCUSSION
Chicks fed the basal diet used in these experiments responded to . 1 % dietary choline to about the same extent as to .2% dietary L-methionine. These results (Table 1) are similar to those of Quillin et al. (1961) and Pesti et al. (1979, 1980), but different from those of Miles et al. (1983) who found a response to choline only in the presence of supplemental sulfate.
3,265 23.00 .38 .74 1,390
Selenium premix: selenium, .02%; calcium carbonate, 99.98%.
National Research Council, 1984.
52.34 37.93 5.87 1.72 1.18 .05 .05 .05 .05 .05
(%>
Calculation 1
22.94 .38 .76 1,089
Experiment 1
Analysis
23.46 .39 .78 1,132
Experiment 2
23.20 .41 .81 1,687
Experiments 3 and 4
Mineral mix provides (milligrams per kilogram of diet): Mn, 60; Zn, 50; Fe, 30; Cu, 5; I, 1.05.
'Bacitracin: BMD 50.
4
Vitamin mix included the following (per kilogram of diet): vitamin A, 5,000 IU; vitamin D 3 , 1,100 ICU; vitamin E, 11 IU; riboflavin, 4.4 mg;Ca pantothenate, 12 mg; nicotinic acid, 44 mg; vitamin B 1 2 , 6.6 Mg; vitamin B 6 , 2.2 mg; menadione, 1.1 mg; folic acid, .55 mg; d-biotin, .11 mg; thiamine, 2.2 mg; ethoxyquin, 125 mg.
3
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1
Ingredient Corn Soybean meal Poultry oil Dicalcium phosphate Limestone Salt Selenium premix 2 Vitamin mix 3 Mineral mix" Bacitracin5 Nutrient Metabolizable energy, kcal/kg Protein, % Methionine, % Methionine plus cystine, % Choline, mg/kg
Diet component
TABLE 1. Ingredient and nutrient composition of basal diet by calculation and analysis
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TABLE 2. Effects of methionine, choline, and Cu supplements1 on copper toxicity: 0 to 20-day weight gain and feed efficiency of male broiler chicks (Experiment i ) 2
Methionine
Cu (mg/kg)
Choline
(%)
-
(g) 541 568 568 558 413 456 498 508
.1 .2 .2 500 500 500 500 1
.1 .1
.2 .2
Feed efficiency3
Gain
.1
± ± ± ± ± ± ± ±
13 3 11 6 9 14 7 5
723 749 759 753 707 733 786 786
± ± ± ± + ± ± ±
6 8 7 4 12 6 8 5
Provided as CuS0 4 - 5HjO, L-methionine, and choline chloride, respectively.
2
Cu, methionine, and choline main effects and the Cu X methionine and methionine X choline interactions significantly contributed to the variation in gain (P<.05); methionine and choline main effects and Cu X methionine and methionine X choline interactions significantly contributed to the variation in feed efficiency (P<.05). 3
Grams gained per kilogram of feed consumed.
The response to choline was much less than the response to methionine in the presence of 500 mg/kg cupric sulfate (Table 1). This indicates that the primary antagonism that exists between copper and methionine is to methionine per se (or rather the homocysteine moeity) and not to labile methyl groups. This conclusion was confirmed by Experiment 2 wherein levels of methionine and choline were doubled (Table 3). In addition, sulfate supplements were without effect when added either alone or with choline. Therefore, the relationship demonstrated by Miles et al. (1983) was not evident under conditions of copper toxicosis.
As copper was added in the form of cupric sulfate in Experiments 1 and 2, it is possible that some of the negative effects of the copper were being overcome by the sulfate added with it. Experiment 3 was conducted to determine if the small amount of sulfate from the copper sulfate could have affected the outcome of Experiments 1 and 2. The small response found by adding sulfate on an isomolar basis (copper provided in the form of cupric acetate) was nearly significant (P = .08) and fairly constant at three levels of methionine. Therefore the growth depression produced by feeding 500 mg/ kg copper may have been slightly less than ex-
TABLE 3. Effects of methionine, choline and sulfate supplements1 on copper toxicity: 0 to 20-day weight gain and feed efficiency of male broiler chicks (Experiment 2) 2
Diet
1 2 3 4 5 7 1
Cu
Methionine
Choline
Sulfate
Gain
(mg/kg)
(g)
500 500 500 500 500
.15 .15
475 394 471 422 391 429
.4 .2 .2
± ± ± + ± ±
Feed efficiency3
20 8 23 21 22 10
699 699 748 714 685 695
± ± ± ± ± ±
10 16 27 22 10 22
Provided as CuSO„ -SH^O, L-methionine, choline chloride, and potassium sulfate, respectively.
