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Tyrosine, Protein and Ascorbic Acid Effects on Egg Shell Thickness from Chickens Subjected to Heat Stress1
1832
R. M.
LEACH, JR., L. C. NOERIS AND M.
industry. Vitamins and Hormones, 12: 235— 275. Miller, G. J., W. W. Ellis, P. G. Rand and R. H. King, 1958. Comparative effects of cottonseed oil, fatty acids and diethylstilbestrol upon
L.
SCOTT
choline-deficient weanling albino rats. J. Nutrition, 66: 425-440. Snedecor, G. W., 1956. Statistical Methods. The Iowa State College Press, Ames, Iowa.
PAUL A.
Department
THORNTON
of Poultry Science, Colorado Agricultural Experiment Fort Collins
Station,
(Received for publication April 2, 1962) REVIEW OF LITERATURE
E
ARLIER it was observed that hens subjected to heat stress appeared to respond favorably to dietary ascorbic acid supplementation in maintaining egg shell thickness when a ration containing 13 percent protein was fed, but not at a 17 percent level (Thornton, 1960). Since all ingredients of those rations were similar with the exception of the corn, milo and soybean oil meal ratio, it seemed probable that the lack of response to ascorbic acid at the higher level may have been due to differences in dietary content of specific amino acids. On calculation it was noted that while the protein ratio of the 13 percent level to the 17 was 0.765, the ratio of tyrosine for the two diets was 0.838 and the phenylalanine and tyrosine levels combined were 0.799. Despite the higher percent of tyrosine and phenylalanine per unit of total protein in the 13 percent diet, the absolute levels of each amino acid in the 17 percent diet were greater. This slight difference in relative tyrosine content was of special interest in view of the accepted 1 Published with the approval of the Director of the Colorado Agricultural Experiment Station as paper number S. S. 769, Journal Series.
relationships between vitamin C and this amino acid. For example, it has been shown that ascorbic acid is involved in the in vitro oxidation of tyrosine (Sealock and Goodland, 1951). The necessity of vitamin C for complete metabolism of phenylalanine and tyrosine is also well illustrated by the urinary excretion of p-hydroxyphenylpyruvic acid by scorbutic animals fed large amounts of these amino acids (Sealock and Silberstein, 1940). Rangneker and Dugal (1958) showed that utilization of both phenylalanine and tyrosine was facilitated by both exposure to cold and ascorbic acid supplementation in rats. Since Thornton (1961) showed that ascorbic acid blood levels in chickens were higher at low environmental temperatures, it seems that some relationship may exist between the animal's metabolic rate and its ability to synthesize ascorbic acid. Further, in view of the observation by Rangneker and Dugal (1958) it seems probable that tyrosine and phenylalanine as well as ascorbic acid may be implicated in thyroid function in cold acclimation, which in turn may have some impact on the rate of endogenous formation of ascorbic acid. Desmarais (1960) presented an excellent
Downloaded from http://ps.oxfordjournals.org/ at University of Nebraka-Lincoln Libraries on April 7, 2015
Tyrosine, Protein and Ascorbic Acid Effects on Egg Shell Thickness from Chickens Subjected to Heat Stress1
1833
TYROSINE, PROTEIN AND ASCORBIC ACID TABLE 1.—Tyrosine and ascorbic acid influence on egg shell maintenance Shell thicknes (microns)
Percent shell
Egg weight (grams)
Addition to control Preliminary
None (12)l Tyrosine (12) Tyrosine+vitamin C (13)
Experimental
Preliminary
Experimental
Preliminary
Experimental
284+7.3 261 + 5.7* 291+4.1 271±4.2**
7.90 + 0.19 7.42 + 0.15* 7.81 + 0.16 7.35 + 0.17*
56.7 + 0.7 54.2 + 0.7* 56.4 + 0.7 55.6 + 0.8
294 + 5.3
8.05 + 0.13 8.12 + 0.12
57.6 + 0.9 56.9 + 0.7
293±5.2
review on the function of ascorbic acid in cold acclimation, and the place of vitamin C is well documented. Since little attention has been given to heat acclimation, basic information concerning biochemical changes in the animal from heat stress and possible means of alleviation are lacking. In spite of a lack of documentary evidence, it does not seem illogical to assume that ascorbic acid may be involved in heat as well as cold acclimation. In the current study emphasis was placed on determining the possible relationship between the dietary levels of tyrosine and ascorbic acid on the maintenance of egg shell thickness under heat stress conditions. PROCEDURE
