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Reproductive Tissue Activity in Hypothyroid or Heat Stressed Hens*
Reproductive Tissue Activity in Hypothyroid or Heat Stressed Hens* M . S. PREMOVICH AND R. B . CHIASSON
Department of Veterinary Science, University of Arizona, Tucson, Arizona 85721 (Received for publication July 22, 1975)
POULTRY SCIENCE 55: 906-910, 1976
T
HE possible role of thyroid hormone in egg production and shell deposition has been investigated by Taylor and Burmester (1940) who reported a decrease in egg production as well as a 9% decrease in shell thickness after thyroidectomy. Berg and Bearse (1951) decreased egg production with the addition of 0.1% thiouracil to the diet of laying hens and Gabuten and Shaffner (1954) reduced shell thickness with a similar diet. Turner et al. (1945) noted an over-all decrease in egg production which coincided with a reduction in thyroxine secretion rates and basal metabolic rates as laying hens grew older. The effects of high environmental temperatures on thyroxine secretion rates and basal metabolic rates are well known (Heninger et al., 1960; Huston 1962a, b; Hahn et al., 1966) and Falconer (1971) has reported a seasonal fluctuation in thyroid secretion rates from an October and November high to a low in April through August. The period of lowest thyroid hormone levels coincides with the period of reduced egg production and shell thickness.
A decrease in productivity, as reported by Taylor and Burmester (1940), seems to indicate hypoactivity of the reproductive organs and this may be correlated with suppressed thyroid activity in high environmental temperatures. Increased uptake of 3 2 P by an organ is generally considered to indicate an increased metabolic activity of the organ (Lamberg, 1953). Comparisons of the amount of 3 2 P deposited in the shells, shell glands, ovaries, and intestinal smooth muscle should therefore serve as an index of metabolic activity of these tissues in heat stressed and thyroid blocked hens.
* Arizona Agricultural Experiment Station technical paper No. 2571.
906
MATERIALS AND METHODS Sixty-nine 18-month-old White Leghorn hens known to be laying regularly were selected at random from a flock at the Poultry Research Station of the University of Arizona. The birds were divided into three groups, nineteen hens (group 1) were heat stressed in an environmental chamber at 36°-40° with a relative humidity between 50% and 80%. Twenty hens (group 2) were fed methimazole, (l-methyl-2-mercaptoimidazole) to block thyroxine synthesis (Tanabe et al., 1965), and 20 hens (group 3) served as controls. Groups 2 and 3 were kept at the
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on May 28, 2015
ABSTRACT The ability of the reproductive tract of the White Leghorn hen under heat stress or methimazole thyroid blockage to assimilate 32P was examined. Both treatments increase 32 the uptake of P by the shell gland and egg shell but only heat stress increases 32P uptake by the ovaries and reduces shell thickness. The greater shell thinness under heat stress is related to an increased deposition of phosphate in the shell. Gilbert (1967) has reported an increased serum calcium, due to calcification inhibitions by phosphate, leading to an increased gonadotropin secretion. Thyroid blockage appears to stimulate TSH and gonadotropin LH secretion but in the absence of phosphate inhibition of calcification shell thickness is not affected. It is also speculated that hypothyroidism does not stimulate FSH secretion and therefore ovarian tissue is not activated by this condition.
907
TISSUE ACTIVITY IN H E N S
Twenty-five mm. 2 sections of uterus and duodenum were removed from each bird and placed on individual planchets. Ovaries from each bird were removed with follicles intact and their volume was determined. The ovaries were then macerated in a Waring Blender and three drops of the homogenate placed on each planchet, dried for 24 h. at 38° C. and counted on a Nuclear-Chicago Geiger counter and scaler. Egg shells were dipped in boiling water to remove the membranes, dried at 38° C. for 24 h., weighed, pulverized and portions of each were placed on dried planchets and their radioactivity was counted and recorded in c.p.m./mg. The remaining five birds in each of the three groups were sacrificed by cervical fracture and their pituitaries removed for TSH assay. The Lamberg (1953) assay technique as modified by Radke and Chiasson (1974) was used to determine relative TSH content which is reported in counts per minute (c.p.m.). Three-day-old chicks were injected with the equivalent of one half a pituitary from the experimental hens macerated in avian saline 6 h. prior to sacrifice. The 3 2 P was injected two h. prior to sacrifice. Controls received only avian saline for the first injection. Chick thyroids were removed, dried at 38° C. for 24 h., and their radioactivity was counted. Student's t-test was applied to determine the significance of difference between all sample means. A probability of 0.05 was selected as the acceptable level of significance. The standard error of the means (SEM) was calculated for each set of data (Steel and Torrie, 1960).
