Heat-stress response of broiler cockerels to manipulation of the gonadal steroids, testosterone and estradiol

Comp. Biochem. Physiol. Vol. 10611, No. 3, pp. 629~533, 1993

0305-0491/93 $6.00 + 0.00 © 1993 Pergamon Press Ltd

Printed in Great Britain

HEAT-STRESS RESPONSE OF BROILER COCKERELS TO MANIPULATION OF THE G O N A D A L STEROIDS, TESTOSTERONE A N D ESTRADIOL SHIYING WANG and F. W. EDENS* North Carolina State University, Department of Poultry Science, Box 7635, Raleigh, NC 27695-7635, U.S.A. (Tel. 919 515-2649; Fax 919 515-2625)

(Received 17 March 1993; accepted 30 April 1993) Al~traet--1. Estradiol supplementation resulted in heat-stress mortality in both intact and caponized cockerels accompanied by depressed plasma corticosterone. 2. Phenotype-seleetion for large comb and high plasma testosterone increased heat tolerance which was attributed to an increased plasma corticosterone. 3. The results suggested that the presence of testosterone had a positive influence on the heat tolerance of broiler cockerels.

INTRODUCTION

Heat-stress mortality is the source of significant revenue loss to the poultry industry in general and especially to the broiler industry. Poultry die in response to heat stress due to dysfunction in many physiological systems allowing cardiovascular collapse (Whittow et al., 1964), acid-base disturbances (Calder and Schmidt-Neilsen, 1966, 1968), adrenal cortical insufficiency (Edens, 1978), and renal failure (Phelps and Edens, 1984). Additionally, there is an apparent sexual dimorphism to heat stress since males tend to be more susceptible and die more quickly than females (Pease, 1947; Leitner et al., 1989). In a previous study, Phelps and Edens (1984) demonstrated that testosterone, injected daily, improved kidney function during acute heat stress but estradiol, given daily to both intact and caponized broilers, decreased kidney function and increased mortality during heat stress. This study was designed to address further the observation that testosterone improved heat tolerance in the broiler cockerel. MATERIALS AND METHODS

Animals Day-old broiler cockerels (Arbor Acres x Arbor Acres) were obtained from the North Carolina Agricultural Research Service poultry research farm at North Carolina State University. The birds were provided with water and feed on an ad libitum basis. They were maintained in thermoneutral environments with continuous lighting for 6 weeks. Thermoneutral environments were established at 35°C during week 1, 30°C during week 2, and 25°C from *To whom correspondence should be addressed. c ~ s ) JO6/~--L

week 3 through the remaining time of the experimental period.

Caponization Two-week-old broiler cockerels were caponized by a procedure similar to that described previously (Holladay and Edens, 1987). Briefly, bilateral incisions were made and both testes removed from 24-hr-fasted males. Skin was closed with stainless steel wound clips. The capons were then allowed to recover from the surgery for 2 weeks. At 5 weeks of age, the cockerels were assigned randomly to different treatments.

Treatments In Experiment 1 and Experiment 2, there were six different treatments (2 x 3) with 25 cockerels used for each treatment. Testosterone and/or estradiol in corn oil were administered intramuscularly (i.m.) on a daily base for 1 week at the level of 5 mg/kg body weight in Experiment 1 and 5 mg/kg of testosterone and 1 mg/kg of estradiol in Experiment 2. Intact and caponized birds, given corn oil vehicle, served as controls in each experiment. At 6 weeks of age the cockerels were subjected to acute heat exposure at 43°C for 90 min. Heat stress mortality was checked continuously in Experiment 1. In Experiment 2, blood samples were taken at 30-min intervals during the heat stress for measurement of plasma levels of corticosterone. In Experiment 3300 broiler cockerels at 3 weeks of age were phenotypically segregated into groups with large (LC) and small combs (SC). Blood samples were taken for determination of testosterone and corticosterone. The birds were further segregated by high (HT) or low testosterone (LT) in circulation. At 6 weeks of age, 50 HT and 50 LT birds were selected and exposed to acute heat stress at 43°C for 120 min.

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Blood samples were take at 0, 30, 60, 90, 120 min for plasma testosterone and corticosterone assays. Heat stress mortality was continuously monitored.

Plasma testosterone and corticosterone assay Plasma testosterone and corticosterone were determined with 125I-labeled commercial kits obtained from DPC (Diagostic Products Corp., Los Angeles, CA) and Ventrex Laboratories (Ventrex Laboratories, Inc., Portland, ME), respectively, by following the manufacturer's procedures.

