Adrenal Function in Chickens Experiencing Mercury Toxicity 123
P. THAXTON, C. R. PARKHURST, L . A. COGBURN AND P. S. YOUNG
Poultry Science Department, North Carolina State University, Raleigh, North Carolina 27607 (Received for publication July 26, 1974)
ABSTRACT Dietary mercury when administered to young chickens via the drinking water depressed growth, increased the rate of mortality and inhibited the normal maturation of the adrenal glands. Additionally, deficiencies of cholesterol and corticosterone were deomonstrated in the adrenals. The exogenous administration of 0.5 or 1.5 mg. of corticosterone/100 gm. of body weight alleviated, in part, the toxic effects of mercury as evidenced by a rapid increase in body weight.
INTRODUCTION
E
XCESSIVE mortality and abnormal growth patterns are primary indicators of mercury toxicity in chickens (Fimreite, 1970; Fimreite and Karstad, 1971; Parkhurst and Thaxton, 1973). Mercuric chloride when administered via the drinking water to juvenile chickens resulted in a severe inhibition of growth which was correlated to the decreased utilization of feed and water (Parkhurst and Thaxton, 1973). Several other physio-pathological changes in chickens, as well as other birds, are attributable to mercury in both organic and inorganic forms. Reproductive deficiencies including decreased egg production, egg shell thinning, and abnormal mating behavior are caused by dietary mercury (Spann etah, 1972; Stoewsand et al., 1971; Tejning, 1967; Thaxton and Parkhurst, 1973b). Neurological dysfunction resulting from demyelination of spi-
nal nerves is an additional symptom of mercury toxicity (Fimreite, 1971; Fimreite and Karstad, 1971; Borg et al, 1969). Ingested mercuric chloride is reported to cause several hematological changes in young cockerels; these including increased packed cell volumes, increased erythrocytes, decreased total leucocytes, and increased heterophils concommitant with decreased lymphocytes (Thaxton et al., 1974). Several gross changes in the internal organs of young chickens are reported to be induced by mercuric chloride. Specifically, these are decreased relative weights of the liver, spleen and bursa of Fabricius, and increased relative weights of the heart and adrenal glands (Thaxton and Parkhurst, 1973a). The present study was conducted to further delineate the toxicity syndrome which is caused by mercury in the domestic chicken. The specific objectives of this study were: (1) to determine functional alterations in the adrenal glands and (2) to relate these changes to mercury-induced growth inhibition.
1. Paper number 4251 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina. 2. The use of trade names in this publication does not imply endorsement by the North Carolina Experiment Station of the products named, nor criticism of ones not mentioned. 3. A preliminary report of this paper was presented at the 73rd Annual Meeting of the Poultry Science Association, Brookings, South Dakota, 1973.
578
MATERIALS AND METHODS Three trials were conducted using commercial broiler cockerels. The chicks were housed for the first three weeks in heated brooding batteries and thereafter in non-heated growing cages. A starter-grower ration which was formulated to fulfill the nutritional
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on May 18, 2015
POULTRY SCIENCE 54: 578-584, 1975
579
ADRENALS AND MERCURY
*A4 - Pregnen-ll(3, 21-diol-3, 20 dione, Sigma Chemical Company, St. Louis, Missouri.
(Snedecor, 1956). Comparisons of means were made using a modification of Duncan's new multiple range test (Kramer, 1956). Statements of significance are based on P < 0.05. RESULTS The data of Trials 1 and 2 illustrating the effects of the mercury-treatments on the growth and livability of the chicks are presented in Table 1. It should be noted that the data of these trials were combined because no significant replicate effect was found. A persistent inhibition of growth occurred in the chicks which received 250 and 500 p.p.m. of mercury. This same effect was apparent in the chicks that received 125 p.p.m. of mercury at 4 and 5 weeks, but not at 3 or 6 weeks. These data confirm our earlier report that 250 and 500 p.p.m of mercury, when administered to broiler cockerels as mercuric chloride via the drinking water, caused a dramatic inhibition of growth coupled with an increased incidence of mortality (Parkhurst and Thaxton, 1973). The total adrenal weights and relative total adrenal weights (mg. adrenal/100 gm. body weight) as influenced by mercury are presented graphically in Figures 1 and 2, respectively. The total adrenal weights of the chicks which received either 250 or 500 p.p.m. of mercury were decreased significantly at each time of measurement. However, differences in total adrenal weights were not significant at the other dose levels of mercury. The relative total adrenal weights were increased significantly as compared to all other dose levels by 500 p.p.m. of mercury at each time interval. Correspondingly, 250 p.p.m. of mercury caused this effect at 4, 5, and 6 weeks of age, but not at 3 weeks. Figure 3 depicts the effects of the mercurytreatments on the levels of adrenal Choi. The adrenal levels of Choi were reduced significantly by 500 p.p.m. of mercury when compared to the controls at each time inter-
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on May 18, 2015
needs of the chicks was employed. Feed and water were available ad libitum in stainless steel troughs. In Trials 1 and 2, 240 chicks were administered mercury in the form of mercuric chloride via the drinking water. Four groups of 10 chicks each received 0, 5, 25, 125, 250, or 500 p,g. /ml. of mercury. To achieve these dosage levels of mercury the calculations were made on the basis of the molecular content of mercury in mercuric chloride and expressed in p.p.m. The chicks were weighed at 3, 4, 5, and 6 weeks of age. Following each body weight determination 10 chicks from each treatmentgroup were killed by cervical dislocation. At necropsy the adrenals were excised, blotted dry, and weighed individually. The glands were then secured in aluminum foil and frozen for later chemical analysis. The concentrations of cholesterol (Choi) and corticosterone (CS) in the thawed adrenals were determined by the procedures of Knobil et al. (1954) and Guillemin et al. (1958), respectively. In Trial 3, three groups of 40 chicks each received 0, 150, or 300 p.p.m. of mercury in the drinking water continuously throughout the experimental period. After five weeks of the mercury-treatments 10 chicks from each mercury-treatment group received either 1 ml. of physiological saline, 0.5, 1.5, or 4.5 mg. corticosterone*/100 gm. of body weight on each of five consecutive days. The CS solutions were prepared by dissolving this hormone in distilled water and adding 0.1 ml. of Tween 80. Body weights were determined prior to the first of the CS injections and again 72 hours after the last of the injections. The injections were made intramuscularly in the pectoral region. All the data collected during the study were analyzed statistically by analysis of variance