RESULTS The results of our experiments are presented in Tables 1, 2 and 3. Heat stressed birds laid eggs with significantly thinner shells than
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on May 28, 2015
University of Arizona Poultry Farm in individual cages and group 1 animals were placed in individual cages in an environmental chamber. All birds were subjected to a 16 h. light, 8 h. dark cycle. Methimazole was added as 0.1% by weight to a University of Arizona poultry laying mash in one experiment and as 0.05% by weight in a second experiment. Food and water were available ad libitum to all birds. All experiments were performed between late October and early April when the birds were assumed to be in euthyroid and optimal laying conditions. Birds to be placed in the environmental chamber were acclimated from two to four days. This period was also used to verify the laying condition of the birds. At the end of the fourth day, birds were subjected to heat stress. Birds on the methimazole diet were started on their feed while the birds of group 1 were being acclimated. The dry weight of the shell without membranes in mg. divided by the product of the greatest width and the greatest length in mm. is designated the "shell index" (Ratcliffe, 1967) and this value was measured and recorded daily. The shell index of birds to be subjected to heat stress was included with that of control group eggs until the second day of acclimation. Shell indices for these birds were calculated again after five days of heat stress. Birds were considered to be heat stressed when shell indices were significantly lower than those of the control group. This occurred within 10 days of the first exposure to constant high temperatures. Following ten days of heat stress, five birds from each of the three groups were injected with 45 |xCi. of 32 P in physiological saline solution. Birds from group 1 were injected sc. first. Birds from group 3 were injected 30 minutes after group 1 and birds from group 2 were injected 30 minutes after group 3. Eggs were collected and birds sacrificed by cervical fracture 8 h. after sc. injections.
908
M. S. PREMOVICH AND R. B. CHIASSON
TABLE 1.—Shell index of eggs (± SEM) laid by experimental hens 1 Group 1 (Heat stressed) 1.504 ± 0.073 (12)* Group 2 (0.05% Methimazole fed) 2.025 ± 0.071 (12) Group 3 (Controls) 2.254 ± 0.057 (12) *( ) = Number of eggs samples. **Egg shell radioactivity for these eggs is presented in Table 2.
2** 1.304 ± 0.102 (6) 2.056 ± 0.093 (5) 2.258 ± 0.085 (7)
TABLE 2.—Radioactive phosphorus uptake by several tissues of the hen Egg shell* Shell gland Duodenal wall Ovaries C.P.M./mg. C.P.M. C.P.M. C.P.M./ovary C.P.M./ml. 44.81 ± 4.37 (6) 1917 ± 162 (19) 979 ± 26 (19) 6612 ± 256 82/7 33.98 ±6.77(5) 1135 ± 147(5)
607 ± 48 (5)
No eggs laid less than 400 (5) 801 ± 103 (5) 10.92+1.27(7) 828 ± 49 (20) 828 ± 73 (20) these eggs are presented in Table 1.
