Adrenocortical responsiveness in southern elephant seal mothers and pups during lactation and the effect of scientific handling

We examined the cortisol responses to chemical and physical restraint stress in southern elephant seal Mirounga leonina females and their pups at three stages during lactation. In anaesthetised females the serum cortisol levels changed moderately during the 45-min sampling period following restraint, with average peaks at 23 min after anaesthetic administration. Overall, cortisol was relatively low 2 days postpartum and increased throughout lactation. In physically restrained pups serum cortisol increased rapidly after capture; the response was milder at age 2 days than at 11 days and 21 days. Levels were higher in female pups than in males. In order to test whether cortisol levels and/or responses became chronically (i.e. days to weeks) altered due to restraint, we compared the cortisol response at a late stage of lactation between three groups of mother-pup pairs previously given different levels of chemical (mothers) or physical (pups) restraint stress: control (not handled previously), moderate treatment (previously handled twice), and high treatment (previously handled 3-4 times). Pups of the three treatment groups showed similar adrenocortical responses suggesting no chronic effect of repeated physical restraint, despite the clear acute effects. Mothers of the control and moderate treatment groups showed similar cortisol responses; however, mothers of the high treatment group showed significantly attenuated responses. This indicated that elephant seals tolerated moderate degrees of handling disturbance; however, repeated (3-4) chemical immobilisations in lactating females may reduce their adrenocortical responsiveness for a period of days or weeks.     

Environmental and seasonal adaptations of the adrenocortical and gonadal responses to capture stress in two populations of the male garter snake, Thamnophis sirtalis

Stress and reproduction are generally thought to work in opposition to one another. This is often manifested as reciprocal relationships between glucocorticoid stress hormones and sex steroid hormones. However, seasonal differences in how animals respond to stressors have been described in extreme environments. We tested the hypothesis that garter snakes, Thamnophis sirtalis, with limited reproductive opportunities will suppress their hormonal stress response during the breeding season relative to conspecifics with an extended breeding season. The red-sided garter snake, T.s. parietalis, of Manitoba, Canada, has a brief breeding season during which males displayed no change in either plasma levels of testosterone or corticosterone, which were both elevated above basal levels, in response to capture stress. During the summer, capture stress resulted in increased plasma corticosterone and decreased testosterone. During the fall, when mating can also occur, males exhibited a significant decrease in testosterone but no increase in corticosterone in response to capture stress. The red-spotted garter snake, T.s. concinnus, of western Oregon, has an extended breeding season during which males displayed a stress response of increased plasma corticosterone and decreased testosterone levels. The corticosterone response to capture stress was similar during the spring, summer, and fall. In contrast, the testosterone response was suppressed during the summer and fall when gametogenesis was occurring. These data suggest that male garter snakes, in both populations, seasonally adapt their stress response but for different reasons and by potentially different mechanisms. J. Exp. Zool. 289:99-108, 2001.     

Seasonal changes in plasma testosterone and glucocorticosteroids in free-living male yellow-pine chipmunks and the response to capture and handling

We measured plasma levels of testosterone, corticosterone, and cortisol in free-living male yellow-pine chipmunks to demonstrate the patterns of seasonal variation and to assess the effects of capture and handling on hormone levels. We achieved the latter by modifying our standard trapping technique (blood samples collected within 1–3 h of capture) to obtain blood samples that allowed measurement of hormone levels within 3 min of capture (basal) and again 30 min later. By alternating the modified and standard trapping techniques over 7 months of the active season we demonstrated that seasonal patterns of variation in steroid hormone levels can be accurately described with the simpler, standard trapping technique. Basal and 30-min post-capture testosterone levels were high during mating and dropped to a persistently low level thereafter. Conversely, both cortisol and corticosterone were at their seasonal low during mating and climbed to peak levels in June following reproduction. Plasma glucocorticosteroid levels increased during the 30 min after capture and handling at all times of the active season, and these elevated levels were similar to the levels obtained by standard trapping. Testosterone levels during the mating period also increased in response to capture and handling. The contrasting patterns of seasonal variation in glucocorticosteroid and testosterone levels and the changes induced by capture and handling suggest that when testosterone concentration is high, adrenocortical activity is suppressed. Accepted: 17 February 2000     