580
P. THAXTON, C. R. PARKHURST, L . A. COGBURN AND P. S.
TABLE I.—Effect Mercury (p.p.m.) 0 5 25 125 250 500
YOUNG
of dietary mercury on the growth and livability of broiler cockerels'-2 Age^weeks) 3
4
399a (80) 388a (80) 449 a (80) 393 a (78) 272" (75) 124= (21)
648ab (80) 612ab(80) 688 a (80) 596 b (78) 318= (74) 150d (21)
5 895a (77) 803" (78) 923 a (80) 720 c (78) 329d (70) 150e (9)
6 1075" (77) 1058" (78) 1130" (80) 920" (78) 417 b (68) 175c (5)
1 Means in a column possessing different superscripts differ significantly at P fi 0.05 and the number of 2birds representing the means are included in parentheses. The dosage of mercury was attained by adding mercuric chloride to the drinking water and the concentrations are correct for the mercury content.
reduced the concentrations of CS in the the adrenals at each measurement interval, except at 5 weeks. At this time no significant differences were found in the levels of CS among the treatment groups. The other doses of mercury were not effectual. The body weights of the controls, i.e. saline-injected chicks, of Trail 3 were determined immediately prior to the first saline-injection and again 72 hours after the fifth saline-injection. These weights are presented
FIG. 1. Total adrenal weights of chicks (10/treatment/week) which received mercury in the form of mercuric chloride via the drinking water. Treatment levels of mercury in p.p.m. were: - 0 - , 0; - # - , 5; -A-, 25; -A-, 125; - • - , 250; - B - , 500.
FIG. 2. Adrenal weights relative to body weights (mg. total adrenal/100 gm. body weight) of chicks (10/treatment/week) which received mercury in the form of mercuric chloride via the drinking water. Treatment levels of mercury in p.p.m. were: -O-, 0; - # - , 5; -A-, 25; -A-, 125; - • - , 250; - B - , 500.
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val. The 250 p.p.m. dose of mercury caused this effect at 5 and 6 weeks, but not at 3 or 4 weeks. The lower doses of 5, 25, and 125 p.p.m. of mercury did not alter significantly the levels of adrenal Choi when compared to the controls at any of the measurement times. The data relevant to the effects of mercury on the adrenal concentrations of CS are presented in Figure 4. The growth inhibitory doses of 250 and 500 p.p.m. of mercury
581
ADRENALS AND MERCURY
in Figure 5. Mercury caused a dose-related inhibition of growth at both times of measurement. Additionally, the rates of growth, albeit inhibited by both 150 and 300 p.p.m. of mercury, increased at near liner rates during the 8 day experimental period. In view of the linearity of the growth curves among the controls and mercury-treated groups (150 and 300 p.p.m.), the final body weights as influenced by exogenous CS administration are presented in Figure 6 as percentage changes from the initial body weights. These percentage changes were converted to arcsin \f% prior to statistical evaluation (Snedecor, 1956). The non-mercury treated chicks which received the 0.5 or 1.5 mg. dosages of CS experienced a significant reduction in body weight as compared to their respective saline-injected counterparts. The opposite effect was observed in the chicks which had received 150 or 300 p.p.m. of mercury and the 0.5 mg. dosage of CS; this was also true for the 300 p.p.m. mercury-treated birds which received either the 0.5 or 1.5 mg. dosages of CS. Specifically, the percentage changes in the body weights of these birds
AGE(wks)
FIG. 4. Adrenal corticosterone concentration of chicks (10/treatment/week) which received mercury in the form of mercuric chloride via the drinking water. Treatment levels of mercury in p.p.m. were: -O-, 0; -%-, 5; -A-, 25; -A-, 125; - • - , 250; - H - , 500. did not differ significantly from their respective saline-injected controls. Additionally, when the dosage of CS was increased to 4.5 mg. /100 gm. body weight/injection the birds which received 0 or 300 p.p.m. of mercury exhibited weight losses. There was no percentage change in the body weight of the 150 p.p.m. mercury-treated birds which received this dosage of CS. DISCUSSION The pivotal role of the pituitary-adrenal axis in an animal's ability to adapt to various environmental stimuli is well known (Selye, 1953). Siegel (1971) has reviewed adrenal responsiveness in the domestic fowl as it is influenced by several environmental parameters. However, the relationship of heavy metals to adrenal function has not been reported previously. The results of this study suggest that dietary inorganic mercury when administered to young cockerels via the drinking water alters
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FIG. 3. Adrenal cholesterol concentrations of chicks (10/treatment/week) which received mercury in the form of mercuric chloride via the drinking water. Treatment levels of mercury in p.p.m. were: -O-, 0; - # - , 5; -A-, 25; -A-, 125; - • - , 250; -H-, 500.