TABLE 3.—TSH content of pituitaries Pituitary source Group 1 (Heat stressed) Group 2 (.05% methimazole) Group 2 (.^methimazole) Group 3 (control) Sham injected
C.P.M./thyroid (± SEM) 1703 ± 315 1786 ± 377 2437 ± 172 1470 ± 244 1170 ± 142
either methimazole fed or control animals (Table 1). The amount of phosphate deposition in the shells of each group may be estimated on the basis of 32 P transferred from the hen to the shell. The shells of birds exposed to high temperatures had approximately four times as much radioactivity as those of the control group (Table 2). The uptake of 32 P by uterine or shell gland tissues in birds subjected to heat stress differed significantly from that of the control group (Table 2). Uptake of 32 P by uterine tissues of the 0.05% methimazole fed birds did not differ significantly from that of the control group (P < 0.05). The radioactivity of total ovarian tissue homogenates from each of the three groups
5258 ± 208
43.5
Not counted 5058 ± 303
45.1
were similar, but the volumes of the three groups ovaries were not equal and the radioactivity of the tissues is therefore not directly comparable. The entire ovary was used in each group since it was not possible to separate endocrine from nonendocrine tissues. The ovaries of hens exposed to high environmental temperatures had fewer large follicles and were therefore much smaller than the ovaries of the control and 0.05% methimazole fed birds. When c.p.m./ml. of ovaries are compared however, the tissue activity of the heat stressed hens is approximately twice as great as that of the other groups (Table 2). The TSH assay did not differ significantly between the various groups although sham injected chicks had the lowest average activity and chicks injected with pituitaries from methimazole fed birds had the greatest (Table 3). Duodenal tissue was of approximately the same magnitude of activity in the three groups, and therefore serves as a good reference of 32 P uptake that is neither stimulated nor depressed. DISCUSSION AND CONCLUSIONS Reproductive tissues of hypothyroid and
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on May 28, 2015
Group Heat stressed (1) Methimazole fed 0.05% (2) Methimazole fed 0.1% (2) Control (3) *Shell indices for
909
TISSUE ACTIVITY IN H E N S
The increased amount of phosphate deposition in the eggs of heat stressed hens may be a major contributing factor to the increased reproductive tissue activity. The role of the phosphate ion as an inhibitor of calcification has been discussed by Smith et al. (1954) and Simkiss (1964). Gilbert (1967) has suggested that an increase in the concentration of calcium in the hen, with a reduction in shell deposition, leads to an increase in secretion of gonadotropins. Taylor (1965) reported hens on a calcium deficient diet ceased egg laying and then resumed ovulation when given injections of FSH. A possible mechanism increasing blood calcium and reducing shell thickness in the heat stressed hen is suggested by earlier studies. Lindsley and Burger (1964) demonstrated a fall in p C 0 2 and a rise in blood pH as soon as body temperatures and respiratory rates increase during exposure to heat stress. Mongin (1968) also reported the increased discharge of C 0 2 during panting may result in respiratory alkalosis and thus decrease the availability of carbonate ion for shell formation. The increased deposition of 32 P in the egg shells of the hens that were heat stressed indicates the phosphate may be substituted for the carbonate ion. This substitution is
also manifest in thinner shells and a higher level of serum calcium (Simkiss, 1964). An explanation of the mechanism whereby hypothyroidism increases the activity of reproductive tissues is less easy to find. Tixier-Vidal et al. (1972) speculated that thyroxine in photostimulated male quail inhibits both gonadotropic beta and thyrotropic delta cells. Lehman's (1970) suggestion that the TSH used in her experiments to stimulate gonadal activity may have been contaminated with gonadotropic hormones, seems probable in view of the findings of Tixier-Vidal et al. (1972). It seems likely that the in vivo hypothyroid stimulation of TSH also stimulates gonadotropes. Our data supports this hypothesis with the exception that methimazole fed hens do not have stimulated ovaries. The levels of LH are known to be much lower in the hen than in the cockerel (Wilson and Sharp, 1975) and the secretory pattern of LH is also different in the two sexes. The lower level of LH in the hen is apparently unable to stimulate the ovary but is sufficient to activate the shell gland. If this conclusion is accepted, it must be assumed that FSH secretion is not stimulated by the hypothyroid condition in the hen. Earlier studies of gonadotropic secretion by Lehman (1970) and Tixier-Vidal et al. (1972) have not distinguished between LH and FSH. These interpretations could be substantiated with the application of more sensitive and more discreet assays for LH and FSH. The assay for LH has been developed (Follett et al., 1972) and that for FSH is anticipated (Scanes and Follett, 1972).