2
The following means were found to be significantly different for gain and feed efficiency by orthogonal comparison (P<.05): Diets 1 vs. 2, 3 vs. 2, and 4 vs. 2. No significant differences were found for Diets 5 vs. 2 or 4 vs. 6 (P>.20). 3
Grams gained per kilogram of feed consumed.
1504
WANG ET AL.
pected had the copper not been accompanied by sulfate in Experiments 1 and 2. However, effect of dietary sulfate appears to be small, if indeed it is real. Copper is normally added to practical diets in the form of the sulfate, so the final experiment was conducted with cupric sulfate. Our original hypothesis was that copper is increasing the chick methionine requirement through the known antagonism between copper and methionine. It could be counterproductive to add cupric sulfate as an inexpensive growth promoter if it is increasing the methionine requirement, and therefore the cost of methionine supplementation. This hypothesis was confirmed in that the methionine requirement was higher with added copper. However, there were also higher body weight gains (Table 5 and 7, Figure 1). This may explain why most commercial producers feed broiler chickens levels of methionine much higher than requirements found under laboratory conditions. Most commercial feeds contain pharmacological levels of copper, but laboratory diets do not. The response surface depicted in Figure 1 contains what economists would call a zone of substitution (the shaded area). In that zone the same response (e.g., 553 g) was obtained by feeding a high level of methionine (.25%) and a low level of copper (.25% methionine, 60 mg/kg Cu), or a low level of methionine and a high level of copper (.18% methionine ,130 mg/ kg Cu). Outside this zone either methionine or copper depresses gains at any point on the surface. The increase in the methionine requirement is evident from the slope of the upper border of the zone of substitution. Response theory holds that it is most important to use experimental data to derive a best-fit surface equation (Box and Wilson, 1951). The surface equation can then be used to predict values of the inputs yielding the maximum response or to predict the point of economic efficiency. The point of economic efficiency is where the value of the return (gain) is just offset by the costs of the inputs (copper and methionine). This is a simple calculation if the response surface equation and costs of gain, methionine, and copper are known. The response surface approach is an alternative to picking and choosing among the experimental points to determine where the maximum response or most profitable feeding levels are. In determining a response surface, all the points are used to determine the shape of the surface. Thus, although the maximum observed response
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methionine and 188 mg/kg supplemental copper. From Figure 1 it is apparent that there is a large area of the surface with responses near the maximum. Criteria for judging whether the surface adequately describes the observed data depend on the objectives. From a statistical viewpoint this surface is a good fit (Table 7). Eighty-six percent of the variability in the gain data were described by the model and the coefficients were all significantly different from zero. If very small differences in response are economically important, the experiment would require repetition until the average response was defined within an economically relevant tolerance. The maximum response in gain (560 g) indicates a total sulfur-containing amino acid requirement of 1.06% in a 23% protein diet, similar to what some practicing nutritionists feel is necessary under field conditions. The value for copper (188 mg/kg) is very similar to Fisher's (1973) estimate from evaluating published values (169 mg/kg Cu). Fisher (1973) also estimated a maximum for feed efficiency at 140 mg/kg copper; in Experiment 4 (Figure 2) no clear maximum was determined within the range of the data. Efficiency was slightly decreased with increased copper at low methionine levels, but slightly increased at higher methionine levels. The results of Experiment 4 are very similar to those of Robbins and Baker (1980), qualitatively if not quantitatively. Requirement estimates from experiments with corn and soybean meal-based diets (Tables 5 to 7) were higher than those of Robbins and Baker (1980) with crystaline amino acid-based diets, as is generally found. The reason chicks in Experiment 4 responded differently than those of Waldroup et al. (1979) is not obvious. Waldroup et al. (1979) examined a smaller portion of the surface and had increased variability from using both male and female chicks. These studies confirm that feeding pharmacological levels of copper is beneficial to the chick. Although the methionine requirement appears to be increased by this practice, the increase is accompanied by an improvement in overall growth rate and feed efficiency. This phenomenon may explain why methionine and total sulfur-containing amino acid requirements appear to be higher under field conditions than under laboratory conditions. Copper should be on the list of dietary factors influencing the methionine requirement of chicks fed practical
METHIONINE AND COPPER IN CHICK DIETS
1507
TABLE 7. Coefficients from multiple linear regression analyses of body weight gain and feed as functions of methionine and Cu supplementation (Experiment 4)1 Source of variation
Weight gain
Intercept Trial Methionine Cu Methionine 2 Cu 2 Methionine X Cu Coefficient of determination (r 2 )
523.3272** -34.9564**
331.0482** .0787* -714.8864**
-.0005** .4205** .86
efficiency
Feed efficiency .6834** -.0081*
.3340** -4.6 X 1 0 ~ 5 * * -.6608** .0003** .68
1 For example: predicted body gain of chicks fed .2% supplemental L-methionine and 250 mg/kg copper in Trial 1 = 523.3 - (35.0) (.5) + 331 (.2) + .0787 (250) - 714.9 (.2) 2 - .0005 (250) 2 + .4205 (.2) (250) = 553; actual = 545 ± 9.
