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Effect of Methionine on Copper-Induced Growth Depression and Gizzard Erosion
Effect of Methionine on Copper-Induced Growth Depression and Gizzard Erosion L. S. JENSEN and D. V. MAURICE Department of Poultry Science, University of Georgia, Athens, Georgia 30602 (Received for publication March 30, 1978)
INTRODUCTION
Supplementary copper (120 to 240 ppm) considerably greater than that needed to meet the nutritional requirements is used in broiler diets in the southeastern United States as an antifungal agent and/or growth stimulator. High levels of dietary copper affect the integrity of the gizzard lining (Fisher et al., 1973; Poupoulis and Jensen, 1976). In studies to investigate if the gizzard erosion could be caused by creation of deficiency of another nutrient, additional selenium slightly reduced, and additional zinc, vitamin E, or vitamin B i 2 had no effect on the gizzard damage produced by high copper levels (Poupoulis and Jensen, 1976). Severity of gizzard erosion was greater when chicks were fed a corn starch-soy diet than when fed a practical diet with the same levels of copper supplementation. In experiments in which crystalline amino acid diets containing 5% isolated soybean protein were fed to chicks, severe erosion of the gizzard lining was observed in chicks fed diets with inadequate methionine (Miller et al, 1975). Condition of the gizzard lining was markedly improved by supplementing methionine. The purpose of the present investigation was to determine if additional methionine would prevent the gizzard erosion associated with high copper levels in a practical diet for broiler chicks. EXPERIMENTAL PROCEDURE
Two experiments were conducted with Cobb broiler chicks maintained in battery brooders to 1978 Poultry Sci 57:1530-1532
four weeks of age. The chicks were vaccinated for Marek's disease at day of age and no diseases were observed during the studies. Composition of the practical ration used as a basal diet for the studies is shown in Table 1. The diet calculated to contain .83% total sulfur amino acids (methionine plus cystine). Three pens of ten chicks each were fed each of the experimental diets ad libitum. Copper was added as copper sulfate pentahydrate (CuSCv 5H 2 0). D,L-methionine (99%) served as the methionine source for the experiments. All birds were killed at the end of the experiments for removal of gizzards which were scored as previously described by Poupoulis and Jensen (1976). Since appearance of the proventriculus was also found to be affected by diets in Experiment 1, the proventriculi were removed from chicks in Experiment 2 and each proventriculus was scored by the following scale: 0, normal size, papillae defined and no mucus; 1, normal size, localized erosion of mucosal surface and papillae not well defined; 2, enlarged and flaccid with wide-spread erosion of mucosal surface, and papillae visable; 3, enlarged and flaccid, wide-spread erosion, no papillae visible and surface covered with mucus. Both the gizzards and proventriculi were individually weighed. Statistical analyses of the data were done by analysis of variance and Duncan's Multiple Range Test. Differences at the 5% level of probability were considered significant. In Experiment 1, a 2 x i factorial design was used with two levels of added methionine (0 and .4%) and three levels of added copper (0, 250, and 500 ppm). In Experiment 2, the
1530
Downloaded from http://ps.oxfordjournals.org/ at NERL on April 12, 2015
ABSTRACT Erosion of the gizzard lining of chicks fed a practical diet supplemented with high levels of copper (250, 500, or 750 ppm) was not prevented by including more methionine in the diet (.4%). Proventriculi of chicks fed high levels of copper were enlarged and abnormal in appearance, and methionine supplementation also failed to prevent this condition. A significant growth depression and reduced feed efficiency caused by 500 ppm copper was completely counteracted by including an additional .4% D,L-methionine in the ration. Adding the same level of methionine to a diet with 750 ppm copper did not significantly counteract the toxicity as measured by growth and feed efficiency.