Statistical analysts Data analysis was performed with analysis of variance (ANOVA) by general linear model (GLM) procedure of the statistical analysis system (SAS, 1986). Differences between means were determined by repeated t-test using probabilities generated by the least-squares means. RESULTS

Experiment 1 Table 1 shows the percent mortality in intact and caponized broiler cockerels given the corn oil vehicle (control), estradiol, or testosterone injections in response to acute heat stress at 43°C for 90 rain. Testosterone treatment either in intact or in caponized birds did not affect the heat stress survivability in comparison to the controls, whereas estradiol administration resulted in significant heat stress mortality. This was accompanied by a short survival time in the estradiol-treated birds during the heat exposure (data not shown). Although the estradiol treatment had a slightly higher mortality rate in caponized cockerels than in intact cockerels, the difference was not significant.

estradiol-treated intact and caponized cockerels were unable to respond to the acute heating episode by elevating plasma corticosterone. The testosteronesupplemented cockerels, both intact and caponized, responded to the heat stress by enhancing plasma corticosterone to a higher level than did their respective controls.

Experiment 3 Interrelationship among plasma concentrations of testosterone, corticosterone, and comb size in phenotype-selected LC and SC cockerels are indicated in Table 2. Broiler cockerels, within a population, were selected on the basis of comb size and plasma samples from those selected birds were analyzed for corticosterone and testosterone before they were subjected to an acute heating episode (43°C for 120 min). Comb size and plasma levels of testosterone were highly correlated (R 2= +0.95). Corticosterone levels were highly correlated also, with testosterone levels in these birds (R 2= +0.97). Following heat exposure, those birds with large combs and associated high levels of testosterone and corticosterone exhibited significantly lower mortality than those with small combs and low levels of testosterone and corticosterone. In addition, there was a divergent corticosterone response between the high- and low-testosteroneselected birds in response to the acute heating episode (Fig. 2). The low testosterone birds showed a peak in plasma corticosterone level at 30 min of heat stress which decreased during the remaining 90 min of the study. On the other hand, the plasma corticosterone in the high-testosterone-selected cockerels increased from 30 min through 120 min of the exposure. DISCUSSION

Experiment 2 In this experiment, there was no mortality observed due to treatment during the heat stress. However, plasma levels of corticosterone were affected by the treatments (Fig. 1). Caponization decreased the corticosterone levels in circulation, while testosterone supplementation induced corticosterone in the caponized birds to return to intact-control levels. The

These results demonstrate that estradiol treatment of both intact and caponized broiler cockerels resuited in high mortality in response to acute heat exposure, whereas no birds died due to testosterone administration (Experiment 1). These data are comparable to an earlier observation by Phelps and Edens (1984) showing that estradiol in cockerels, regardless

Table I. Cumulativemortality(%) after90 minof heat exposureat 43°Cin both intactand caponizedbroiler cockerels supplementedwith testosterone,estradiol, or corn oil vehicle(ExperimentI) Capon Intact Time (rain) Control Estradiol Testosterone Control Estradiol Testosterone 0 0 0 0 0 0 0 30 0 0b 0 0 16 (4)''B 0 60 0 68 (17) *'B 0 0 52 (13) ''B 0 90

0

92 (23) ~'s

0

0

84 (21 ).,s

0

N ffi25 for each treatment. Numbersin parentheses indicate numberof cockerels that died duringthe heat exposure. "bMeanswith different lower case superscriptswithinestradioltreatmentsindicatingsignificantdifferences (P ~ 0.05).

BMeanswiththe upperease superscriptsare significantlydifferentfrom other treatmentmeanswithincapon or intact groupingwithinindividualtimes(P ~ 0.05),

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of the presence or absence of the testes, rendered the birds more susceptible to heat stress as indicated by cumulative mortality. It has been indicated that the ultimate cause of heat-induced mortality in the chicken could be related to cardiovascular failure (Whittow et aL, 1964) and adrenal corticosterone insufficiency as well (Edens and Siegel, 1975, 1976; Edens, 1978). There are reports which support the hypothesis that human males who had higher than normal levels of circulating estradiol were more at risk of having a heart attack (e.g. Sewdarsen et al., 1990) although many other studies showed no such role of estradiol in causing heart disease in men (e.g. Cauley et al., 1987). Nevertheless, this same scenario has not been demonstrated in the chicken, but steroid interaction with cardiovascular involvement in the mortality reported here cannot be ruled out at this time. Since the estradiol-related mortality was so great in Experiment 1, Experiment 2 was conducted to examine plasma ~orticosterone which determines resistance of animals to heat stress (Edens and Siegel, 1975, 1976, Edens, 1978). It was found that in Table 2. Levels of plasma testosterone and corticosterone in broiler cockerels with different comb phenotypes (Experiment 3)

Av. comb length (cm) Av. comb height (cm) Plasma testosterone (ng/ml)* Plasma corticosterone (ng/ml)" Heat-stress mortality (%)b

T

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30 60 90 Minutes of Exposure to Heat

120

Fig. 2.

Fig. 1.