582
P. THAXTON, C. R. PARKHURST, L. A. COGBURN AND P. S. YOUNG
lOOOr
40 30 20 10
0
1
1, -fi_ 150
-10
300
FIG. 5. Growth curves of chicks (40/treatment) which received 0, 150, or 300 p.p.m. of mercury in the form of mercuric chloride added to the drinking water. The -O- represents body weights at 5 weeks of age and -A- represents the weights of the same chicks 8 days later. As stated in the text this time corresponds to 72 hours after the last of the 5 saline injections.
the development and function of the adrenals. The gross adrenal weights were decreased (Fig. 1), while the adrenal weights, when adjusted for body weights (Fig. 2), were increased significantly by 250 and 500 p.p.m. of mercury. This apparent hypertrophy of the adrenals can be interpreted as a positive indication that the toxic levels of mercury induced a classic physiological stress (Selye, 1953; Siegel, 1971). The finding that Choi, which is the only known precursor for the adrenal steroids, and CS, the primary cortical hormone in birds, were depleted from the glands adds credence to this interpretation (Frankel, 1970). Additionally, in an earlier study it was reported that the chronic administration of mercury caused atrophy of the bursa of Fabricius and spleen (Thaxton and Parkhurst, 1973a) and an increase in the absolute circulating number of heterophils concomitant with a decrease in the absolute number of lymphocytes (Thaxton et al.,
300
-20 L
FIG. 6. The relationship of exogenous corticosterone administration to growth in cockerels which received 0, 150, or 300 p.p.m. of mercury in the form of mercuric chloride via the drinking water. The corticosterone and saline dosages were administered IM daily for 5 days on the basis of the particular dose level/100 gm. of body weight. The following symbols identify the dose levels: Q saline controls; 0 0.5 mg.; § 1.5 mg.; and HI 4.5 mg. 1974). The data again are suggestive that the syndrome of mercury toxicity in chickens is attributable, at least in part, to physiological stress. An alternate hypothesis to explain the toxic effects of mercury in chickens, and thus the results of this study, may be equally tenable. It is possible that mercury limits the synthesis of the adrenocortical hormones. Thus, the adrenal hypertrophy and depletion of adrenal Choi and CS could reflect inhibited steroid metabolism within the gland. The finding that the exogenous dosages of 0.5 and 1.5 mg. of CS caused a percentage increase in body weight in the birds experiencing an inhibition of normal growth supports this alternate hypothesis. Specifically, if the toxic birds were experiencing a physiological deficiency of CS due to inhibited biosynthesis, it is logical that correction of this deficiency by exogenous hormone therapy would enhance the homeostatic condition. The increased body weights observed in the birds receiving
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150 MERCURY(ppm)
MERCURY (ppm)
1
ADRENALS AND MERCURY
ACKNOWLEDGEMENTS We thank Ms. J. C. Bibb for excellent technical assistance. REFERENCES Adams, B. M., 1968. Effect of Cortisol on growth and uric acid excretion in the chick. J. Endocr. 40: 145-151. Bellany, D., and R. Leonard, 1965. Effect of Cortisol on the growth of chicks. Gen. Comp. Endocr. 5: 402-410. Borg, K., H. Wanntorp, K. Erne and E. Hanko, 1969. Alkyl mercury poisoning in terrestrial Swedish wildlife. Viltrevy, 6: 301-379. Brown, K. I., R. K. Meyer and D. J. Brown, 1958.
A study of adrenalectomized male chickens with and without adrenal hormone treatment. Poultry Sci. 37: 680-684. Fimreite, N., 1970. Effects of methyl mercury treated feed on the mortality and growth of Leghorn cockerels. Can. J. Anim. Sci. 50: 387-389. Fimreite, N., 1971. Effects of dietary methylmercury on Ring-Necked Pheasants. Occasional Paper No. 9. Can. Wildlife Service, pp. 3-39. Fimreite, N., and L. Karstad, 1971. Effects of dietary methyl mercury on Red-tailed Hawks. J. Wildlife Man. 35: 293-300. Frankel, A. I., 1970. Symposium: Recent advances in avian endocrinology. 4. Neurohumoral control of the avian adrenal: A review. Poultry Sci. 49: 869-921. Guillemin, R., G. W. Clayton, J. E. Smith and H. S. Lipscomb, 1958. Measurement of free corticosteroids in rat plasma and physiological validation of a method. Endocrinology, 63: 349-358. Knobil, E., M. C. Haney, E. I. Wilder and F. N. Briggs, 1954. Simplified method for determination of total adrenal cholesterol. Proc. Soc. Exp. Biol. Med. 87: 48-50. Kramer, C. Y., 1956. Extension of multiple range tests to group means with unequal numbers of replications. Biometrics, 12: 307-310. Parkhurst, C. R., and P. Thaxton, 1973. Toxicity of mercury to young chickens. 1. Effect on growth and mortality. Poultry Sci. 52: 273-276. Selye, H., 1953. Stress. Explorations, 1: 57-76. Siegel, H. S., 1971. Adrenals, stress and the environment. World's Poultry Sci. J. 27: 327-349. Sendecor, G. W., 1956. Statistical Methods, 5th ed., The Iowa State University Press, Ames, Iowa. Spann, J. W., R. G. Heath, J. F. Kreitzer and L. M. Locke, 1972. Ethyl mercury p-toluene sulfonanilide: Lethal and reproductive effects on pheasants. Science, 175: 328-331. Stoewsand, G. S., J. L. Anderson, W. H. Gutewmann, G. A. Bache and D. J. Lish, 1971. Eggshell thinning in Japanese quail fed mercuric chloride. Science, 173: 1030-1031. Tejning, S., 1967. Biological effects of methyl mercury dicyandiamide treated grain in the domestic fowl Gallus gallus L. I. Studies on food consumption, egg production and general health. Oikos Suppl. 8:7-17. Thaxton, P., and C. R. Parkhurst, 1973a. Toxicity of mercury to young chickens. 2. Gross changes in organs. Poultry Sci. 52: 277-281. Thaxton, P., and C. R. Parkhurst, 1973b. Abnormal mating behavior and reproductive dysfunction caused by mercury in Japanese quail. Proc. Soc. Exp. Biol. Med. 144: 252-255.