INTERACTION OF METHIONINE AND COPPER TABLE 1.—Composition of basal diet
(%)
Ingredient Corn Soybean meal (48.5% protein) Poultry by-product meal Animal fat Alfalfa meal (17% protein) Ground limestone Defluorinated phosphate Salt DL-methionine Vitamin premix a Trace mineral mix
57.25 30.5 5.0 3.0 1.0 .65 1.75 .4 .15 .25 .05
u n s u p p l e m e n t e d basal diet and diets with 5 0 0 or 7 5 0 p p m copper with and w i t h o u t . 4 % m e t h i o n i n e were fed t o t h e chicks.
significantly increased b y adding 5 0 0 p p m copper, b u t m e t h i o n i n e failed t o significantly reduce t h e index. In E x p e r i m e n t 2, adding 5 0 0 a n d 7 5 0 p p m copper significantly depressed growth rate a n d increased feed conversion ratios c o m p a r e d t o t h a t of t h e u n s u p p l e m e n t e d basal diet (Table 3). M e t h i o n i n e c o m p l e t e l y c o u n t e r a c t e d t h e growth a n d feed efficiency effect of 5 0 0 p p m copper. Performance of chicks fed t h e higher level of copper was slightly improved b u t n o t significantly b y t h e a d d e d m e t h i o n i n e (Table 3). Gizzard erosion index values were increased b y b o t h levels of a d d e d c o p p e r a n d m e t h i o n i n e did n o t significantly reduce t h e m . C o p p e r also m a r k e d l y affected t h e appearance of t h e proventriculus a n d a d d e d m e t h i o n i n e also failed t o c o u n t e r a c t this effect. Absolute gizzard weights were n o t significantly affected b y t h e dietary t r e a t m e n t s b u t were significantly increased per u n i t of b o d y weight in t h e groups in which growth was depressed. On t h e o t h e r hand, absolute weight of t h e proventriculus was significantly increased b y a d d e d c o p p e r a n d this was n o t c o u n t e r a c t e d b y m e t h i o n i n e supplementation.
RESULTS
DISCUSSION
In E x p e r i m e n t 1, b o t h levels of a d d e d copper significantly depressed g r o w t h rate, while feed t o gain ratio was significantly increased b y adding 5 0 0 p p m c o p p e r (Table 2). Including .4% m e t h i o n i n e in t h e diets prevented b o t h the- g r o w t h depression and r e d u c t i o n in feed efficiency. Gizzard erosion index was
Evidence was n o t o b t a i n e d in these studies for a modifying effect of s u p p l e m e n t a l m e t h i o nine on i m p a i r m e n t of t h e gizzard lining of chicks fed diets with high levels of c o p p e r . T h e erosion of gizzard linings observed b y Miller et al. ( 1 9 7 5 ) with m e t h i o n i n e deficient diets, therefore, a p p e a r e d t o be different from t h a t
TABLE 2.—Effect of copper and methionine on gizzard integrity and performance of chicks (Experiment I) Added to basal diet Cu (ppm)
Methionine (%)
0 250 500 0 250 500 a
.4 .4 .4
Body weight 4 wk (g)
Feed/gain (0 to 4 wk)
654 a 591bc
1.83 a b 1.88 b
564 c
2.07 c
.10 a .20ab .77 d
640 a 658a 647a
1.75 a b 1.75 a b 1.68 a
.20ab # 44abc .48bcd
' ' c ' Means within each column without a common letter are significantly different (P<.05). Gizzards were scored from 0 (normal) to 3 (severe erosion).
Gizzard erosion index e
Downloaded from http://ps.oxfordjournals.org/ at NERL on April 12, 2015
Vitamin premix provided in the practical diet (per kg feed): 4400 IU vitamin A, 880 ICU vitamin D 3 , 11 IU vitamin E, 4.4 mg riboflavin, 8.8 mg pantothenic acid, 44 mg nicotinic acid, 220 mg choline chloride, 2.2 mg vitamin B 6 , 2.2 mg menadione sodium bisulfite, 2.2 mg thiamine, .0066 mg vitamin B 1 2 , .55 mg folic acid, .11 mg biotin, 125.4 mg ethoxyquin.