*P<.05. **P<.01.
diets along with protein, energy, cystine, sulfate, choline, and vitamin B 12 . REFERENCES Baker, D. H., and K. R. Robins, 1979. Sulfur amino acid utilization in chicks fed supplemental monensin and copper. Pages 39-44 in: Proc. 1979 Maryland Nutr. Conf. Box, G.E.P., and K. B. Wilson, 1951. On the experimental attainment of optimum conditions (with discussion). J. R. Stat. Soc. B13:1^15. Fisher, C., 1973. Use of copper sulfate as a growth promoter for broilers. Feedstuffs: July 16, 24-25. Fisher, C , D. Wise, and D. G. Filmer, 1970. The effect of copper on the growth of broilers and the interaction of copper with zinc and iron. Proc. 14th World's Poult. Congr. Madrid, Spain 2:759-764. Jensen, L. S., and D. V. Maurice, 1976. Alleviation of copper-induced growth depression by methionine. Poultry Sci. 55:2048-2049. (Abstr.) Lim, F., and E. D. Schall, 1963. Determination of choline in feeds. Seventy-seventh Annu. Meet. Assoc. Off. Agric. Chem. Purdue Univ., Agric. Exp. Stn., West Lafayette, IN. Miles, R. D., N. Ruiz, and R. H. Harms, 1983. The interrelationship between methionine, choline, and sulfate in broiler diets. Poultry Sci. 62:495^198.
Moore, S., D. H. Spackman, and W. N. Stein, 1958. Chromatography of amino acids on sulfonated polystyrene resins. Anal. Chem. 30:1185-1190. National Research Council, 1984. Nutrient Requirements of Domestic Animals. 1. Nutrient Requirements of Poultry. Natl. Acad. Sci., Washington, DC. 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. 59:10731081. 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. 40:639645. Robbins, K. R., andD. H. Baker, 1980. Effect of high-level copper feeding on the sulfur amino acid need of chicks fed corn-soybean meal and purified crystalline amino acid diets. Poultry Sci. 59:1099-1108. Statistical Analysis System Institute, Inc., 1982. SAS User's Guide: Basic. 1982 ed. SAS Institute, Inc., Cary, NC. Waldroup, P. W., C. J. Mabray, J. R. Blackman, and F. Z. Johnson, 1979. The influence of copper sulfate on the methionine requirement of the young chick. Nutr. Rep. Int. 20:303-308.
The chick's nutritional requirement for copper is approximately 8 mg/kg (National Research Council, 1984). Copper is often fed at much higher pharmacological levels (100 to 300 mg/kg Cu) because of its growth-promoting properties (Fisher et al., 1970; Fisher, 1973). Although copper and methionine have been demonstrated to be antagonists (Jensen and Maurice, 1976), quantification of the relationship between them at levels common in feeds has not been performed. Baker and Robbins (1979) and Robbins and Baker (1980) reported that the addition of 125 mg Cu per kilogram diet to a crystalline amino acid diet significantly influenced the total sulphur-containing amino acid requirement of chicks. However, Waldroup et al. (1979) did not observe this with chicks fed a corn and soybean meal basal diet. The question of how much (if any) increase in methionine requirement results from the addi-
' Supported by State and Hatch funds allocated to the Georgia Agricultural Experiment Stations of the University of Georgia. 2 To whom correspondence should be addressed.