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150 and 300 p.p.m. of mercury and either 0.5 or 1.5 mg. of CS would be expected. Brown et al. (1958) demonstrated that adrenalectomized birds which received deoxycorticosterone acetate or cortisone acetate experienced increases in body weight as compared to the adrenalectomized controls. Of equal importance to this interpretation is the finding that in the non-mercury treated birds all the dosages of CS caused a lower percentage increase in body weight as compared to the saline controls and the 4.5 mg. dosage resulted in body weight losses in all the birds which received 300 p.p.m. of mercury (Fig. 6). This level of CS apparently exceeded the physiological needs of the birds and thus induced catabolic effects which are attributable to non-physiological levels of adrenocortical steroids. Bellany and Leonard (1965) and Adams (1968) have demonstrated that exogenous adrenocortical hormones caused a marked depression in the body weight of young chickens. The question of the relationship of mercury toxicity in chickens to physiological stress remains unanswered. However, the results herein indicate that the development and function of the adrenal glands, which are unquestionably important in the physiological adaptation responses, are altered radically by toxic levels of dietary mercury.
583
584
P. THAXTON, C. R. PARKHURST, L. A. COGBURN AND P. S. YOUNG
Thaxton, P., P. S. Young, L. A. Cogburn and C. R. Parkhurst, 1974. Hematology of mercury toxicity
in young chickens. Bull. Environ. Contamin. Toxicol. 12: 46-52.
Factors Associated with Utilization of the Calcium Salt of Methionine Hydroxy Analogue by the Young Chick R. S. K A T Z AND D . H . BAKER
Department of Animal Science, University of Illinois, Urbana, Illinois 61801 (Received for publication July 26, 1974)
POULTRY SCIENCE 54: 584-591, 1975
INTRODUCTION HE efficacy of methionine hydroxy analogue (OH-M) as a source of sulfur amino acids (SAA) in practical-type diets is fairly well established (Bird, 1952; Gordon etal, 1954; Scott era/., 1966). Its metabolism, however, is less well documented. OH-M has been reported to be equivalent in biological activity to DL-methionine by some (Scott et
T
toglutarate amino transferase was not influenced by source of SAA (i.e. methionine or OH-M), and growth performance of chicks fed two levels of branched-chain amino acids, inadequate or superadequate, was also unaffected by source of SAA. The mechanism proposed by Gordon and Sizer (1965) for OH-M conversion to methionine is shown below. amino donor
OH-M-^methionine keto analogue
\
methionine cysteine
al., 1966; Chow et al., 1974) and inferior by others (Smith, 1966; Featherston and Horn, 1974). The branched-chain amino acids are reputed to be the most active amino donors in the conversion of OH-M to methionine (Gordon and Sizer, 1965), but recent work from Purdue (Featherston and Horn, 1974) has not substantiated this concept. In their study, the activity of L-leucine: a-ke-
It would appear from this scheme that OH-M could serve equally well as a source of either methionine or cysteine. However, OH-M must be converted to methionine before it may serve as a source of cysteine. Thus, the ratio of methionine to cystine in a diet could conceivably influence the efficacy of OH-M. The studies reported herein were designed
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ABSTRACT Six growth assays were conducted using crystalline amino acid diets to investigate the efficacy of methionine hydroxy analogue (OH-M) as a precursor of sulfur bearing amino acids (SAA). The influence on OH-M utilization of dietary modifications of the methionine-cystine ratio, inorganic sulfate as K 2 S0 4 and supplemental mixtures of the branched-chain amino acids (leucine, isoleucine and valine) was explored. Performance of chicks fed OH-M was inferior to those fed an equivalent molar quantity of DL-methionine. Additional dietary sulfate or supplemental mixtures of the branched-chain amino acids did not improve OH-M utilization. OH-M was found to support growth equal to that of 0.35% methionine and 0.35% cystine when fed at a level 25% above the SAA level provided by methionine and cystine. On a weight basis the calcium salt of OH-M has 88.15% OH-M activity. From assays reported herein the calcium salt of OH-M was calculated to have approximately 70% methionine activity.