Two separate studies were conducted to investigate the possible relationship between ascorbic acid and tyrosine in egg shell formation. In the first study, 45 Single Comb White Leghorn hens which had been in production for 10 months and given the 13 percent protein basal diet (Thornton, 1960) were used. These animals were maintained in individual cages prior to and throughout the experimental period with feed and water supplied ad libitum. The preliminary studies were conducted in July, thus the hens had been exposed to a rather high environmental temperature (mean maximum 84°F., mean low 72°F.) for 30 days prior to egg shell measurements. After
at least six consecutively laid eggs were collected from each hen, the birds were divided into three equal groups and placed on experimental diets as shown in Table 1. The tyrosine was added at a level of 2.71 grams per kilogram of feed, which increased the absolute amount of tyrosine and phenylaline to that level calculated to be in the 17 percent protein diet (Thornton, 1960). This procedure was followed since it seemed remotely possible that a lack of response to ascorbic acid at that protein level could have resulted from the higher actual level of these amino acids. The ascorbic acid in this experiment as well as the second was added at a level of 44 mg. per kilogram of ration. After the eggs were weighed and broken, shell thickness and percent shell were determined in a manner previously described by Thornton (1960). The birds were then subjected to a daily average maximum temperature of 92.0 ± 0.6°F. and a daily minimum of 74.1 ± 0.7°F. for 21 days. After this time an additional six consecutively laid egg sample was collected and treated in a manner similar to that used in the preliminary study. Any hen which did not produce six eggs at each collection was discarded. In the second study, 72 ten-month old hens which had been in production for approximately four months were used. These pullets were selected on a basis of egg production rate. The animals were divided into
Downloaded from http://ps.oxfordjournals.org/ at University of Nebraka-Lincoln Libraries on April 7, 2015
* Significant decrease ** Highly significant decrease 1 Values in parentheses refer to number of hens in each group
1834
P. A. THORNTON TABLE 2.—Protein level and the response to vitamin C Shell thickness (microns)
17% Protein Basal Tyrosine Tyrosine+vitamin C 13% Protein Basal Tyrosine Tyrosine+vitamin C
Percent
Egg weight (grams) Preliminary Experimental
Preliminary
Experimental
maintenance
320 + 2.4 321 + 2.8 316 + 4.0 318 + 4.0 320 + 3.8 321+3.7
298 + 3.1* 306+2.5* 303 + 2.6* 298 + 5.2* 304 + 3.0* 315 + 3.5
93.1 95.3 95.9 93.7 95.2 98.2
60.3 61.4 59.1 58.6 61.4 59.3
60.5 61.7 59.5 58.9 61.3 59.7
RESULTS AND DISCUSSION In the first study it was evident that the
birds had been heat stressed prior to initiation of the study as shown by the low shell thickness values (Table 1). Despite the preliminary thin-shell values, a further significant decrease resulted when the birds were given the control ration and subjected to a higher temperature. The addition of tyrosine appeared to have no influence. When both tyrosine and vitamin C were added, there was nearly complete maintenance of egg shell thickness. Since Thornton (1960) showed that vitamin C added alone to this diet produced a similar response, it may be assumed that the improved shell thickness maintenance was due to the added vitamin in this case. Percent shell values were very similar in nature to the shell thickness values. Egg weight also was decreased to a significant degree in the control group when the environmental temperature was raised (Table 1). In the second study the egg weight was slightly greater in all but one group in the higher temperature environment, with no particular advantage shown by any of the dietary treatments (Table 2). The difference in response between the two studies could have resulted from differences in age as well as the degree of stress imposed. The shell thickness results obtained in the second study for the 13 percent protein diet were very similar to those of the first trial (Table 2). Although it was observed that the addition of tyrosine at both protein levels resulted in a slightly greater
Downloaded from http://ps.oxfordjournals.org/ at University of Nebraka-Lincoln Libraries on April 7, 2015
* Highly significant decrease.