1531
JENSEN AND MAURICE
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REFERENCES Fisher, C, A. P. Laursen-Jones, K. J. Hill, and W. S. Hardy, 1973. The effect of copper sulfate on performance and the structure of the gizzard in broilers. Brit. Poultry Sci. 14:55-68. Harry, E. G., J. F. Tucker, and A. P. Laursen-Jones, 1975. The role of histamine and fish meal in the incidence of gizzard erosion and pro-ventricular abnormalities in the fowl. Brit. Poultry Sci. 16:69-78. Huck, D. W., and A. J. Clawson, 1976. Excess dietary colbalt in pigs. J. Anim. Sci. 43:1231—1246. Miller, C, P. E. Bauersfeld, Jr., G. N. Biddle, and A. Former, 1975. Effect of sulfur-containing dietary supplements on gizzard lining erosions. Poultry Sci. 54:428-435. Poupoulis, C, and L. S. Jensen, 1976. Effect of high dietary copper on gizzard integrity of the chick. Poultry Sci. 55:113-121.
Downloaded from http://ps.oxfordjournals.org/ at NERL on April 12, 2015
caused by high copper supplementation. In the first experiment in the present study, abnormal appearance of the proventriculus was also observed in chicks receiving a high copper level. Severely affected proventriculi were enlarged, flaccid, covered with mucus and showed no visible papillae. Use of a scoring index to quantify changes in the proventriculus in Experiment 2 showed that copper induced abnormalities were not prevented by supplemented methionine. Abnormalities of the proventriculus were observed by Harry et al. (1975) with chicks fed high levels offish meal. Fisher et al. (1973) also indicated an effect of copper on appearance of the proventriculus. An interesting observation in the present study was the complete counteraction by D,L-methionine of the toxicity of 500 ppm copper. Although sulfur-containing amino acids have been shown to reduce the toxicity of other micro-elements in animals, (e.g. in pigs, Huck and Clawson, 1976), no previous reports of counteraction of copper toxicity by these amino acids in animals have apparently appeared. The level of methionine used in the present study failed to counteract the toxicity of 750 ppm copper. Further experiments will be needed to determine the quantitative aspects of copper-methionine interaction and to investigate the mechanism of action of methionine alleviating the toxicity. Since additional methionine affects metabolism of high levels of copper, effect of high levels of copper on sulfur-amino acid requirement of chicks becomes of interest. The inclusion of .05 to . 1 % copper sulfate in commercial broiler rations may possibly increase the requirement of broiler chicks for sulfur-containing amino acids.
INTRODUCTION
Supplementary copper (120 to 240 ppm) considerably greater than that needed to meet the nutritional requirements is used in broiler diets in the southeastern United States as an antifungal agent and/or growth stimulator. High levels of dietary copper affect the integrity of the gizzard lining (Fisher et al., 1973; Poupoulis and Jensen, 1976). In studies to investigate if the gizzard erosion could be caused by creation of deficiency of another nutrient, additional selenium slightly reduced, and additional zinc, vitamin E, or vitamin B i 2 had no effect on the gizzard damage produced by high copper levels (Poupoulis and Jensen, 1976). Severity of gizzard erosion was greater when chicks were fed a corn starch-soy diet than when fed a practical diet with the same levels of copper supplementation. In experiments in which crystalline amino acid diets containing 5% isolated soybean protein were fed to chicks, severe erosion of the gizzard lining was observed in chicks fed diets with inadequate methionine (Miller et al, 1975). Condition of the gizzard lining was markedly improved by supplementing methionine. The purpose of the present investigation was to determine if additional methionine would prevent the gizzard erosion associated with high copper levels in a practical diet for broiler chicks. EXPERIMENTAL PROCEDURE
Two experiments were conducted with Cobb broiler chicks maintained in battery brooders to 1978 Poultry Sci 57:1530-1532