P. THAXTON, C. R. PARKHURST, L . A. COGBURN AND P. S. YOUNG
Poultry Science Department, North Carolina State University, Raleigh, North Carolina 27607 (Received for publication July 26, 1974)
ABSTRACT Dietary mercury when administered to young chickens via the drinking water depressed growth, increased the rate of mortality and inhibited the normal maturation of the adrenal glands. Additionally, deficiencies of cholesterol and corticosterone were deomonstrated in the adrenals. The exogenous administration of 0.5 or 1.5 mg. of corticosterone/100 gm. of body weight alleviated, in part, the toxic effects of mercury as evidenced by a rapid increase in body weight.
INTRODUCTION
E
XCESSIVE mortality and abnormal growth patterns are primary indicators of mercury toxicity in chickens (Fimreite, 1970; Fimreite and Karstad, 1971; Parkhurst and Thaxton, 1973). Mercuric chloride when administered via the drinking water to juvenile chickens resulted in a severe inhibition of growth which was correlated to the decreased utilization of feed and water (Parkhurst and Thaxton, 1973). Several other physio-pathological changes in chickens, as well as other birds, are attributable to mercury in both organic and inorganic forms. Reproductive deficiencies including decreased egg production, egg shell thinning, and abnormal mating behavior are caused by dietary mercury (Spann etah, 1972; Stoewsand et al., 1971; Tejning, 1967; Thaxton and Parkhurst, 1973b). Neurological dysfunction resulting from demyelination of spi-
nal nerves is an additional symptom of mercury toxicity (Fimreite, 1971; Fimreite and Karstad, 1971; Borg et al, 1969). Ingested mercuric chloride is reported to cause several hematological changes in young cockerels; these including increased packed cell volumes, increased erythrocytes, decreased total leucocytes, and increased heterophils concommitant with decreased lymphocytes (Thaxton et al., 1974). Several gross changes in the internal organs of young chickens are reported to be induced by mercuric chloride. Specifically, these are decreased relative weights of the liver, spleen and bursa of Fabricius, and increased relative weights of the heart and adrenal glands (Thaxton and Parkhurst, 1973a). The present study was conducted to further delineate the toxicity syndrome which is caused by mercury in the domestic chicken. The specific objectives of this study were: (1) to determine functional alterations in the adrenal glands and (2) to relate these changes to mercury-induced growth inhibition.
1. Paper number 4251 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina. 2. The use of trade names in this publication does not imply endorsement by the North Carolina Experiment Station of the products named, nor criticism of ones not mentioned. 3. A preliminary report of this paper was presented at the 73rd Annual Meeting of the Poultry Science Association, Brookings, South Dakota, 1973.
578
MATERIALS AND METHODS Three trials were conducted using commercial broiler cockerels. The chicks were housed for the first three weeks in heated brooding batteries and thereafter in non-heated growing cages. A starter-grower ration which was formulated to fulfill the nutritional
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on May 18, 2015
POULTRY SCIENCE 54: 578-584, 1975
579
ADRENALS AND MERCURY
*A4 - Pregnen-ll(3, 21-diol-3, 20 dione, Sigma Chemical Company, St. Louis, Missouri.
(Snedecor, 1956). Comparisons of means were made using a modification of Duncan's new multiple range test (Kramer, 1956). Statements of significance are based on P < 0.05. RESULTS The data of Trials 1 and 2 illustrating the effects of the mercury-treatments on the growth and livability of the chicks are presented in Table 1. It should be noted that the data of these trials were combined because no significant replicate effect was found. A persistent inhibition of growth occurred in the chicks which received 250 and 500 p.p.m. of mercury. This same effect was apparent in the chicks that received 125 p.p.m. of mercury at 4 and 5 weeks, but not at 3 or 6 weeks. These data confirm our earlier report that 250 and 500 p.p.m of mercury, when administered to broiler cockerels as mercuric chloride via the drinking water, caused a dramatic inhibition of growth coupled with an increased incidence of mortality (Parkhurst and Thaxton, 1973). The total adrenal weights and relative total adrenal weights (mg. adrenal/100 gm. body weight) as influenced by mercury are presented graphically in Figures 1 and 2, respectively. The total adrenal weights of the chicks which received either 250 or 500 p.p.m. of mercury were decreased significantly at each time of measurement. However, differences in total adrenal weights were not significant at the other dose levels of mercury. The relative total adrenal weights were increased significantly as compared to all other dose levels by 500 p.p.m. of mercury at each time interval. Correspondingly, 250 p.p.m. of mercury caused this effect at 4, 5, and 6 weeks of age, but not at 3 weeks. Figure 3 depicts the effects of the mercurytreatments on the levels of adrenal Choi. The adrenal levels of Choi were reduced significantly by 500 p.p.m. of mercury when compared to the controls at each time inter-
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on May 18, 2015
needs of the chicks was employed. Feed and water were available ad libitum in stainless steel troughs. In Trials 1 and 2, 240 chicks were administered mercury in the form of mercuric chloride via the drinking water. Four groups of 10 chicks each received 0, 5, 25, 125, 250, or 500 p,g. /ml. of mercury. To achieve these dosage levels of mercury the calculations were made on the basis of the molecular content of mercury in mercuric chloride and expressed in p.p.m. The chicks were weighed at 3, 4, 5, and 6 weeks of age. Following each body weight determination 10 chicks from each treatmentgroup were killed by cervical dislocation. At necropsy the adrenals were excised, blotted dry, and weighed individually. The glands were then secured in aluminum foil and frozen for later chemical analysis. The concentrations of cholesterol (Choi) and corticosterone (CS) in the thawed adrenals were determined by the procedures of Knobil et al. (1954) and Guillemin et al. (1958), respectively. In Trial 3, three groups of 40 chicks each received 0, 150, or 300 p.p.m. of mercury in the drinking water continuously throughout the experimental period. After five weeks of the mercury-treatments 10 chicks from each mercury-treatment group received either 1 ml. of physiological saline, 0.5, 1.5, or 4.5 mg. corticosterone*/100 gm. of body weight on each of five consecutive days. The CS solutions were prepared by dissolving this hormone in distilled water and adding 0.1 ml. of Tween 80. Body weights were determined prior to the first of the CS injections and again 72 hours after the last of the injections. The injections were made intramuscularly in the pectoral region. All the data collected during the study were analyzed statistically by analysis of variance