ACKNOWLEDGMENTS White Leghorn hens, assay chicks, and feed mixes were supplied through the generosity of Dr. B. L. Reid and the University of Arizona Poultry Farm.
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on May 28, 2015
heat stressed hens are considerably more active than those of control animals. Lehman (1970) also found a greater uptake of 32 P by the testes of hypothyroid cockerels and expressed surprise with these results in consideration of the "usual role of thyroxine in elevating metabolic rate." Our results indicate the ovary is unaffected by hypothyroidism although the shell gland uptake of 32 P is increased 37% and the egg shells of methimazole fed hens have three times the 32 P activity of those of control hens. The ovaries as well as accessory tissues are more active in heat stressed hens than in controls so heat stressed hens appear most like hypothyroid cockerels in this regard.
910
M.S.
PREMOVICH AND R. B. CHIASSON
REFERENCES
JUNE 6-9, 1976. FIFTY-NINTH CHEMICAL CONFERENCE, CHEMICAL INSTITUTE OF CANADA, LONDON, ONTARIO JULY 6-8, 1976. ANNUAL MEETING AND TECHNICAL SESSIONS, CANADIAN SOCIETY OF ANIMAL SCIENCE, ST. MARY'S UNIVERSITY, HALIFAX, NOVA SCOTIA
AUGUST 8-11, 1976. JOINT MEETING OF THE AMERICAN INSTITUTE OF NUTRITION, THE AMERICAN SOCIETY OF CLINICAL NUTRITION, AND THE NUTRITION SOCIETY OF CANADA, MICHIGAN STATE UNIVERSITY, EAST LANSING, MICHIGAN
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on May 28, 2015
Berg, L. R., and G. E. Bearse, 1951. Effect of iodinated casein and thiouracil on the performance of laying birds. Poultry Sci. 30: 21-28. Falconer, I. R., 1971. The Thyroid Glands. In: Physiology and Biochemistry of the Domestic Fowl. (D. J. Bell and B. M. Freeman, ed.). Vol. II, Chap. 17. Academic Press, New York. Follett, B. K., C. G. Scanes and F. J. Cunningham, 1972. A radioimmunoassay for avian luteinizing hormone. J. Endocr. 52: 359-378. Gabuten, A. R., and C. S. Shaffner, 1954. A study of the physiological mechanisms affecting specific gravity of chicken eggs. Poultry Sci. 33: 47-70. Gilbert, A. B., 1967. Formation of the egg in the domestic chicken. Chapt. 3. In: Advances in Reproductive Physiology. (A. McLaren ed.). Vol. 2, pp. 111-180. Logos Press, London. Hahn, D. W., T. Ishibashi and C. W. Turner, 1966. Alteration of thyroid hormone secretion rate in fowls changes from a cold to a warm environment. Poultry Sci. 45: 31-33 Heninger, R. W., W. S. Newcomer and R. H. Thayer, 1960. The effect of elevated ambient temperatures on the thyroxine secretion rate of chickens. Poultry Sci. 39: 1332-1337. Huston, T. M., 1962a. The influence of high environmental temperatures on thyroid size of domestic fowl. Poultry Sci. 41: 175-179. Huston, T. M., 1962b. Effects of high environmental temperature on thyroid secretion rate of domestic fowl. Poultry Sci. 41: 640-645. Lamberg, B. A., 1953. Radioactive phosphorus as indicator in a chick assay of thyrotropic hormone. Acta. Med. Scand. Suppl. 279, pp. 1-79. Lehman, G. C , 1970. The effects of hypo- and hyperthyroidism on testes and anterior pituitary glands of cockerels. Gen. Comp. Endocrinol. 14: 567-577. Lindsley, J. G., and R. E. Burger, 1964. Respiratory and cardiovascular responses in the hyperthermic domestic cock. Poultry Sci. 43: 291-305.