tion of pharmacological levels of cupric sulfate (400 to 1,000 mg/kg) to a diet has not been answered. Choline has also been shown to influence, by donating labile methyl groups, the methionine requirement of chicks fed practical diets (Quillin et al, 1961; Pesti et al, 1979, 1980). The first experiments reported herein were designed to determine if the primary antagonism is between cupric sulfate and methionine or between cupric sulfate and labile methyl groups. The influence of sulfate and methionine supplements was then determined in the presence of 500 mg/kg Cu. Finally a response surface depicting the response to various levels of methionine and cupric sulfate supplements was determined. MATERIALS AND METHODS
Day-old male broiler chicks (Peterson x Arbor Acres) from a commercial hatchery were used in each experiment and grown to 3 wk of age in temperature-controlled Petersime battery brooders with raised wire floors and constant illumination. Feed and water were supplied ad libitum. A corn soybean meal poultry oil-based
1500
METHIONINE AND COPPER IN CHICK DIETS
diet was fed in each experiment. Mortality was recorded daily. Feed consumption data were adjusted to account for any deaths on a chick day basis. Analyses of variance and orthogonal comparisons were calculated using the general linear models procedures of the Statistical Analysis System (SAS) Institute, Inc. (1982). The ingredient and nutrient composition of the basal diets are given in Table 1. In Experiment 1, two levels of L-methionine (basal and basal plus .2%) and two levels of choline (basal and basal plus .1%) were fed in the presence of 0 or 500 mg/kg Cu (from cupric sulfate). Each diet was fed to five randomly assigned pens of 10 male chicks each. In Experiment 2, two levels of methionine (basal and basal plus .4%), two levels of choline (basal and basal plus .2%) and two levels of sulfate (basal and basal plus .15% from potassium sulfate) in addition to two levels of copper were fed to five randomly assigned pens of 10 male chicks each. Experiment 3 had a 3 x 2 factorial design with three levels of L-methionine (basal and basal plus .2 or .4%) and two levels of potassium sulfate (basal and basal plus 758 mg/kg) in the presence of 500 mg/kg Cu in the form of cupric acetate. A negative control diet without copper, methionine, or sulfate was also fed. Each diet was fed to three pens of 10 chicks each. In Experiment 4, four levels of methionine (basal and basal plus . 1 , .2, or .4%) and four levels of copper as cupric sulfate (basal and basal plus 125, 250, or 500 mg/kg) were fed in each of two trials. Data were pooled to fit the response surface. Each diet was fed to two or three randomly assigned pens of 10 male chicks each in each trial, for a total of five replicates for each diet. The basal diet was supplemented to contain 2,000 mg/kg choline. The following response model was fitted: Y = b 0 + T + b. Met + b 2 Met2 + b 3 Cu + b4XSuB cu2 + b 5 Cu • Met where Y represents the weight gain, feed efficiency, or feed consumption, T = trial (0 or 1), Met = methionine concentration (%), and Cu = copper concentration in milligrams per kilogram. Terms where coefficients were not significantly different from zero (P>.10) were removed from the models. Models with all coefficients significantly different from zero were then calculated. The general linear models procedure (SAS Institute Inc., 1982) was used for the analyses.