Behavioural and physiological stress responses to handling in wild guanacos

Both ethology and physiology can provide insights about stress conditions in wild animals during handling. Lama guanicoe (guanaco) is the major large herbivore inhabiting arid zones in Argentina. Since 2005, the Payún Matrú Cooperative, a business established and run by native people, has captured and handled guanacos for shearing at Payunia Reserve, Mendoza Province, Argentina, to market the species’ fibre for clothing and other products. The aim of our study was to evaluate the behavioural and physiological stress responses to handling of wild guanacos during these shearing activities. We evaluated stress conditions in guanacos by monitoring the frequency of agonistic behaviour in the holding corrals, following capture, the rate of vocalisation and agonistic behaviour during handling, and by estimating cortisol levels in handled animals. Crowding increased agonistic behaviour; this appeared to be related to dominance conflicts between males in the corrals. Time of corralling increased rate of agonistic behaviour and vocalisations during handling. The rate of behavioural stress responses was negatively related to cortisol levels in males; which may be due to animal fatigue or because behavioural stress responses may help mitigate physiological stress responses. Incorporating our results into animal welfare practices could help reduce stress conditions in wild guanacos. Our results suggest that both behavioural and physiological responses are important measures of stress in handled animals, and that behavioural stress responses cannot be used as a simple surrogate for glucocorticoid levels.     

粪便激素水平反映不同出生时期雄性布氏田鼠的繁殖策略

布氏田鼠是我国内蒙古草原的主要害鼠之一,具有明显的季节繁殖特征,不同季节出生个体可能具有不同的繁殖策略,但尚缺乏内分泌证据支持。本研究采用标志重捕法,连续监测大型自然围栏中不同年龄雄性布氏田鼠的繁殖状态和应激水平的季节变化,分析各年龄组雄鼠的繁殖发育策略。结果表明,越冬雄鼠可保持较高的睾丸下降率和睾酮水平至8月初;而部分5月生雄鼠的繁殖期睾酮水平较接近越冬鼠,但8月初已降至年内最低水平,而6月及以后出生雄鼠睾酮始终处于较低水平;这说明越冬鼠的繁殖状态可贯穿繁殖期始终,只有部分5月生雄鼠可能参与当年繁殖,而6月及以后出生雄鼠则不能在当年繁殖。越冬鼠皮质醇水平高于当年鼠,繁殖期高于非繁殖期,这可能是越冬鼠由于繁殖需要而保持较高应激状态,从而造成繁殖盛期后死亡率升高。这些结果说明,不同时期出生的雄性布氏田鼠具有不同的出生后性腺发育模式和繁殖策略,反映出生存与繁殖之间的权衡。     

Arctic spring: hormone-behavior interactions in a severe environment

Arctic breeding birds arrive on their nesting grounds in spring when weather conditions may still be extreme (low temperature, snow). The brief Arctic summer requires that they begin breeding as early as possible to take advantage of the ephemeral abundance of food to feed young. Failure to adjust to the local phenology results in drastically reduced reproductive success. Hormone-behavior adaptations may have evolved that maximize survival and reproductive success in the Arctic. It has been shown that the interrelationship of testosterone and territorial aggression, as birds arrive on the Arctic breeding grounds, varies according to species and locality. In some, territoriality is extremely brief following which birds become apparently refractory to the effects of testosterone. Others are territorial throughout the breeding season, but the dependence of these behaviors upon activation by testosterone is lost. Extensive data also indicate that Arctic birds modulate the adrenocortical response to acute stress. Secretion of corticosterone in response to a standardized capture stress protocol, used to mimic acute stress as a function of local environmental conditions, varies with the stage in the breeding cycle. Arctic breeding birds modulate the sensitivity of the adrenocortical response to acute stress at both the population and individual levels. These modulations are thought to be adaptations to allow the onset of territorial behavior and breeding in the face of potentially stressful conditions. Behavioral and physiological responses to corticosterone treatment are also diminished. A combination of these two hormone-behavior interrelationships can form important components of the proximate mechanisms by which birds, and other vertebrates, breed successfully in a severe and often capricious environment.     

Reproduction in shark-attacked sea turtles is supported by stress-reduction mechanisms

Vertebrates exhibit varied behavioural and physiological tactics to promote reproductive success. We examined mechanisms that could enable female loggerhead turtles to undertake nesting activities and maintain seasonal reproduction despite recent shark injuries of varying severity. We proposed that endocrinal mechanisms that regulate both a turtle's stress response and reproductive ability are modified to promote successful and continued reproduction. Irrespective of the degree of injury, females did not exhibit increased levels of the stress hormone corticosterone, nor decreased levels of the reproductive steroid testosterone; hormone responses consistent with stress. When exposed to a capture stressor, females with shark injury did not exhibit any greater corticosterone response than controls. In addition, breeding females showed a reduced corticosterone stress response compared to non-breeding females. Reduced endocrinal responses following shark injury, and during breeding in general may, in part, enable females to maintain behavioural and physiological commitment to reproduction.     