Mongin, P., 1968. Role of acid-base balance in the physiology of egg shell formation. Worlds Poultry Sci. J. 24: 200-230. Radke, W. J., and R. B. Chiasson, 1974. Thyroidstimulating hormone location in the chicken pars distalis. J. Endocr. 60: 187-188. Ratcliffe, D. A., 1967. Decrease in egg shell weight in certain birds of prey. Nature, 215: 208-210. Scanes, C. G., and B. K. Follett, 1972. Fractionation and assay of chicken pituitary hormones. Br. Poultry Sci. 13: 603-610. Simkiss, K., 1964. Phosphates as crystal poisons of calcification. Bio. Rev. 39: 487-505. Smith, A. H., C. M. Winget and J. R. Blackard, 1954. The transfer of phosphorus to the hen's egg, under controlled environment, as traced with radiophosphorus (P52). Poultry Sci. 33: 908-919. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics (with Special Reference to the Biological Sciences). McGraw Hill Book Company, Inc., New York, Toronto, London. Tanabe, Y., T. Kamiyama, D. Kubota and Y. Tamaki, 1965. Comparison of the effects of thiouracil, propylthiouracil and methimazole on 1131 metabolism by the chick thyroid and measurements of thyroxine secretion rates. Gen. Comp. Endocrinol. 5: 60-68. Taylor, T. G., 1965. Calcium-endocrine relationships in the laying hen. Proc. Nutr. Soc. 24: 49-54. Taylor, T. G., and B. R. Burmester, 1940. Effect of thyroidectomy on production, quality, and composition of chicken eggs. Poultry Sci. 19: 326331. Tixier-Vidal, A., A. ChandolaandF. Franquelin, 1972. "Cellules de thyroidectomie" et "cellules de castration" chez la Caille Japonaise, Coturnix coturnix japonica. Etude ultrastructurale et cytoenzymologique. Z. Zellforsch. 125: 506-531. Turner, C. W., M. R. Irwin and E. P. Reineke, 1945. Effect of thyroid hormone on egg production of White Leghorn hens. Poultry Sci. 24: 171-180. Wilson, S. C , and P. J. Sharp, 1975. Episodic release of luteinizing hormone in the domestic fowl. J. Endocr. 64: 77-86.
Department of Veterinary Science, University of Arizona, Tucson, Arizona 85721 (Received for publication July 22, 1975)
POULTRY SCIENCE 55: 906-910, 1976
T
HE possible role of thyroid hormone in egg production and shell deposition has been investigated by Taylor and Burmester (1940) who reported a decrease in egg production as well as a 9% decrease in shell thickness after thyroidectomy. Berg and Bearse (1951) decreased egg production with the addition of 0.1% thiouracil to the diet of laying hens and Gabuten and Shaffner (1954) reduced shell thickness with a similar diet. Turner et al. (1945) noted an over-all decrease in egg production which coincided with a reduction in thyroxine secretion rates and basal metabolic rates as laying hens grew older. The effects of high environmental temperatures on thyroxine secretion rates and basal metabolic rates are well known (Heninger et al., 1960; Huston 1962a, b; Hahn et al., 1966) and Falconer (1971) has reported a seasonal fluctuation in thyroid secretion rates from an October and November high to a low in April through August. The period of lowest thyroid hormone levels coincides with the period of reduced egg production and shell thickness.
A decrease in productivity, as reported by Taylor and Burmester (1940), seems to indicate hypoactivity of the reproductive organs and this may be correlated with suppressed thyroid activity in high environmental temperatures. Increased uptake of 3 2 P by an organ is generally considered to indicate an increased metabolic activity of the organ (Lamberg, 1953). Comparisons of the amount of 3 2 P deposited in the shells, shell glands, ovaries, and intestinal smooth muscle should therefore serve as an index of metabolic activity of these tissues in heat stressed and thyroid blocked hens.