six numerically equal groups and placed on the diets shown in Table 2. Tyrosine was added to the 17 percent protein diets so that the total tyrosine-phenylalanine level would equal that amount per unit of protein in the 13 percent protein diets (14 grams per kilogram feed). Addition of tyrosine to the 13 percent protein level was made as previously stated. The animals were maintained in a cool environment (max. = 68.2 ± 0.6, min. = 58.9 ± 0.4), for four weeks. Eight eggs were then collected from each hen and subjected to shell thickness and total egg weight determinations. Following this, the environmental temperature was gradually increased to a daily average of 84.2 ± 0.2. This environment was maintained for 48 days and a second egg sample collected for shell thickness and weight determinations. Only those birds which laid at least eight eggs during each sampling period were included in the data. At least nine individuals in each group were retained under this stipulation. In both studies each bird acted as its own control; thus, the data were analyzed on both an individual and group basis. There appeared to be little or no difference in the final values, as between the two procedures, particularly in percent maintenance of egg shell thickness, therefore, the values pertaining only to the group are given.
TYROSINE, PROTEIN AND ASCORBIC ACID
SUMMARY Two experiments were conducted to determine why hens given a 13 percent protein diet responded favorably to ascorbic acid supplementation in egg shell maintenance under heat stress while those given a 17 percent diet did not. Since the two rations differed only in the corn, milo and soybean oil meal ratio, it was theorized that differences in amino acid composition possibly could be implicated. In view of the known metabolic relationships between tyrosine and ascorbic acid and the slight difference of these amino acids relative to the
total protein level, tyrosine was chosen for study. The shell thickness values were decreased at both protein levels by heat stress whether tyrosine was added or not, therefore, it was evident that this amino acid was not involved under the experimental conditions. The addition of tyrosine with ascorbic acid to the 17 percent diet also appeared to have no influence. Conversely, the addition of ascorbic acid and tyrosine to the 13 percent diet resulted in a high degree of shellthickness maintenance. From these results it was concluded that the failure of response to ascorbic acid at the 17 percent protein level was not influenced by the different tyrosine level' of the ration. REFERENCES Desmarais, A., 1960. Ascorbic acid in cold acclimation. Proceedings of a symposium on cold acclimation. Federation Proc. 19: 88-90. Rangneker, P. V., and L. P. Dugal, 1958. A note on the effect of cold and vitamin C on the blood levels of phenylalanine and tyrosine in rats. Canadian J. Biochem. Physiol. 36: 185186. Sealock, R. R., and R. L. Goodland, 1951. Ascorbic acid, a coenzyme in tyrosine oxidation. Science, 114: 645-646. Sealock, R. R., and H. E. Silberstein, 1940. The excretion of homogentisic and other tyrosine metabolites by the vitamin C-deficient guinea pig. J. Biol. Chem. 135: 251-258. Thornton, P. A., 1960. The influence of dietary protein level on the response of Single Comb White Leghorns to supplementary ascorbic acid. Poultry Sci. 39: 1072-1076. Thornton, P. A., 1961. Increased environmental temperature influences on ascorbic acid ac 1 tivity in the domestic fowl. Federation Prdc. 20: 210A.
Downloaded from http://ps.oxfordjournals.org/ at University of Nebraka-Lincoln Libraries on April 7, 2015
maintenance of shell thickness, actual thickness values were reduced to a highly significant degree. Thus, the addition of this amino acid alone appeared to have little 01 no influence. When ascorbic acid and tyrosine were added jointly to the 17 percent diet, a highly significant decrease in shell thickness also resulted. From this it appears that the response to vitamin C was different at the two protein levels. Since the response to ascorbic acid was also shown earlier to be different in these two diets (Thornton, 1960) in the absence of supplementary tyrosine, it may be assumed that no synergism existed between the two compounds in this case. From these results, it appears that the failure of added vitamin C to maintain shell thickness in the higher protein diet (Thornton, 1960) was due neither to an excess of the absolute level of tyrosine and phenylalanine nor to a deficiency of these amino acids relative to others.