An Enhanced Intestinal Absorption of Calcium in the Rat Directly Attributed to Dietary Casein

The whole-body burdens and concentrations of ZDDT (the sum of p,p,′-DDE and p,p′-DDT) and PCBs in the Weddell seal, Leptonychotes weddelli, caught near Syowa Station, Antarctica, were determined by detailed biometric measurements of their organs and tissues and analyses of ZDDT and PCBs in them. The concentration levels of ZDDT and PCBs in Weddell seals were much lower than those in various species of marine mammals from other oceans. The low levels may be attributable to the low concentration of these chemicals in the food of Weddell seals and in the sea water under antarctic fast ice. However, the concentration ratio of ZDDT between the food organisms of seals and sea water under antarctic fast ice was higher than those of other ocean ecosystems.     

Diurnal and annual variation of adrenocortical activity in the squirrel monkey

Diurnal changes in basal cortisol levels and stress reactivity were assessed in male and female squirrel monkeys. Blood samples were collected at four-hour intervals throughout the day and night in the mating and nonmating seasons. Basal cortisol levels in females were similar in both seasons, but males tended to have higher cortisol levels during the mating season, especially at night. For both sexes, cortisol secretion was highest between 0400 and 0800 preceding the onset of the diurnal activity period. Assessment of cortisol responses following brief handling and anesthesia indicated that stress responses were relatively stable across the year, but cortisol increments were slightly higher in the nonmating season. Cortisol levels post-stress were generally related to prior baseline values. Thus, a knowledge of biorhythmic changes in basal hormone levels was important for predicting hormone levels after acute stressors. Males also underwent marked seasonal variation in their basal testosterone levels, which markedly altered the nature of their testosterone responses 30-minutes poststress. © 1995 Wiley-Liss, Inc.     

Wood mice aggressiveness and flight response to human handling: Effect of individual and environmental factors

Numerous studies have examined human disturbance repercussions on wildlife, mainly focused on the effects on behaviour, reproductive success and population dynamics. However, few studies have addressed the behaviour of prey species during and after human capture and handling and how this may correlate to individual characteristics or variation in their physical environment they inhabit. We explored wood mouse’s fleeing and aggressive behaviours in response to captures by human in their natural habitat. Eighty‐seven wood mice were caught using Sherman live traps. For each trapped individual, aggressiveness was measured as the total number of bites inflicted upon the investigator during handling time. Afterwards, each mouse was released in a two‐metre radius partially covered vegetation area that allowed visual mouse tracking by the observer and flight behaviour was registered by individual one‐zero focal sampling technique during 2 min. Both aggressiveness and fleeing behaviour were analysed regarding individual (sex, reproductive status, age) and environmental factors (habitat and season). Males, adults and breeding individuals showed heightened aggression levels. Higher aggressiveness levels were found in wood mice occupying scrubland and during summer and autumn. The flight response was exclusively explained by reproductive status, whereby breeding individuals spent more time on fast escape than nonbreeding ones. These results indicate that both individual and environmental factors seem to influence defensive behaviours in the wood mouse during and after being captured by a human. Since human disturbance shares many aspects with the predation risk, behavioural responses found to captures may likely be influenced by previous experience of individuals with predators as well as to seasonal and habitat features conditioning predators’ densities but also protection against them.     

Variation in probability of first reproduction of Weddell seals

1. For many species, when to begin reproduction is an important life-history decision that varies by individual and can have substantial implications for lifetime reproductive success and fitness. 2. We estimated age-specific probabilities of first-time breeding and modelled variation in these rates to determine age at first reproduction and understand why it varies in a population of Weddell seals in Erebus Bay, Antarctica. We used multistate mark-recapture modelling methods and encounter histories of 4965 known-age female seals to test predictions about age-related variation in probability of first reproduction and the effects of annual variation, cohort and population density. 3. Mean age at first reproduction in this southerly located study population (7.62 years of age, SD=1.71) was greater than age at first reproduction for a Weddell seal population at a more northerly and typical latitude for breeding Weddell seals (mean=4-5 years of age). This difference suggests that age at first reproduction may be influenced by whether a population inhabits the core or periphery of its range. 4. Age at first reproduction varied from 4 to 14 years, but there was no age by which all seals recruited to the breeding population, suggesting that individual heterogeneity exists among females in this population. 5. In the best model, the probability of breeding for the first time varied by age and year, and the amount of annual variation varied with age (average variance ratio for age-specific rates=4.3%). 6. Our results affirmed the predictions of life-history theory that age at first reproduction in long-lived mammals will be sensitive to environmental variation. In terms of life-history evolution, this variability suggests that Weddell seals display flexibility in age at first reproduction in order to maximize reproductive output under varying environmental conditions. Future analyses will attempt to test predictions regarding relationships between environmental covariates and annual variation in age at first reproduction and evaluate the relationship between age at first reproduction and lifetime reproductive success.     