Parameter

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Large comb group

Small comb group

A

3.19 1.24 3.72 4.72 2.00

2.34 0.64 0.90 2.88 52.0

* * ** ** **

'Plasma levels before acute heat stress. bMortality (N = 50) after exposure to 43°C for 120min. *P ~<0.05. **P ~ 0.01.

both intact and caponized cockerels, the plasma levels of corticosterone were elevated by testosterone treatment but depressed by estradiol treatment. This suggested that the estradiol-associated heat stress mortality of male chickens could be due to the depletion of corticosterone by administration of the female sex hormone. Based on the concept that increased comb growth and size are indicative of elevated concentrations of plasma testosterone in chicks (Breneman and Mason, 1951), high and low testosterone phenotypes were selected from an existing population. The chicks selected phenotypically by comb size were further segregated by plasma testosterone level. An unselected characteristic of these birds, high and low plasma corticosterone concentrations, was correlated with high and low plasma testosterone, respectively, in normal conditions as well as during heating episodes. When these birds were subjected to the acute heat stress, the mortality was negligible in the chicks selected for large comb size whereas over 50% of the small-comb birds died. Recently, in a simulated field trial with 4500 cockerels, segregation of dead birds after a heat stress episode revealed that large-comb birds were more resistant to heat stressors than were small-comb birds (Davis et aL, 1991). Wang (1992) reported that large-comb cockerels synthesized higher levels of beat-shock proteins (HSPs) than the small-comb cockerels, and the increased expression of HSPs was stimulated by testosterone. The HSPs have been demonstrated to play a crucial role in development of resistance of cells to heat exposures (for reviews, see Li and Laszlo, 1985; Lindquist, 1986; Hahn and Li, 1990).

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StaYING W A N G and F. W. EDENS

The findings of this study provide more evidence to support the concept that it is high plasma testosterone with an associated high plasma corticosterone, that creates a condition in which cockerels are resistant to high environmental temperature. These responses were very reminiscent to those of chickens selected for high and low adrenal responsiveness to ACTH and subjected to acute heat stress (Edens and Siegel, 1975). There is little information available about the relationship between plasma testosterone and corticosterone in non-heat-stressed or heat-stressed domestic fowl. In studies of behavior and reproductive physiology of male birds with free-running rhythms, corticosterone was found to depress plasma testosterone levels (for review, see Edens, 1983), which is similar to that reported among nonmammalian and non-avian vertebrates (Moore and Zoeller, 1985; Wingfield and Silverin, 1986; Tokarz, 1987). In adult male geese, plasma levels of corticosterone decreased significantly as mating activity and androgens increased (Akesson and Raveling, 1981). This reverse relationship between the adrenal corticosterone and gonadal testosterone levels was also observed in handling-stressed male reptiles (Moore et al., 1991). It was believed that the stressinduced elevated cortieosterone depressed testosterone secretion in amphibians and reptiles (for reviews, see Grcenberg and Wingtield, 1987; Moore and Deviche, 1988; Wingfield, 1988). However, the reverse relationship does not always exist between the adrenal glucocorticoid hormones and the gonadal hormones. For example, in Bufo marinus corticosterone stimulated plasma testosterone secretion (Orchinik et al., 1988), but corticosterone was not affected by administration of gonadal steroid in castrated ducks (Deviche et al., 1980). In this study the positive correlation of plasma testosterone with corticosterone was noted through the phenotypic selection as well as during the heat stress episode. These divergent findings could be due to differences in species, type of stressors applied, and physiological conditions of animals. In summary, the presence of testosterone appeared to have a positive influence on the heat tolerance of broiler cockerels. Increased concentrations of testosterone associated with phenotypic selection, or through injection, was also associated with increased concentrations of corticosterone. Estradiol in either the capon or intact cockerel was associated with decreased tolerance to heat stress. Decreased heat tolerance in the estradiol-treated cockerel was associated with decreased ability to respond to heat stress via increased corticosterone levels. REFERENCES

Akesson T. R. and Raveling D. R. (1981) Endocrine and body weight changes of nesting and non-nesting Canadian geese. Biol. Reprod. 25, 792-804.