1835
R. M.
LEACH, JR., L. C. NOERIS AND M.
industry. Vitamins and Hormones, 12: 235— 275. Miller, G. J., W. W. Ellis, P. G. Rand and R. H. King, 1958. Comparative effects of cottonseed oil, fatty acids and diethylstilbestrol upon
L.
SCOTT
choline-deficient weanling albino rats. J. Nutrition, 66: 425-440. Snedecor, G. W., 1956. Statistical Methods. The Iowa State College Press, Ames, Iowa.
PAUL A.
Department
THORNTON
of Poultry Science, Colorado Agricultural Experiment Fort Collins
Station,
(Received for publication April 2, 1962) REVIEW OF LITERATURE
E
ARLIER it was observed that hens subjected to heat stress appeared to respond favorably to dietary ascorbic acid supplementation in maintaining egg shell thickness when a ration containing 13 percent protein was fed, but not at a 17 percent level (Thornton, 1960). Since all ingredients of those rations were similar with the exception of the corn, milo and soybean oil meal ratio, it seemed probable that the lack of response to ascorbic acid at the higher level may have been due to differences in dietary content of specific amino acids. On calculation it was noted that while the protein ratio of the 13 percent level to the 17 was 0.765, the ratio of tyrosine for the two diets was 0.838 and the phenylalanine and tyrosine levels combined were 0.799. Despite the higher percent of tyrosine and phenylalanine per unit of total protein in the 13 percent diet, the absolute levels of each amino acid in the 17 percent diet were greater. This slight difference in relative tyrosine content was of special interest in view of the accepted 1 Published with the approval of the Director of the Colorado Agricultural Experiment Station as paper number S. S. 769, Journal Series.
relationships between vitamin C and this amino acid. For example, it has been shown that ascorbic acid is involved in the in vitro oxidation of tyrosine (Sealock and Goodland, 1951). The necessity of vitamin C for complete metabolism of phenylalanine and tyrosine is also well illustrated by the urinary excretion of p-hydroxyphenylpyruvic acid by scorbutic animals fed large amounts of these amino acids (Sealock and Silberstein, 1940). Rangneker and Dugal (1958) showed that utilization of both phenylalanine and tyrosine was facilitated by both exposure to cold and ascorbic acid supplementation in rats. Since Thornton (1961) showed that ascorbic acid blood levels in chickens were higher at low environmental temperatures, it seems that some relationship may exist between the animal's metabolic rate and its ability to synthesize ascorbic acid. Further, in view of the observation by Rangneker and Dugal (1958) it seems probable that tyrosine and phenylalanine as well as ascorbic acid may be implicated in thyroid function in cold acclimation, which in turn may have some impact on the rate of endogenous formation of ascorbic acid. Desmarais (1960) presented an excellent
Downloaded from http://ps.oxfordjournals.org/ at University of Nebraka-Lincoln Libraries on April 7, 2015
Tyrosine, Protein and Ascorbic Acid Effects on Egg Shell Thickness from Chickens Subjected to Heat Stress1
1833
TYROSINE, PROTEIN AND ASCORBIC ACID TABLE 1.—Tyrosine and ascorbic acid influence on egg shell maintenance Shell thicknes (microns)
Percent shell
Egg weight (grams)
Addition to control Preliminary
None (12)l Tyrosine (12) Tyrosine+vitamin C (13)
Experimental
Preliminary
Experimental
Preliminary
Experimental
284+7.3 261 + 5.7* 291+4.1 271±4.2**
7.90 + 0.19 7.42 + 0.15* 7.81 + 0.16 7.35 + 0.17*
56.7 + 0.7 54.2 + 0.7* 56.4 + 0.7 55.6 + 0.8
294 + 5.3
8.05 + 0.13 8.12 + 0.12
57.6 + 0.9 56.9 + 0.7
293±5.2
review on the function of ascorbic acid in cold acclimation, and the place of vitamin C is well documented. Since little attention has been given to heat acclimation, basic information concerning biochemical changes in the animal from heat stress and possible means of alleviation are lacking. In spite of a lack of documentary evidence, it does not seem illogical to assume that ascorbic acid may be involved in heat as well as cold acclimation. In the current study emphasis was placed on determining the possible relationship between the dietary levels of tyrosine and ascorbic acid on the maintenance of egg shell thickness under heat stress conditions. PROCEDURE