* Arizona Agricultural Experiment Station technical paper No. 2571.
906
MATERIALS AND METHODS Sixty-nine 18-month-old White Leghorn hens known to be laying regularly were selected at random from a flock at the Poultry Research Station of the University of Arizona. The birds were divided into three groups, nineteen hens (group 1) were heat stressed in an environmental chamber at 36°-40° with a relative humidity between 50% and 80%. Twenty hens (group 2) were fed methimazole, (l-methyl-2-mercaptoimidazole) to block thyroxine synthesis (Tanabe et al., 1965), and 20 hens (group 3) served as controls. Groups 2 and 3 were kept at the
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on May 28, 2015
ABSTRACT The ability of the reproductive tract of the White Leghorn hen under heat stress or methimazole thyroid blockage to assimilate 32P was examined. Both treatments increase 32 the uptake of P by the shell gland and egg shell but only heat stress increases 32P uptake by the ovaries and reduces shell thickness. The greater shell thinness under heat stress is related to an increased deposition of phosphate in the shell. Gilbert (1967) has reported an increased serum calcium, due to calcification inhibitions by phosphate, leading to an increased gonadotropin secretion. Thyroid blockage appears to stimulate TSH and gonadotropin LH secretion but in the absence of phosphate inhibition of calcification shell thickness is not affected. It is also speculated that hypothyroidism does not stimulate FSH secretion and therefore ovarian tissue is not activated by this condition.
907
TISSUE ACTIVITY IN H E N S
Twenty-five mm. 2 sections of uterus and duodenum were removed from each bird and placed on individual planchets. Ovaries from each bird were removed with follicles intact and their volume was determined. The ovaries were then macerated in a Waring Blender and three drops of the homogenate placed on each planchet, dried for 24 h. at 38° C. and counted on a Nuclear-Chicago Geiger counter and scaler. Egg shells were dipped in boiling water to remove the membranes, dried at 38° C. for 24 h., weighed, pulverized and portions of each were placed on dried planchets and their radioactivity was counted and recorded in c.p.m./mg. The remaining five birds in each of the three groups were sacrificed by cervical fracture and their pituitaries removed for TSH assay. The Lamberg (1953) assay technique as modified by Radke and Chiasson (1974) was used to determine relative TSH content which is reported in counts per minute (c.p.m.). Three-day-old chicks were injected with the equivalent of one half a pituitary from the experimental hens macerated in avian saline 6 h. prior to sacrifice. The 3 2 P was injected two h. prior to sacrifice. Controls received only avian saline for the first injection. Chick thyroids were removed, dried at 38° C. for 24 h., and their radioactivity was counted. Student's t-test was applied to determine the significance of difference between all sample means. A probability of 0.05 was selected as the acceptable level of significance. The standard error of the means (SEM) was calculated for each set of data (Steel and Torrie, 1960).
RESULTS The results of our experiments are presented in Tables 1, 2 and 3. Heat stressed birds laid eggs with significantly thinner shells than
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on May 28, 2015
University of Arizona Poultry Farm in individual cages and group 1 animals were placed in individual cages in an environmental chamber. All birds were subjected to a 16 h. light, 8 h. dark cycle. Methimazole was added as 0.1% by weight to a University of Arizona poultry laying mash in one experiment and as 0.05% by weight in a second experiment. Food and water were available ad libitum to all birds. All experiments were performed between late October and early April when the birds were assumed to be in euthyroid and optimal laying conditions. Birds to be placed in the environmental chamber were acclimated from two to four days. This period was also used to verify the laying condition of the birds. At the end of the fourth day, birds were subjected to heat stress. Birds on the methimazole diet were started on their feed while the birds of group 1 were being acclimated. The dry weight of the shell without membranes in mg. divided by the product of the greatest width and the greatest length in mm. is designated the "shell index" (Ratcliffe, 1967) and this value was measured and recorded daily. The shell index of birds to be subjected to heat stress was included with that of control group eggs until the second day of acclimation. Shell indices for these birds were calculated again after five days of heat stress. Birds were considered to be heat stressed when shell indices were significantly lower than those of the control group. This occurred within 10 days of the first exposure to constant high temperatures. Following ten days of heat stress, five birds from each of the three groups were injected with 45 |xCi. of 32 P in physiological saline solution. Birds from group 1 were injected sc. first. Birds from group 3 were injected 30 minutes after group 1 and birds from group 2 were injected 30 minutes after group 3. Eggs were collected and birds sacrificed by cervical fracture 8 h. after sc. injections.