Breneman W. R. and Mason R. C. (1951) Androgen influenceon pituitary-gonad inter-relationship. Endocrinology 48, 752-762. Calder W. A. and Sehmidt-Neilsen K. (1966) Evaporative cooling and respiratory alkalosis in the pigeon. Proc. Soc. exp. Biol. Med. 55, 750-758. Calder W. A. and Schmidt-Neilsen K. (1968) Panting and blood carbon dioxide in birds. Am. J. Physiol. 215, 477-482. Cauley J. A., Gutai J. P., Kuller L. H. and Dai W. S. (1987) Uses of sex steroid hormone levels in predicting coronary artery disease in men. Am. J. Cardiol. 60, 771-777. Davis G. S., Edens F. W. and Parkhurst C. R. (1991) Computer-aided heat acclimation in broiler cockerels. Poult. Sci. 70, 302-306. Deviche P., Balthazart J. Heyns W. and Hendrick J.-C. (1980) Endocrine effects of castration followed by androgen replacement and ACTH injections in the male domestic duck (Anas platyrhynchos). Gen. comp. Endocr. 41, 53-61. Edens F. W. (1978) Adrenal cortical insufficiencyin young chickens exposed to a high ambient temperature. Poult. Sci. 57, 1746-1750. Edens F. W. (1983) Effect of environmental stressors on male reproduction. Poult. Sci. 62, 1676--1689. Edens F. W. and Siegel H. S. (1975) Adrenal response in high and low response line of chickens during acute heat stress. Gen. comp. Endocr. 25, 64-73. Edens F. W. and Siegel H. S. (1976) Modification of corticosterone and glucose responses by sympatholytic agents in young chickens during acute heat exposure. Poult. Sci. 55, 1704--1712. Greenberg N. and Wingfield J. C. (1987) Stress and reproduction: reciprocal relationships. In Hormones and Reproduction in Fishes, Amphibians and Reptiles (Edited by Morris D. O. and Jones R. E.), pp. 461-505. Plenum Press, New York. Hahn G. M. and Li G. C. (1990) Thermotolerance, thermoresistance and thermosensitization. In Stress Proteins in Biology and Medicine (Edited by Morimoto R. I., Tissieres A. and Geogopoulos C.), pp. 79-100. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Holladay S. D. and Edens F. W. (1987) Effect of caponization on central nervous system monoamines in the Japanese quail. Comp. Biochem. Physiol. 86A, 465-468. Leitner G., Heller E. D. and Friedman A. (1989) Sex-related differences in immune response and survival rate of broiler chickens. Vet. Immunol. Immunopathol. 21, 249-260. Li G. C. and Laszlo A. (1985) Thermotolerance in mammalian cells: a possible role from heat shock proteins. In Changes in Eukaryotic Gene Expression in Response to Environmental Stress (Edited by Atkinson B. G. and Walden D. B.), pp. 227-254. Academic Press, Orlando, FL. Lindquist S. (1986) The heat-shock responses. A. Rev. Biochem. 55, 1151-1191. Moore M. C. and Deviche P. (1988) Neuroendocrine pocessing of environmental information in amphibians. In Processing of Environmental Information in Vertebrates (Edited by Stetson M. H.), pp. 19-45. Springer, New York. Moore M. C. and Zoeller R. T. (1985) Stress-induced inhibition of reproduction: evidence of suppressed secretion of LH-RH in an amphibian. Gen. comp. Endocr. 60, 225-258. Moore M. C., Thompson C. W. and Marler C. A. (1991) Reciprocal changes in corticosterone and testosterone levels following acute and chronic handling stress in the tree lizard, Urosaurus ornatus. Gen. comp. Endocr. 81, 217-226.

Gonadal steroids and heat-stress response Orchinik M., Licht P. and Crews D. (1988) Plasma steroid concentrations change in response to sexual behavior in Bufo marinus. Horm. Behav. 22, 338-350. Pease M. (1947) How long do poultry breeding stock live? J. Ministry Agric. (G.B.) 54, 263-2268. Phelps J. L. and Edens F. W. (1984) Effect of sex steroid supplementation on heat stress responses of capons and intact broiler cockerels. Poult. Sci. 63, (Suppl. 1) 30 (Abstract). SAS. (1986) A User's Guide to Statistical Analysis System. SAS Institute Inc., Cary, NC 27511. Sewdarsen M., Vythilingum S., Jialal I., Desai R. K. and Beeker P. (1990) Abnormalities in sex hormones are a risk factor for premature manifestation of coronary artery disease in South African Indian men. Atherosclerosis 83, 111-117. Tokarz R. R. (1987) Effects of corticosterone treatment on

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male aggressive behavior in a lizard (Anolis sagrei). Horm. Behav. 21, 358-370. Wang S. (1992) Steroidal modulation of gene expression for heat-shock proteins in domestic chickens. Ph.D. Dissertation, The Graduate School, North Carolina State University, Raleigh, NC. Whittow G. C., Sturkie P. D. and Stein G. (1964) Cardiovascular changes associated with thermal polypnea in the chicken. Am. J. Physiol. 207, 1349-1353. Wingfield J. C. and Silverin B. (1986) Effects of corticosterone on territorial behavior of free-living male song sparrows Melospiza melodia. Horm. Behav. 20, 405-417. Wingheld J. C. (1988) Changes in reproductive functions of free-living birds in direct response to environmental perturbations. In Processing of Environmental Information in Vertebrates (Edited by Stetson M. H.), pp. 121-148. Springer, New York.