Two separate studies were conducted to investigate the possible relationship between ascorbic acid and tyrosine in egg shell formation. In the first study, 45 Single Comb White Leghorn hens which had been in production for 10 months and given the 13 percent protein basal diet (Thornton, 1960) were used. These animals were maintained in individual cages prior to and throughout the experimental period with feed and water supplied ad libitum. The preliminary studies were conducted in July, thus the hens had been exposed to a rather high environmental temperature (mean maximum 84°F., mean low 72°F.) for 30 days prior to egg shell measurements. After
at least six consecutively laid eggs were collected from each hen, the birds were divided into three equal groups and placed on experimental diets as shown in Table 1. The tyrosine was added at a level of 2.71 grams per kilogram of feed, which increased the absolute amount of tyrosine and phenylaline to that level calculated to be in the 17 percent protein diet (Thornton, 1960). This procedure was followed since it seemed remotely possible that a lack of response to ascorbic acid at that protein level could have resulted from the higher actual level of these amino acids. The ascorbic acid in this experiment as well as the second was added at a level of 44 mg. per kilogram of ration. After the eggs were weighed and broken, shell thickness and percent shell were determined in a manner previously described by Thornton (1960). The birds were then subjected to a daily average maximum temperature of 92.0 ± 0.6°F. and a daily minimum of 74.1 ± 0.7°F. for 21 days. After this time an additional six consecutively laid egg sample was collected and treated in a manner similar to that used in the preliminary study. Any hen which did not produce six eggs at each collection was discarded. In the second study, 72 ten-month old hens which had been in production for approximately four months were used. These pullets were selected on a basis of egg production rate. The animals were divided into
Downloaded from http://ps.oxfordjournals.org/ at University of Nebraka-Lincoln Libraries on April 7, 2015
* Significant decrease ** Highly significant decrease 1 Values in parentheses refer to number of hens in each group
1834
P. A. THORNTON TABLE 2.—Protein level and the response to vitamin C Shell thickness (microns)
17% Protein Basal Tyrosine Tyrosine+vitamin C 13% Protein Basal Tyrosine Tyrosine+vitamin C
Percent
Egg weight (grams) Preliminary Experimental
Preliminary
Experimental
maintenance
320 + 2.4 321 + 2.8 316 + 4.0 318 + 4.0 320 + 3.8 321+3.7
298 + 3.1* 306+2.5* 303 + 2.6* 298 + 5.2* 304 + 3.0* 315 + 3.5
93.1 95.3 95.9 93.7 95.2 98.2
60.3 61.4 59.1 58.6 61.4 59.3
60.5 61.7 59.5 58.9 61.3 59.7
RESULTS AND DISCUSSION In the first study it was evident that the
birds had been heat stressed prior to initiation of the study as shown by the low shell thickness values (Table 1). Despite the preliminary thin-shell values, a further significant decrease resulted when the birds were given the control ration and subjected to a higher temperature. The addition of tyrosine appeared to have no influence. When both tyrosine and vitamin C were added, there was nearly complete maintenance of egg shell thickness. Since Thornton (1960) showed that vitamin C added alone to this diet produced a similar response, it may be assumed that the improved shell thickness maintenance was due to the added vitamin in this case. Percent shell values were very similar in nature to the shell thickness values. Egg weight also was decreased to a significant degree in the control group when the environmental temperature was raised (Table 1). In the second study the egg weight was slightly greater in all but one group in the higher temperature environment, with no particular advantage shown by any of the dietary treatments (Table 2). The difference in response between the two studies could have resulted from differences in age as well as the degree of stress imposed. The shell thickness results obtained in the second study for the 13 percent protein diet were very similar to those of the first trial (Table 2). Although it was observed that the addition of tyrosine at both protein levels resulted in a slightly greater
Downloaded from http://ps.oxfordjournals.org/ at University of Nebraka-Lincoln Libraries on April 7, 2015
* Highly significant decrease.