580
P. THAXTON, C. R. PARKHURST, L . A. COGBURN AND P. S.
TABLE I.—Effect Mercury (p.p.m.) 0 5 25 125 250 500
YOUNG
of dietary mercury on the growth and livability of broiler cockerels'-2 Age^weeks) 3
4
399a (80) 388a (80) 449 a (80) 393 a (78) 272" (75) 124= (21)
648ab (80) 612ab(80) 688 a (80) 596 b (78) 318= (74) 150d (21)
5 895a (77) 803" (78) 923 a (80) 720 c (78) 329d (70) 150e (9)
6 1075" (77) 1058" (78) 1130" (80) 920" (78) 417 b (68) 175c (5)
1 Means in a column possessing different superscripts differ significantly at P fi 0.05 and the number of 2birds representing the means are included in parentheses. The dosage of mercury was attained by adding mercuric chloride to the drinking water and the concentrations are correct for the mercury content.
reduced the concentrations of CS in the the adrenals at each measurement interval, except at 5 weeks. At this time no significant differences were found in the levels of CS among the treatment groups. The other doses of mercury were not effectual. The body weights of the controls, i.e. saline-injected chicks, of Trail 3 were determined immediately prior to the first saline-injection and again 72 hours after the fifth saline-injection. These weights are presented
FIG. 1. Total adrenal weights of chicks (10/treatment/week) which received mercury in the form of mercuric chloride via the drinking water. Treatment levels of mercury in p.p.m. were: - 0 - , 0; - # - , 5; -A-, 25; -A-, 125; - • - , 250; - B - , 500.
FIG. 2. Adrenal weights relative to body weights (mg. total adrenal/100 gm. body weight) of chicks (10/treatment/week) which received mercury in the form of mercuric chloride via the drinking water. Treatment levels of mercury in p.p.m. were: -O-, 0; - # - , 5; -A-, 25; -A-, 125; - • - , 250; - B - , 500.
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val. The 250 p.p.m. dose of mercury caused this effect at 5 and 6 weeks, but not at 3 or 4 weeks. The lower doses of 5, 25, and 125 p.p.m. of mercury did not alter significantly the levels of adrenal Choi when compared to the controls at any of the measurement times. The data relevant to the effects of mercury on the adrenal concentrations of CS are presented in Figure 4. The growth inhibitory doses of 250 and 500 p.p.m. of mercury
581
ADRENALS AND MERCURY
in Figure 5. Mercury caused a dose-related inhibition of growth at both times of measurement. Additionally, the rates of growth, albeit inhibited by both 150 and 300 p.p.m. of mercury, increased at near liner rates during the 8 day experimental period. In view of the linearity of the growth curves among the controls and mercury-treated groups (150 and 300 p.p.m.), the final body weights as influenced by exogenous CS administration are presented in Figure 6 as percentage changes from the initial body weights. These percentage changes were converted to arcsin \f% prior to statistical evaluation (Snedecor, 1956). The non-mercury treated chicks which received the 0.5 or 1.5 mg. dosages of CS experienced a significant reduction in body weight as compared to their respective saline-injected counterparts. The opposite effect was observed in the chicks which had received 150 or 300 p.p.m. of mercury and the 0.5 mg. dosage of CS; this was also true for the 300 p.p.m. mercury-treated birds which received either the 0.5 or 1.5 mg. dosages of CS. Specifically, the percentage changes in the body weights of these birds
AGE(wks)
FIG. 4. Adrenal corticosterone concentration of chicks (10/treatment/week) which received mercury in the form of mercuric chloride via the drinking water. Treatment levels of mercury in p.p.m. were: -O-, 0; -%-, 5; -A-, 25; -A-, 125; - • - , 250; - H - , 500. did not differ significantly from their respective saline-injected controls. Additionally, when the dosage of CS was increased to 4.5 mg. /100 gm. body weight/injection the birds which received 0 or 300 p.p.m. of mercury exhibited weight losses. There was no percentage change in the body weight of the 150 p.p.m. mercury-treated birds which received this dosage of CS. DISCUSSION The pivotal role of the pituitary-adrenal axis in an animal's ability to adapt to various environmental stimuli is well known (Selye, 1953). Siegel (1971) has reviewed adrenal responsiveness in the domestic fowl as it is influenced by several environmental parameters. However, the relationship of heavy metals to adrenal function has not been reported previously. The results of this study suggest that dietary inorganic mercury when administered to young cockerels via the drinking water alters
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FIG. 3. Adrenal cholesterol concentrations of chicks (10/treatment/week) which received mercury in the form of mercuric chloride via the drinking water. Treatment levels of mercury in p.p.m. were: -O-, 0; - # - , 5; -A-, 25; -A-, 125; - • - , 250; -H-, 500.