908
M. S. PREMOVICH AND R. B. CHIASSON
TABLE 1.—Shell index of eggs (± SEM) laid by experimental hens 1 Group 1 (Heat stressed) 1.504 ± 0.073 (12)* Group 2 (0.05% Methimazole fed) 2.025 ± 0.071 (12) Group 3 (Controls) 2.254 ± 0.057 (12) *( ) = Number of eggs samples. **Egg shell radioactivity for these eggs is presented in Table 2.
2** 1.304 ± 0.102 (6) 2.056 ± 0.093 (5) 2.258 ± 0.085 (7)
TABLE 2.—Radioactive phosphorus uptake by several tissues of the hen Egg shell* Shell gland Duodenal wall Ovaries C.P.M./mg. C.P.M. C.P.M. C.P.M./ovary C.P.M./ml. 44.81 ± 4.37 (6) 1917 ± 162 (19) 979 ± 26 (19) 6612 ± 256 82/7 33.98 ±6.77(5) 1135 ± 147(5)
607 ± 48 (5)
No eggs laid less than 400 (5) 801 ± 103 (5) 10.92+1.27(7) 828 ± 49 (20) 828 ± 73 (20) these eggs are presented in Table 1.
TABLE 3.—TSH content of pituitaries Pituitary source Group 1 (Heat stressed) Group 2 (.05% methimazole) Group 2 (.^methimazole) Group 3 (control) Sham injected
C.P.M./thyroid (± SEM) 1703 ± 315 1786 ± 377 2437 ± 172 1470 ± 244 1170 ± 142
either methimazole fed or control animals (Table 1). The amount of phosphate deposition in the shells of each group may be estimated on the basis of 32 P transferred from the hen to the shell. The shells of birds exposed to high temperatures had approximately four times as much radioactivity as those of the control group (Table 2). The uptake of 32 P by uterine or shell gland tissues in birds subjected to heat stress differed significantly from that of the control group (Table 2). Uptake of 32 P by uterine tissues of the 0.05% methimazole fed birds did not differ significantly from that of the control group (P < 0.05). The radioactivity of total ovarian tissue homogenates from each of the three groups
5258 ± 208
43.5
Not counted 5058 ± 303
45.1
were similar, but the volumes of the three groups ovaries were not equal and the radioactivity of the tissues is therefore not directly comparable. The entire ovary was used in each group since it was not possible to separate endocrine from nonendocrine tissues. The ovaries of hens exposed to high environmental temperatures had fewer large follicles and were therefore much smaller than the ovaries of the control and 0.05% methimazole fed birds. When c.p.m./ml. of ovaries are compared however, the tissue activity of the heat stressed hens is approximately twice as great as that of the other groups (Table 2). The TSH assay did not differ significantly between the various groups although sham injected chicks had the lowest average activity and chicks injected with pituitaries from methimazole fed birds had the greatest (Table 3). Duodenal tissue was of approximately the same magnitude of activity in the three groups, and therefore serves as a good reference of 32 P uptake that is neither stimulated nor depressed. DISCUSSION AND CONCLUSIONS Reproductive tissues of hypothyroid and
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on May 28, 2015
Group Heat stressed (1) Methimazole fed 0.05% (2) Methimazole fed 0.1% (2) Control (3) *Shell indices for
909
TISSUE ACTIVITY IN H E N S
The increased amount of phosphate deposition in the eggs of heat stressed hens may be a major contributing factor to the increased reproductive tissue activity. The role of the phosphate ion as an inhibitor of calcification has been discussed by Smith et al. (1954) and Simkiss (1964). Gilbert (1967) has suggested that an increase in the concentration of calcium in the hen, with a reduction in shell deposition, leads to an increase in secretion of gonadotropins. Taylor (1965) reported hens on a calcium deficient diet ceased egg laying and then resumed ovulation when given injections of FSH. A possible