Coping with intense reproductive aggression in male arctic ground squirrels: the stress axis and its signature tell divergent stories

We tested the adaptive stress hypothesis that male arctic ground squirrels (Urocitellus parryii) exhibit a stress response over the course of the breeding season that is characterized by increasing free cortisol concentrations, increasing mobilization of stored energy, and decreasing physical condition. We assessed the functioning of the hypothalamic-pituitary-adrenal axis by measuring cortisol levels in response to the stress of capture and in response to a hormone challenge protocol (dexamethasone suppression and adrenocorticotropic hormone stimulation). We measured blood glucose levels, free fatty acids, white blood cells, and hematocrit to assess the downstream physiological responses to cortisol. Immediately after spring emergence, male arctic ground squirrels had ample free abdominal fat and few signs of wounding. By the end of the breeding season 3 wk later, visible fat reserves were almost entirely gone, and most males had extensive wounds. Total plasma cortisol concentrations increased over this period, but so did corticosteroid-binding capacity, resulting in no change in the free cortisol response to capture. We found no significant changes in how the animals responded to our hormone challenges, contrary to our prediction that the stress axis should increase free cortisol production. Even though we found no change in the functioning of the stress axis, all of the downstream measures suggested that male arctic ground squirrels are chronically exposed to high cortisol concentrations. Over the breeding season, blood glucose increased, fat stores and circulating free fatty acids were depleted, and both hematocrit levels and white blood cell counts decreased significantly. Our data suggest that a more complex relationship between the stress axis and downstream measures of stress exists than that proposed by the adaptive stress hypothesis. We propose several nonexclusive, testable mechanisms that could explain our observations.     

Ignoring the challenge? Male black redstarts (Phoenicurus ochruros) do not increase testosterone levels during territorial conflicts but they do so in response to gonadotropin-releasing hormone

Competition elevates plasma testosterone in a wide variety of vertebrates, including humans. The ‘challenge hypothesis’ proposes that seasonal peaks in testosterone during breeding are caused by social challenges from other males. However, during experimentally induced male–male conflicts, testosterone increases only in a minority of songbird species tested so far. Why is this so? Comparative evidence suggests that species with a short breeding season may not elevate testosterone levels during territory defence. These species may even be limited in their physiological capability to increase testosterone levels, which can be tested by injecting birds with gonadotropin-releasing hormone (GnRH). We studied two populations of black redstarts that differ in breeding altitude, morphology and the length of their breeding season. Unexpectedly, males of neither population increased testosterone in response to a simulated territorial intrusion, but injections with GnRH resulted in a major elevation of testosterone. Thus, black redstarts would have been capable of mounting a testosterone response during the male–male challenge. Our data show, for the first time, that the absence of an androgen response to male–male challenges is not owing to physiological limitations to increase testosterone. Furthermore, in contrast to comparative evidence between species, populations of black redstarts with a long breeding season do not show the expected elevation in testosterone during male–male challenges.     

Territorial aggression does not feed back on testosterone in a multiple-brooded songbird species with breeding and non-breeding season territoriality,the European stonechat