582
P. THAXTON, C. R. PARKHURST, L. A. COGBURN AND P. S. YOUNG
lOOOr
40 30 20 10
0
1
1, -fi_ 150
-10
300
FIG. 5. Growth curves of chicks (40/treatment) which received 0, 150, or 300 p.p.m. of mercury in the form of mercuric chloride added to the drinking water. The -O- represents body weights at 5 weeks of age and -A- represents the weights of the same chicks 8 days later. As stated in the text this time corresponds to 72 hours after the last of the 5 saline injections.
the development and function of the adrenals. The gross adrenal weights were decreased (Fig. 1), while the adrenal weights, when adjusted for body weights (Fig. 2), were increased significantly by 250 and 500 p.p.m. of mercury. This apparent hypertrophy of the adrenals can be interpreted as a positive indication that the toxic levels of mercury induced a classic physiological stress (Selye, 1953; Siegel, 1971). The finding that Choi, which is the only known precursor for the adrenal steroids, and CS, the primary cortical hormone in birds, were depleted from the glands adds credence to this interpretation (Frankel, 1970). Additionally, in an earlier study it was reported that the chronic administration of mercury caused atrophy of the bursa of Fabricius and spleen (Thaxton and Parkhurst, 1973a) and an increase in the absolute circulating number of heterophils concomitant with a decrease in the absolute number of lymphocytes (Thaxton et al.,
300
-20 L
FIG. 6. The relationship of exogenous corticosterone administration to growth in cockerels which received 0, 150, or 300 p.p.m. of mercury in the form of mercuric chloride via the drinking water. The corticosterone and saline dosages were administered IM daily for 5 days on the basis of the particular dose level/100 gm. of body weight. The following symbols identify the dose levels: Q saline controls; 0 0.5 mg.; § 1.5 mg.; and HI 4.5 mg. 1974). The data again are suggestive that the syndrome of mercury toxicity in chickens is attributable, at least in part, to physiological stress. An alternate hypothesis to explain the toxic effects of mercury in chickens, and thus the results of this study, may be equally tenable. It is possible that mercury limits the synthesis of the adrenocortical hormones. Thus, the adrenal hypertrophy and depletion of adrenal Choi and CS could reflect inhibited steroid metabolism within the gland. The finding that the exogenous dosages of 0.5 and 1.5 mg. of CS caused a percentage increase in body weight in the birds experiencing an inhibition of normal growth supports this alternate hypothesis. Specifically, if the toxic birds were experiencing a physiological deficiency of CS due to inhibited biosynthesis, it is logical that correction of this deficiency by exogenous hormone therapy would enhance the homeostatic condition. The increased body weights observed in the birds receiving
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150 MERCURY(ppm)
MERCURY (ppm)
1
ADRENALS AND MERCURY
ACKNOWLEDGEMENTS We thank Ms. J. C. Bibb for excellent technical assistance. REFERENCES Adams, B. M., 1968. Effect of Cortisol on growth and uric acid excretion in the chick. J. Endocr. 40: 145-151. Bellany, D., and R. Leonard, 1965. Effect of Cortisol on the growth of chicks. Gen. Comp. Endocr. 5: 402-410. Borg, K., H. Wanntorp, K. Erne and E. Hanko, 1969. Alkyl mercury poisoning in terrestrial Swedish wildlife. Viltrevy, 6: 301-379. Brown, K. I., R. K. Meyer and D. J. Brown, 1958.
A study of adrenalectomized male chickens with and without adrenal hormone treatment. Poultry Sci. 37: 680-684. Fimreite, N., 1970. Effects of methyl mercury treated feed on the mortality and growth of Leghorn cockerels. Can. J. Anim. Sci. 50: 387-389. Fimreite, N., 1971. Effects of dietary methylmercury on Ring-Necked Pheasants. Occasional Paper No. 9. Can. Wildlife Service, pp. 3-39. Fimreite, N., and L. Karstad, 1971. Effects of dietary methyl mercury on Red-tailed Hawks. J. Wildlife Man. 35: 293-300. Frankel, A. I., 1970. Symposium: Recent advances in avian endocrinology. 4. Neurohumoral control of the avian adrenal: A review. Poultry Sci. 49: 869-921. Guillemin, R., G. W. Clayton, J. E. Smith and H. S. Lipscomb, 1958. Measurement of free corticosteroids in rat plasma and physiological validation of a method. Endocrinology, 63: 349-358. Knobil, E., M. C. Haney, E. I. Wilder and F. N. Briggs, 1954. Simplified method for determination of total adrenal cholesterol. Proc. Soc. Exp. Biol. Med. 87: 48-50. Kramer, C. Y., 1956. Extension of multiple range tests to group means with unequal numbers of replications. Biometrics, 12: 307-310. Parkhurst, C. R., and P. Thaxton, 1973. Toxicity of mercury to young chickens. 1. Effect on growth and mortality. Poultry Sci. 52: 273-276. Selye, H., 1953. Stress. Explorations, 1: 57-76. Siegel, H. S., 1971. Adrenals, stress and the environment. World's Poultry Sci. J. 27: 327-349. Sendecor, G. W., 1956. Statistical Methods, 5th ed., The Iowa State University Press, Ames, Iowa. Spann, J. W., R. G. Heath, J. F. Kreitzer and L. M. Locke, 1972. Ethyl mercury p-toluene sulfonanilide: Lethal and reproductive effects on pheasants. Science, 175: 328-331. Stoewsand, G. S., J. L. Anderson, W. H. Gutewmann, G. A. Bache and D. J. Lish, 1971. Eggshell thinning in Japanese quail fed mercuric chloride. Science, 173: 1030-1031. Tejning, S., 1967. Biological effects of methyl mercury dicyandiamide treated grain in the domestic fowl Gallus gallus L. I. Studies on food consumption, egg production and general health. Oikos Suppl. 8:7-17. Thaxton, P., and C. R. Parkhurst, 1973a. Toxicity of mercury to young chickens. 2. Gross changes in organs. Poultry Sci. 52: 277-281. Thaxton, P., and C. R. Parkhurst, 1973b. Abnormal mating behavior and reproductive dysfunction caused by mercury in Japanese quail. Proc. Soc. Exp. Biol. Med. 144: 252-255.