1501
Choline was determined by the Reineckate technique described by Lim and Schall (1963). Methionine and cystine analyses were conducted by another laboratory using a modification of the method of Moore et al. (1958). RESULTS
Experiment 1. In the absence of supplemental copper, gain and feed efficiency responses to . 1 % supplemental choline were equivalent to responses from the .2% L-methionine supplement (Table 2). However, in the presence of 500 mg/kg Cu, response to L-methionine was much greater than response to choline. This resulted in the significant methionine x copper and methionine x choline interactions, and lack of a copper x choline interaction (P>.05). Experiment 2. The supplementation of .4% L-methionine completely overcame the adverse effects of feeding 500 mg/kg Cu from cupric sulfate (Table 3). Supplementation of .2% choline resulted in a significant response, as in Experiment 1. Supplemental sulfate, either alone or in addition to choline, was without effect. Experiment 3. In the presence of 500 mg/kg Cu from cupric acetate there was a large and significant response in gain and feed efficiency to L-methionine (Table 4). A much smaller, nearly significant (P = .08) response in gain to sulfate supplementation was observed. Experiment 4. At low copper levels, chick gains were higher but were lower with increased supplemental methionine (Table 5, Figure 1). At high copper levels, gains were higher with higher methionine levels. Conversely, at low methionine levels gains were lower with increased copper; but at high methionine levels gains were higher and then lower with higher copper levels. Feed efficiency trends were similar although efficiency gains were smaller (Table 6, Figure 2). Response surface equations accounted for 86% of the variation in weight gain and 68% of the variation in feed efficiency (Table 7). DISCUSSION
Chicks fed the basal diet used in these experiments responded to . 1 % dietary choline to about the same extent as to .2% dietary L-methionine. These results (Table 1) are similar to those of Quillin et al. (1961) and Pesti et al. (1979, 1980), but different from those of Miles et al. (1983) who found a response to choline only in the presence of supplemental sulfate.
3,265 23.00 .38 .74 1,390
Selenium premix: selenium, .02%; calcium carbonate, 99.98%.
National Research Council, 1984.
52.34 37.93 5.87 1.72 1.18 .05 .05 .05 .05 .05
(%>
Calculation 1
22.94 .38 .76 1,089
Experiment 1
Analysis
23.46 .39 .78 1,132
Experiment 2
23.20 .41 .81 1,687
Experiments 3 and 4
Mineral mix provides (milligrams per kilogram of diet): Mn, 60; Zn, 50; Fe, 30; Cu, 5; I, 1.05.
'Bacitracin: BMD 50.
4
Vitamin mix included the following (per kilogram of diet): vitamin A, 5,000 IU; vitamin D 3 , 1,100 ICU; vitamin E, 11 IU; riboflavin, 4.4 mg;Ca pantothenate, 12 mg; nicotinic acid, 44 mg; vitamin B 1 2 , 6.6 Mg; vitamin B 6 , 2.2 mg; menadione, 1.1 mg; folic acid, .55 mg; d-biotin, .11 mg; thiamine, 2.2 mg; ethoxyquin, 125 mg.
3
2
1
Ingredient Corn Soybean meal Poultry oil Dicalcium phosphate Limestone Salt Selenium premix 2 Vitamin mix 3 Mineral mix" Bacitracin5 Nutrient Metabolizable energy, kcal/kg Protein, % Methionine, % Methionine plus cystine, % Choline, mg/kg
Diet component
TABLE 1. Ingredient and nutrient composition of basal diet by calculation and analysis
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METHIONINE AND COPPER IN CHICK DIETS
1503
TABLE 2. Effects of methionine, choline, and Cu supplements1 on copper toxicity: 0 to 20-day weight gain and feed efficiency of male broiler chicks (Experiment i ) 2
Methionine
Cu (mg/kg)
Choline
(%)
-
(g) 541 568 568 558 413 456 498 508
.1 .2 .2 500 500 500 500 1
.1 .1
.2 .2
Feed efficiency3
Gain
.1
± ± ± ± ± ± ± ±
13 3 11 6 9 14 7 5
723 749 759 753 707 733 786 786
± ± ± ± + ± ± ±
6 8 7 4 12 6 8 5
Provided as CuS0 4 - 5HjO, L-methionine, and choline chloride, respectively.
2
Cu, methionine, and choline main effects and the Cu X methionine and methionine X choline interactions significantly contributed to the variation in gain (P<.05); methionine and choline main effects and Cu X methionine and methionine X choline interactions significantly contributed to the variation in feed efficiency (P<.05). 3
Grams gained per kilogram of feed consumed.
The response to choline was much less than the response to methionine in the presence of 500 mg/kg cupric sulfate (Table 1). This indicates that the primary antagonism that exists between copper and methionine is to methionine per se (or rather the homocysteine moeity) and not to labile methyl groups. This conclusion was confirmed by Experiment 2 wherein levels of methionine and choline were doubled (Table 3). In addition, sulfate supplements were without effect when added either alone or with choline. Therefore, the relationship demonstrated by Miles et al. (1983) was not evident under conditions of copper toxicosis.