Testosterone mediates reproductive behaviours in male vertebrates. For example, breeding season territoriality depends on testosterone in many species of birds and in some, territorial interactions feed back on testosterone concentrations. However, the degree to which territorial behaviour and testosterone are associated differs even between species with seemingly similar life histories, especially between species that also defend territories outside the breeding season. Here, we investigate the link between territorial behaviour and testosterone in European stonechats. Previous studies found that territorial aggression in stonechats depends on testosterone in a breeding, but not in a non-breeding context. We investigated whether stonechats show a rise in testosterone during simulated territorial intrusions (STI) during the breeding season. Post-capture testosterone concentrations of males caught after an STI were not higher than those of males caught in a control situation regardless of breeding stage. However, most of the males would have been able to mount a testosterone response because the same individuals that did not increase testosterone during the STI showed a substantial increase in testosterone after injections of gonadotropin-releasing hormone (GnRH). GnRH-induced and post-capture testosterone concentrations were positively correlated and both decreased with successive breeding stages. Further, territory owners with a short latency to attack the decoy expressed higher post-capture testosterone concentrations than males with a longer latency to attack the decoy. Thus, there is no evidence for behavioural feedback on testosterone concentrations during male-male interactions in stonechats. In combination with previous studies our data suggest that testosterone functions as an on/off switch of high intensity territorial aggression during the breeding season in stonechats. The among-species variation in the androgen control of territorial behaviour may be only partly a result of environmental differences. Instead, potential differences in how territoriality evolved in different species may have influenced whether and how a reproductive hormone such as testosterone was co-opted into the mechanistic control of territorial behaviour.     

The implications of stress on male mating behavior and success in a sexually dimorphic polygynous mammal, the grey seal

Studies on primates and other taxa have shown that the physiological response of an individual to stress reflects their social status. We combined behavioral observations with measures of stress to test the hypothesis that stress is an important physiological determinant of mating behavior and success in the male grey seal. Known-age males (N=19) were studied during the breeding seasons of 2004 and 2005 at Sable Island, Canada. The stressor was a capture and restraint period of 35 min and serial samples of cortisol and testosterone were taken as measures of stress. The mean baseline concentrations of cortisol and testosterone were 9.7+/-0.5 ug/dl and 6.2+/-0.6 ng/mL, respectively. The baseline cortisol concentration was negatively correlated with the duration of time a male spent at a site (r=-0.507, P=0.027), which was a strong correlate of mating success (r=0.659, P=0.002). All males experienced an increase in the concentration of cortisol during the restraint period (79.1+/-8.4%; CV=46.1%). The percentage rise in cortisol during restraint was correlated with the mean duration of time spent at a site (r=0.544, P=0.016) and thus success. The concentration of testosterone also increased during the restraint period (32.8+/-9.7%). This might be an adaptive response to maintaining the ability to reproduce while under stress. Our study indicates that stress is an important determinant of success in male grey seals. More successful males might exhibit an adaptive response to stress by maintaining low concentrations of cortisol during breeding.     

Maternal buffering of stress response in free‐ranging Pacific harbor seal pups in Alaska

We examined the effects of maternal buffering in free‐ranging Pacific harbor seal pups during capture and handling research procedures. We predicted that pups held with their mother would benefit from social buffering and exhibit lower cortisol concentrations resulting from capture and handling than dependent pups caught without their mothers and weaned pups. We expected that pups captured with their mother that experienced a short separation would exhibit increased stress‐induced vocal behavior and activity level compared to dependent weaned pups caught alone. The results showed that the presence of the mother significantly buffered the stress response, as measured by reduced serum cortisol concentrations, in pups captured with their mothers as compared to dependent pups captured alone. Cortisol concentrations of mothers with pups initially were higher than nonlactating females, then diminished. Pups showed a significantly higher rate of vocalization soon after maternal separation compared to single pups separated for a longer period of time. Newly separated pups, especially males, showed a high level of activity compared to the other pups. The results provide unique quantitative evidence of the physiology underlying the maternal‐pup bond in a marine mammal, and the role that maternal buffering may play on the stress response of the offspring.     

Influence of age and individual identity in the use of breeding colony habitat by male Weddell seals in Erebus Bay,Antarctica

Male–male contest behavior can contribute to spatial distributions of male pinnipeds during breeding seasons. To maximize breeding opportunities, the most competitive males would be expected to be surrounded by the highest numbers of reproductive-age females. As information regarding fine-scale spatial ecology of Weddell seals is lacking, we performed an exploratory study using kernel density analyses to evaluate age-specific habitat use of male Weddell seals in Erebus Bay, Antarctica. Additionally, we investigated the relationship between age and number of surrounding reproductive-age females using a competing set of regression models in a Bayesian framework that considered different functional forms of age while incorporating individual heterogeneity. As male adult Weddell seals aged, to at least 20 years, they were more likely to be found in areas associated with the greatest densities of reproductive-age females, but individual heterogeneity also influenced the number of reproductive-age female neighbors. The youngest males tended to haul out in offshore areas associated with better hunting, and older males tended to settle in more nearshore areas associated with more pup production. Our findings from this preliminary investigation indicate that male Weddell seal spatial behavior during the breeding season varies with age and individual and might be related to reproductive activity.     