six numerically equal groups and placed on the diets shown in Table 2. Tyrosine was added to the 17 percent protein diets so that the total tyrosine-phenylalanine level would equal that amount per unit of protein in the 13 percent protein diets (14 grams per kilogram feed). Addition of tyrosine to the 13 percent protein level was made as previously stated. The animals were maintained in a cool environment (max. = 68.2 ± 0.6, min. = 58.9 ± 0.4), for four weeks. Eight eggs were then collected from each hen and subjected to shell thickness and total egg weight determinations. Following this, the environmental temperature was gradually increased to a daily average of 84.2 ± 0.2. This environment was maintained for 48 days and a second egg sample collected for shell thickness and weight determinations. Only those birds which laid at least eight eggs during each sampling period were included in the data. At least nine individuals in each group were retained under this stipulation. In both studies each bird acted as its own control; thus, the data were analyzed on both an individual and group basis. There appeared to be little or no difference in the final values, as between the two procedures, particularly in percent maintenance of egg shell thickness, therefore, the values pertaining only to the group are given.
TYROSINE, PROTEIN AND ASCORBIC ACID
SUMMARY Two experiments were conducted to determine why hens given a 13 percent protein diet responded favorably to ascorbic acid supplementation in egg shell maintenance under heat stress while those given a 17 percent diet did not. Since the two rations differed only in the corn, milo and soybean oil meal ratio, it was theorized that differences in amino acid composition possibly could be implicated. In view of the known metabolic relationships between tyrosine and ascorbic acid and the slight difference of these amino acids relative to the
total protein level, tyrosine was chosen for study. The shell thickness values were decreased at both protein levels by heat stress whether tyrosine was added or not, therefore, it was evident that this amino acid was not involved under the experimental conditions. The addition of tyrosine with ascorbic acid to the 17 percent diet also appeared to have no influence. Conversely, the addition of ascorbic acid and tyrosine to the 13 percent diet resulted in a high degree of shellthickness maintenance. From these results it was concluded that the failure of response to ascorbic acid at the 17 percent protein level was not influenced by the different tyrosine level' of the ration. REFERENCES Desmarais, A., 1960. Ascorbic acid in cold acclimation. Proceedings of a symposium on cold acclimation. Federation Proc. 19: 88-90. Rangneker, P. V., and L. P. Dugal, 1958. A note on the effect of cold and vitamin C on the blood levels of phenylalanine and tyrosine in rats. Canadian J. Biochem. Physiol. 36: 185186. Sealock, R. R., and R. L. Goodland, 1951. Ascorbic acid, a coenzyme in tyrosine oxidation. Science, 114: 645-646. Sealock, R. R., and H. E. Silberstein, 1940. The excretion of homogentisic and other tyrosine metabolites by the vitamin C-deficient guinea pig. J. Biol. Chem. 135: 251-258. Thornton, P. A., 1960. The influence of dietary protein level on the response of Single Comb White Leghorns to supplementary ascorbic acid. Poultry Sci. 39: 1072-1076. Thornton, P. A., 1961. Increased environmental temperature influences on ascorbic acid ac 1 tivity in the domestic fowl. Federation Prdc. 20: 210A.
Downloaded from http://ps.oxfordjournals.org/ at University of Nebraka-Lincoln Libraries on April 7, 2015
maintenance of shell thickness, actual thickness values were reduced to a highly significant degree. Thus, the addition of this amino acid alone appeared to have little 01 no influence. When ascorbic acid and tyrosine were added jointly to the 17 percent diet, a highly significant decrease in shell thickness also resulted. From this it appears that the response to vitamin C was different at the two protein levels. Since the response to ascorbic acid was also shown earlier to be different in these two diets (Thornton, 1960) in the absence of supplementary tyrosine, it may be assumed that no synergism existed between the two compounds in this case. From these results, it appears that the failure of added vitamin C to maintain shell thickness in the higher protein diet (Thornton, 1960) was due neither to an excess of the absolute level of tyrosine and phenylalanine nor to a deficiency of these amino acids relative to others.
1835