As copper was added in the form of cupric sulfate in Experiments 1 and 2, it is possible that some of the negative effects of the copper were being overcome by the sulfate added with it. Experiment 3 was conducted to determine if the small amount of sulfate from the copper sulfate could have affected the outcome of Experiments 1 and 2. The small response found by adding sulfate on an isomolar basis (copper provided in the form of cupric acetate) was nearly significant (P = .08) and fairly constant at three levels of methionine. Therefore the growth depression produced by feeding 500 mg/ kg copper may have been slightly less than ex-
TABLE 3. Effects of methionine, choline and sulfate supplements1 on copper toxicity: 0 to 20-day weight gain and feed efficiency of male broiler chicks (Experiment 2) 2
Diet
1 2 3 4 5 7 1
Cu
Methionine
Choline
Sulfate
Gain
(mg/kg)
(g)
500 500 500 500 500
.15 .15
475 394 471 422 391 429
.4 .2 .2
± ± ± + ± ±
Feed efficiency3
20 8 23 21 22 10
699 699 748 714 685 695
± ± ± ± ± ±
10 16 27 22 10 22
Provided as CuSO„ -SH^O, L-methionine, choline chloride, and potassium sulfate, respectively.
2
The following means were found to be significantly different for gain and feed efficiency by orthogonal comparison (P<.05): Diets 1 vs. 2, 3 vs. 2, and 4 vs. 2. No significant differences were found for Diets 5 vs. 2 or 4 vs. 6 (P>.20). 3
Grams gained per kilogram of feed consumed.
1504
WANG ET AL.
pected had the copper not been accompanied by sulfate in Experiments 1 and 2. However, effect of dietary sulfate appears to be small, if indeed it is real. Copper is normally added to practical diets in the form of the sulfate, so the final experiment was conducted with cupric sulfate. Our original hypothesis was that copper is increasing the chick methionine requirement through the known antagonism between copper and methionine. It could be counterproductive to add cupric sulfate as an inexpensive growth promoter if it is increasing the methionine requirement, and therefore the cost of methionine supplementation. This hypothesis was confirmed in that the methionine requirement was higher with added copper. However, there were also higher body weight gains (Table 5 and 7, Figure 1). This may explain why most commercial producers feed broiler chickens levels of methionine much higher than requirements found under laboratory conditions. Most commercial feeds contain pharmacological levels of copper, but laboratory diets do not. The response surface depicted in Figure 1 contains what economists would call a zone of substitution (the shaded area). In that zone the same response (e.g., 553 g) was obtained by feeding a high level of methionine (.25%) and a low level of copper (.25% methionine, 60 mg/kg Cu), or a low level of methionine and a high level of copper (.18% methionine ,130 mg/ kg Cu). Outside this zone either methionine or copper depresses gains at any point on the surface. The increase in the methionine requirement is evident from the slope of the upper border of the zone of substitution. Response theory holds that it is most important to use experimental data to derive a best-fit surface equation (Box and Wilson, 1951). The surface equation can then be used to predict values of the inputs yielding the maximum response or to predict the point of economic efficiency. The point of economic efficiency is where the value of the return (gain) is just offset by the costs of the inputs (copper and methionine). This is a simple calculation if the response surface equation and costs of gain, methionine, and copper are known. The response surface approach is an alternative to picking and choosing among the experimental points to determine where the maximum response or most profitable feeding levels are. In determining a response surface, all the points are used to determine the shape of the surface. Thus, although the maximum observed response
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methionine and 188 mg/kg supplemental copper. From Figure 1 it is apparent that there is a large area of the surface with responses near the maximum. Criteria for judging whether the surface adequately describes the observed data depend on the objectives. From a statistical viewpoint this surface is a good fit (Table 7). Eighty-six percent of the variability in the gain data were described by the model and the coefficients were all significantly different from zero. If very small differences in response are economically important, the experiment would require repetition until the average response was defined within an economically