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150 and 300 p.p.m. of mercury and either 0.5 or 1.5 mg. of CS would be expected. Brown et al. (1958) demonstrated that adrenalectomized birds which received deoxycorticosterone acetate or cortisone acetate experienced increases in body weight as compared to the adrenalectomized controls. Of equal importance to this interpretation is the finding that in the non-mercury treated birds all the dosages of CS caused a lower percentage increase in body weight as compared to the saline controls and the 4.5 mg. dosage resulted in body weight losses in all the birds which received 300 p.p.m. of mercury (Fig. 6). This level of CS apparently exceeded the physiological needs of the birds and thus induced catabolic effects which are attributable to non-physiological levels of adrenocortical steroids. Bellany and Leonard (1965) and Adams (1968) have demonstrated that exogenous adrenocortical hormones caused a marked depression in the body weight of young chickens. The question of the relationship of mercury toxicity in chickens to physiological stress remains unanswered. However, the results herein indicate that the development and function of the adrenal glands, which are unquestionably important in the physiological adaptation responses, are altered radically by toxic levels of dietary mercury.
583
584
P. THAXTON, C. R. PARKHURST, L. A. COGBURN AND P. S. YOUNG
Thaxton, P., P. S. Young, L. A. Cogburn and C. R. Parkhurst, 1974. Hematology of mercury toxicity
in young chickens. Bull. Environ. Contamin. Toxicol. 12: 46-52.
Factors Associated with Utilization of the Calcium Salt of Methionine Hydroxy Analogue by the Young Chick R. S. K A T Z AND D . H . BAKER
Department of Animal Science, University of Illinois, Urbana, Illinois 61801 (Received for publication July 26, 1974)
POULTRY SCIENCE 54: 584-591, 1975
INTRODUCTION HE efficacy of methionine hydroxy analogue (OH-M) as a source of sulfur amino acids (SAA) in practical-type diets is fairly well established (Bird, 1952; Gordon etal, 1954; Scott era/., 1966). Its metabolism, however, is less well documented. OH-M has been reported to be equivalent in biological activity to DL-methionine by some (Scott et
T
toglutarate amino transferase was not influenced by source of SAA (i.e. methionine or OH-M), and growth performance of chicks fed two levels of branched-chain amino acids, inadequate or superadequate, was also unaffected by source of SAA. The mechanism proposed by Gordon and Sizer (1965) for OH-M conversion to methionine is shown below. amino donor
OH-M-^methionine keto analogue
\
methionine cysteine
al., 1966; Chow et al., 1974) and inferior by others (Smith, 1966; Featherston and Horn, 1974). The branched-chain amino acids are reputed to be the most active amino donors in the conversion of OH-M to methionine (Gordon and Sizer, 1965), but recent work from Purdue (Featherston and Horn, 1974) has not substantiated this concept. In their study, the activity of L-leucine: a-ke-
It would appear from this scheme that OH-M could serve equally well as a source of either methionine or cysteine. However, OH-M must be converted to methionine before it may serve as a source of cysteine. Thus, the ratio of methionine to cystine in a diet could conceivably influence the efficacy of OH-M. The studies reported herein were designed
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ABSTRACT Six growth assays were conducted using crystalline amino acid diets to investigate the efficacy of methionine hydroxy analogue (OH-M) as a precursor of sulfur bearing amino acids (SAA). The influence on OH-M utilization of dietary modifications of the methionine-cystine ratio, inorganic sulfate as K 2 S0 4 and supplemental mixtures of the branched-chain amino acids (leucine, isoleucine and valine) was explored. Performance of chicks fed OH-M was inferior to those fed an equivalent molar quantity of DL-methionine. Additional dietary sulfate or supplemental mixtures of the branched-chain amino acids did not improve OH-M utilization. OH-M was found to support growth equal to that of 0.35% methionine and 0.35% cystine when fed at a level 25% above the SAA level provided by methionine and cystine. On a weight basis the calcium salt of OH-M has 88.15% OH-M activity. From assays reported herein the calcium salt of OH-M was calculated to have approximately 70% methionine activity.