mechanism increasing blood calcium and reducing shell thickness in the heat stressed hen is suggested by earlier studies. Lindsley and Burger (1964) demonstrated a fall in p C 0 2 and a rise in blood pH as soon as body temperatures and respiratory rates increase during exposure to heat stress. Mongin (1968) also reported the increased discharge of C 0 2 during panting may result in respiratory alkalosis and thus decrease the availability of carbonate ion for shell formation. The increased deposition of 32 P in the egg shells of the hens that were heat stressed indicates the phosphate may be substituted for the carbonate ion. This substitution is
also manifest in thinner shells and a higher level of serum calcium (Simkiss, 1964). An explanation of the mechanism whereby hypothyroidism increases the activity of reproductive tissues is less easy to find. Tixier-Vidal et al. (1972) speculated that thyroxine in photostimulated male quail inhibits both gonadotropic beta and thyrotropic delta cells. Lehman's (1970) suggestion that the TSH used in her experiments to stimulate gonadal activity may have been contaminated with gonadotropic hormones, seems probable in view of the findings of Tixier-Vidal et al. (1972). It seems likely that the in vivo hypothyroid stimulation of TSH also stimulates gonadotropes. Our data supports this hypothesis with the exception that methimazole fed hens do not have stimulated ovaries. The levels of LH are known to be much lower in the hen than in the cockerel (Wilson and Sharp, 1975) and the secretory pattern of LH is also different in the two sexes. The lower level of LH in the hen is apparently unable to stimulate the ovary but is sufficient to activate the shell gland. If this conclusion is accepted, it must be assumed that FSH secretion is not stimulated by the hypothyroid condition in the hen. Earlier studies of gonadotropic secretion by Lehman (1970) and Tixier-Vidal et al. (1972) have not distinguished between LH and FSH. These interpretations could be substantiated with the application of more sensitive and more discreet assays for LH and FSH. The assay for LH has been developed (Follett et al., 1972) and that for FSH is anticipated (Scanes and Follett, 1972).
ACKNOWLEDGMENTS White Leghorn hens, assay chicks, and feed mixes were supplied through the generosity of Dr. B. L. Reid and the University of Arizona Poultry Farm.
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on May 28, 2015
heat stressed hens are considerably more active than those of control animals. Lehman (1970) also found a greater uptake of 32 P by the testes of hypothyroid cockerels and expressed surprise with these results in consideration of the "usual role of thyroxine in elevating metabolic rate." Our results indicate the ovary is unaffected by hypothyroidism although the shell gland uptake of 32 P is increased 37% and the egg shells of methimazole fed hens have three times the 32 P activity of those of control hens. The ovaries as well as accessory tissues are more active in heat stressed hens than in controls so heat stressed hens appear most like hypothyroid cockerels in this regard.
910
M.S.
PREMOVICH AND R. B. CHIASSON
REFERENCES
JUNE 6-9, 1976. FIFTY-NINTH CHEMICAL CONFERENCE, CHEMICAL INSTITUTE OF CANADA, LONDON, ONTARIO JULY 6-8, 1976. ANNUAL MEETING AND TECHNICAL SESSIONS, CANADIAN SOCIETY OF ANIMAL SCIENCE, ST. MARY'S UNIVERSITY, HALIFAX, NOVA SCOTIA
AUGUST 8-11, 1976. JOINT MEETING OF THE AMERICAN INSTITUTE OF NUTRITION, THE AMERICAN SOCIETY OF CLINICAL NUTRITION, AND THE NUTRITION SOCIETY OF CANADA, MICHIGAN STATE UNIVERSITY, EAST LANSING, MICHIGAN
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