relevant tolerance. The maximum response in gain (560 g) indicates a total sulfur-containing amino acid requirement of 1.06% in a 23% protein diet, similar to what some practicing nutritionists feel is necessary under field conditions. The value for copper (188 mg/kg) is very similar to Fisher's (1973) estimate from evaluating published values (169 mg/kg Cu). Fisher (1973) also estimated a maximum for feed efficiency at 140 mg/kg copper; in Experiment 4 (Figure 2) no clear maximum was determined within the range of the data. Efficiency was slightly decreased with increased copper at low methionine levels, but slightly increased at higher methionine levels. The results of Experiment 4 are very similar to those of Robbins and Baker (1980), qualitatively if not quantitatively. Requirement estimates from experiments with corn and soybean meal-based diets (Tables 5 to 7) were higher than those of Robbins and Baker (1980) with crystaline amino acid-based diets, as is generally found. The reason chicks in Experiment 4 responded differently than those of Waldroup et al. (1979) is not obvious. Waldroup et al. (1979) examined a smaller portion of the surface and had increased variability from using both male and female chicks. These studies confirm that feeding pharmacological levels of copper is beneficial to the chick. Although the methionine requirement appears to be increased by this practice, the increase is accompanied by an improvement in overall growth rate and feed efficiency. This phenomenon may explain why methionine and total sulfur-containing amino acid requirements appear to be higher under field conditions than under laboratory conditions. Copper should be on the list of dietary factors influencing the methionine requirement of chicks fed practical
METHIONINE AND COPPER IN CHICK DIETS
1507
TABLE 7. Coefficients from multiple linear regression analyses of body weight gain and feed as functions of methionine and Cu supplementation (Experiment 4)1 Source of variation
Weight gain
Intercept Trial Methionine Cu Methionine 2 Cu 2 Methionine X Cu Coefficient of determination (r 2 )
523.3272** -34.9564**
331.0482** .0787* -714.8864**
-.0005** .4205** .86
efficiency
Feed efficiency .6834** -.0081*
.3340** -4.6 X 1 0 ~ 5 * * -.6608** .0003** .68
1 For example: predicted body gain of chicks fed .2% supplemental L-methionine and 250 mg/kg copper in Trial 1 = 523.3 - (35.0) (.5) + 331 (.2) + .0787 (250) - 714.9 (.2) 2 - .0005 (250) 2 + .4205 (.2) (250) = 553; actual = 545 ± 9.
*P<.05. **P<.01.
diets along with protein, energy, cystine, sulfate, choline, and vitamin B 12 . REFERENCES Baker, D. H., and K. R. Robins, 1979. Sulfur amino acid utilization in chicks fed supplemental monensin and copper. Pages 39-44 in: Proc. 1979 Maryland Nutr. Conf. Box, G.E.P., and K. B. Wilson, 1951. On the experimental attainment of optimum conditions (with discussion). J. R. Stat. Soc. B13:1^15. Fisher, C., 1973. Use of copper sulfate as a growth promoter for broilers. Feedstuffs: July 16, 24-25. Fisher, C , D. Wise, and D. G. Filmer, 1970. The effect of copper on the growth of broilers and the interaction of copper with zinc and iron. Proc. 14th World's Poult. Congr. Madrid, Spain 2:759-764. Jensen, L. S., and D. V. Maurice, 1976. Alleviation of copper-induced growth depression by methionine. Poultry Sci. 55:2048-2049. (Abstr.) Lim, F., and E. D. Schall, 1963. Determination of choline in feeds. Seventy-seventh Annu. Meet. Assoc. Off. Agric. Chem. Purdue Univ., Agric. Exp. Stn., West Lafayette, IN. Miles, R. D., N. Ruiz, and R. H. Harms, 1983. The interrelationship between methionine, choline, and sulfate in broiler diets. Poultry Sci. 62:495^198.
Moore, S., D. H. Spackman, and W. N. Stein, 1958. Chromatography of amino acids on sulfonated polystyrene resins. Anal. Chem. 30:1185-1190. National Research Council, 1984. Nutrient Requirements of Domestic Animals. 1. Nutrient Requirements of Poultry. Natl. Acad. Sci., Washington, DC. 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. 59:10731081. 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. 40:639645. Robbins, K. R., andD. H. Baker, 1980. Effect of high-level copper feeding on the sulfur amino acid need of chicks fed corn-soybean meal and purified crystalline amino acid diets. Poultry Sci. 59:1099-1108. Statistical Analysis System Institute, Inc., 1982. SAS User's Guide: Basic. 1982 ed. SAS Institute, Inc., Cary, NC. Waldroup, P. W., C. J. Mabray, J. R. Blackman, and F. Z. Johnson, 1979. The influence of copper sulfate on the methionine requirement of the young chick. Nutr. Rep. Int. 20:303-308.