Response to capture stress involves multiple corticosteroids and is associated with serum thyroid hormone concentrations in Guadalupe fur seals (Arctocephalus philippii townsendi)

Guadalupe fur seals are a threatened species with few breeding locations, which potentially makes them sensitive to environmental or anthropogenic stressors. We present the first study to quantify adrenal and thyroid function in this species in an effort to measure their stress response to capture. We analyzed a suite of corticosteroid hormones released over time during capture in both adult females (n = 10) and weanling pups (n = 26) during March 2016. Multiple corticosteroids were released during capture, and aldosterone was associated with the response to stress in adults only. These results suggest the regulation of aldosterone secretion in association with the HPA axis in otariids as reported in other marine mammals. Individuals varied markedly in the magnitude of their endocrine response to capture. A lower total integrated stress response to capture for both cortisol and corticosterone was associated with decreased concentrations of thyroid hormone T3 and elevated concentrations of reverse T3 (rT3), suggesting parallel downregulation of adrenal and thyroid endocrine axes in some individuals. A scaled body condition index was negatively associated with T3 and positively associated with rT3 in adults. Together these findings suggest utility in using endocrine responses to capture stress to evaluate individual and population health.     

The breeding season duration hypothesis: acute handling stress and total plasma concentrations of corticosterone and androgens in male and female striped plateau lizards (Sceloporus virgatus)

Acute glucocorticoid elevations can be adaptations to short-term stressors. The breeding season hypothesis predicts reduced glucocorticoid responsiveness to acute stressors in populations or species with short breeding seasons. The striped plateau lizard (Sceloporus virgatus) has a short breeding season in Arizona. We measured plasma corticosterone and total androgen levels (dihydrotestosterone and testosterone) following one of the four stress-handling treatments (0, 10, 60, or 180 min). In both sexes, longer handling stress yielded higher corticosterone; females had higher corticosterone than males at all time points. Androgens did not vary with handling duration, in either sex. Combining treatments, plasma androgens correlated positively with corticosterone (CORT) in females but not in males; plasma CORT and body mass residuals were negatively correlated in both sexes, suggesting lizards in poor body condition and/or not investing heavily in reproduction (follicle mass) have higher acute corticosterone. Total plasma androgens and body mass residuals were positively associated in males, but showed no association in females. The maximal CORT elevation after handling stress in this single-clutching species was of comparable magnitude to responses in related multi-clutching lizard species with longer breeding seasons. Using data from studies of multiple populations of three Sceloporus species, we found no relationship between the relative magnitude of the CORT increase and either latitude or elevation, two variables in the literature correlated with duration of the breeding season, and only weak relationships with geographic elevation and actual (not relative) stress-elevated CORT values in this multi-population comparison.     

Sex differences in cortisol secretion after administration of an ACTH analogue in sheep during the breeding and non-breeding season

The aim of this study was to compare the response of cortisol in sheep of different sex and gonadal status to adrenal cortex stimulation by an ACTH analogue in the breeding and non-breeding season. Twenty-four adult Corriedale sheep were used in the non-breeding season, and 19 in the breeding season. Three weeks prior to the first trial (non-breeding season), six rams and six ewes were gonadectomised. In each trial, blood was obtained every 15min for 9h and the animals received 0.5mg of ACTH (Tetracosactid, Synacthen Depot i.m., after 1.5h of sampling. Sampling began at 10:00a.m. in the non-breeding season and at 9:00a.m. in the breeding season. Three main effects (sex, gonadal status and season) were evaluated, each with two levels (male and female, intact and gonadectomised, breeding and non-breeding season, respectively). In both seasons, the females showed higher cortisol levels after ACTH than males (P<0.001), though the difference seemed less marked in the non-breeding season. The cortisol response in the ewes was not affected by season. The rams, however, showed a lower response in the breeding season (P<0.03). Gonadectomy reduced the response in the ewes (P<0.001) but had no effect in the rams. Nevertheless, gonadectomy also eliminated the differences between the ewes and the rams, such that the intact rams had lower levels of cortisol compared to the intact females, with those of the gonadectomised animals of both sexes being intermediate between the gonad-intact groups. The results of this study confirm sex differences in ACTH induced cortisol secretion in intact sheep in vivo. Furthermore, by applying exogenous ACTH we have directly stimulated the adrenal cortex, indicating the existence of sex differences also at this level. The circulating gonadal steroids, which are responsible at least in part for the sex differences in the responses to stress, may influence cortisol secretion from the adrenal gland by direct action at the cortex.