four weeks of age. The chicks were vaccinated for Marek's disease at day of age and no diseases were observed during the studies. Composition of the practical ration used as a basal diet for the studies is shown in Table 1. The diet calculated to contain .83% total sulfur amino acids (methionine plus cystine). Three pens of ten chicks each were fed each of the experimental diets ad libitum. Copper was added as copper sulfate pentahydrate (CuSCv 5H 2 0). D,L-methionine (99%) served as the methionine source for the experiments. All birds were killed at the end of the experiments for removal of gizzards which were scored as previously described by Poupoulis and Jensen (1976). Since appearance of the proventriculus was also found to be affected by diets in Experiment 1, the proventriculi were removed from chicks in Experiment 2 and each proventriculus was scored by the following scale: 0, normal size, papillae defined and no mucus; 1, normal size, localized erosion of mucosal surface and papillae not well defined; 2, enlarged and flaccid with wide-spread erosion of mucosal surface, and papillae visable; 3, enlarged and flaccid, wide-spread erosion, no papillae visible and surface covered with mucus. Both the gizzards and proventriculi were individually weighed. Statistical analyses of the data were done by analysis of variance and Duncan's Multiple Range Test. Differences at the 5% level of probability were considered significant. In Experiment 1, a 2 x i factorial design was used with two levels of added methionine (0 and .4%) and three levels of added copper (0, 250, and 500 ppm). In Experiment 2, the
1530
Downloaded from http://ps.oxfordjournals.org/ at NERL on April 12, 2015
ABSTRACT Erosion of the gizzard lining of chicks fed a practical diet supplemented with high levels of copper (250, 500, or 750 ppm) was not prevented by including more methionine in the diet (.4%). Proventriculi of chicks fed high levels of copper were enlarged and abnormal in appearance, and methionine supplementation also failed to prevent this condition. A significant growth depression and reduced feed efficiency caused by 500 ppm copper was completely counteracted by including an additional .4% D,L-methionine in the ration. Adding the same level of methionine to a diet with 750 ppm copper did not significantly counteract the toxicity as measured by growth and feed efficiency.
INTERACTION OF METHIONINE AND COPPER TABLE 1.—Composition of basal diet
(%)
Ingredient Corn Soybean meal (48.5% protein) Poultry by-product meal Animal fat Alfalfa meal (17% protein) Ground limestone Defluorinated phosphate Salt DL-methionine Vitamin premix a Trace mineral mix
57.25 30.5 5.0 3.0 1.0 .65 1.75 .4 .15 .25 .05
u n s u p p l e m e n t e d basal diet and diets with 5 0 0 or 7 5 0 p p m copper with and w i t h o u t . 4 % m e t h i o n i n e were fed t o t h e chicks.
significantly increased b y adding 5 0 0 p p m copper, b u t m e t h i o n i n e failed t o significantly reduce t h e index. In E x p e r i m e n t 2, adding 5 0 0 a n d 7 5 0 p p m copper significantly depressed growth rate a n d increased feed conversion ratios c o m p a r e d t o t h a t of t h e u n s u p p l e m e n t e d basal diet (Table 3). M e t h i o n i n e c o m p l e t e l y c o u n t e r a c t e d t h e growth a n d feed efficiency effect of 5 0 0 p p m copper. Performance of chicks fed t h e higher level of copper was slightly improved b u t n o t significantly b y t h e a d d e d m e t h i o n i n e (Table 3). Gizzard erosion index values were increased b y b o t h levels of a d d e d c o p p e r a n d m e t h i o n i n e did n o t significantly reduce t h e m . C o p p e r also m a r k e d l y affected t h e appearance of t h e proventriculus a n d a d d e d m e t h i o n i n e also failed t o c o u n t e r a c t this effect. Absolute gizzard weights were n o t significantly affected b y t h e dietary t r e a t m e n t s b u t were significantly increased per u n i t of b o d y weight in t h e groups in which growth was depressed. On t h e o t h e r hand, absolute weight of t h e proventriculus was significantly increased b y a d d e d c o p p e r a n d this was n o t c o u n t e r a c t e d b y m e t h i o n i n e supplementation.
RESULTS
DISCUSSION
In E x p e r i m e n t 1, b o t h levels of a d d e d copper significantly depressed g r o w t h rate, while feed t o gain ratio was significantly increased b y adding 5 0 0 p p m c o p p e r (Table 2). Including .4% m e t h i o n i n e in t h e diets prevented b o t h the- g r o w t h depression and r e d u c t i o n in feed efficiency. Gizzard erosion index was
Evidence was n o t o b t a i n e d in these studies for a modifying effect of s u p p l e m e n t a l m e t h i o nine on i m p a i r m e n t of t h e gizzard lining of chicks fed diets with high levels of c o p p e r . T h e erosion of gizzard linings observed b y Miller et al. ( 1 9 7 5 ) with m e t h i o n i n e deficient diets, therefore, a p p e a r e d t o be different from t h a t
TABLE 2.—Effect of copper and methionine on gizzard integrity and performance of chicks (Experiment I) Added to basal diet Cu (ppm)
Methionine (%)
0 250 500 0 250 500 a
.4 .4 .4
Body weight 4 wk (g)
Feed/gain (0 to 4 wk)
654 a 591bc
1.83 a b 1.88 b
564 c
2.07 c
.10 a .20ab .77 d
640 a 658a 647a
1.75 a b 1.75 a b 1.68 a
.20ab # 44abc .48bcd
' ' c ' Means within each column without a common letter are significantly different (P<.05). Gizzards were scored from 0 (normal) to 3 (severe erosion).
Gizzard erosion index e
Downloaded from http://ps.oxfordjournals.org/ at NERL on April 12, 2015
Vitamin premix provided in the practical diet (per kg feed): 4400 IU vitamin A, 880 ICU vitamin D 3 , 11 IU vitamin E, 4.4 mg riboflavin, 8.8 mg pantothenic acid, 44 mg nicotinic acid, 220 mg choline chloride, 2.2 mg vitamin B 6 , 2.2 mg menadione sodium bisulfite, 2.2 mg thiamine, .0066 mg vitamin B 1 2 , .55 mg folic acid, .11 mg biotin, 125.4 mg ethoxyquin.
1531
JENSEN AND MAURICE
1532
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REFERENCES Fisher, C, A. P. Laursen-Jones, K. J. Hill, and W. S. Hardy, 1973. The effect of copper sulfate on performance and the structure of the gizzard in broilers. Brit. Poultry Sci. 14:55-68. Harry, E. G., J. F. Tucker, and A. P. Laursen-Jones, 1975. The role of histamine and fish meal in the incidence of gizzard erosion and pro-ventricular abnormalities in the fowl. Brit. Poultry Sci. 16:69-78. Huck, D. W., and A. J. Clawson, 1976. Excess dietary colbalt in pigs. J. Anim. Sci. 43:1231—1246. Miller, C, P. E. Bauersfeld, Jr., G. N. Biddle, and A. Former, 1975. Effect of sulfur-containing dietary supplements on gizzard lining erosions. Poultry Sci. 54:428-435. Poupoulis, C, and L. S. Jensen, 1976. Effect of high dietary copper on gizzard integrity of the chick. Poultry Sci. 55:113-121.
Downloaded from http://ps.oxfordjournals.org/ at NERL on April 12, 2015
caused by high copper supplementation. In the first experiment in the present study, abnormal appearance of the proventriculus was also observed in chicks receiving a high copper level. Severely affected proventriculi were enlarged, flaccid, covered with mucus and showed no visible papillae. Use of a scoring index to quantify changes in the proventriculus in Experiment 2 showed that copper induced abnormalities were not prevented by supplemented methionine. Abnormalities of the proventriculus were observed by Harry et al. (1975) with chicks fed high levels offish meal. Fisher et al. (1973) also indicated an effect of copper on appearance of the proventriculus. An interesting observation in the present study was the complete counteraction by D,L-methionine of the toxicity of 500 ppm copper. Although sulfur-containing amino acids have been shown to reduce the toxicity of other micro-elements in animals, (e.g. in pigs, Huck and Clawson, 1976), no previous reports of counteraction of copper toxicity by these amino acids in animals have apparently appeared. The level of methionine used in the present study failed to counteract the toxicity of 750 ppm copper. Further experiments will be needed to determine the quantitative aspects of copper-methionine interaction and to investigate the mechanism of action of methionine alleviating the toxicity. Since additional methionine affects metabolism of high levels of copper, effect of high levels of copper on sulfur-amino acid requirement of chicks becomes of interest. The inclusion of .05 to . 1 % copper sulfate in commercial broiler rations may possibly increase the requirement of broiler chicks for sulfur-containing amino acids.