ACTH Challenge Research Articles

Validating the use of dermal secretion as a matrix for monitoring glucocorticoid concentrations in African amphibian species.

The complex interaction between factors leading to amphibian declines is responsible for the inability to develop robust, standardized conservation action plans. Monitoring physiological stress responses in amphibians may provide an ideal tool to assist conservationists in this regard. This study aimed to validate dermal secretions as a robust matrix for monitoring glucocorticoid alterations in an African amphibian, the edible bullfrog (Pyxicephalus edulis). Both a biological (handling) and physiological (ACTH challenge) validation were conducted to determine which of five available enzyme immunoassays (EIAs) tested is most suited for monitoring alterations in dermal glucocorticoid (dGC) concentrations. Additionally, the most optimal body region for monitoring dGC concentrations in P. edulis was identified. To confirm the activation of the hypothalamic-pituitary-interrenal (HPI) axis following ACTH administration, urinary glucocorticoid metabolites (uGCM) were also quantified. The tested corticosterone EIA was the only assay able to monitor alterations in dGC concentrations following the handling event in P. edulis. Further validation during the ACTH challenge supported the corticosterone EIA in this regard. Dermal secretions collected from both the ventral and dorsal regions were suitable for monitoring dGC concentrations in both sexes. A considerable increase in uGCM concentrations following ACTH administration was found, suggesting that the dGC concentrations observed were derived from the HPI axis. This study offers further support for the use of dermal secretions as an important matrix for monitoring physiological stress, and thus general health, in amphibian species.

Growing without a mother during rearing affects the response to stressors in rams

Early maternal care contributes to the normal development of the physiological, social and emotional functions of the offspring. As sheep are a strongly gregarious species, separation and isolation of an individual from the flock provokes fear and, consequently a stress response. The first aim of this study was to compare the stress response to social isolation of rams that were reared by their mothers or artificially reared. A complementary objective was to compare the cortisol response to an ACTH challenge in both groups of rams. Rams were: 1) separated from their dams 24–36 h after birth (Week 0) and artificially fed with sheep milk until Week 10 (group AR, n = 14); or 2) reared by their dams until Week 10 (group DR, n = 13). The rams were then kept in separate, adjacent paddocks for the rest of the study. When they were 8 months old, 10 animals randomly selected from each group were isolated from the group in an unfamiliar pen during 10 min. Behavioural (the number of lines crossed, frequencies of vocalizations, eliminations, sniffing, escape attempts and freezing), physiological (heart and respiratory rate, and rectal temperature), biochemical (total blood proteins, globulins and albumin), and endocrine (serum cortisol concentrations) variables were recorded. Dam-reared rams eliminated (defecations + urinations) more times and crossed more lines during the social isolation test than AR lambs (P < 0.05). The increase in total proteins and albumin concentration after the social isolation test was also greater in DR than AR rams (P = 0.01 and P < 0.01 respectively). Artificially-reared rams had greater globulin concentration than DR rams after the test (P = 0.04). When rams were 2 years old, ACTH was administered to 10 rams randomly selected from each group, and the cortisol response was measured. Dam-reared rams had greater cortisol concentrations than AR rams at 150 and 180 min after the administration of ACTH (P < 0.05). In conclusion, artificially reared rams were less affected by social isolation and had lower adrenal response to an ACTH challenge than rams reared by their mothers. This study suggests that the relationship with the mother plays a key role in the development of rams’ social behaviour.

Fecal glucocorticoid metabolites and assay validation: Stress response evaluation in captive brown howler monkeys (Alouatta clamitans).

The advent of non-invasive methods provides a powerful alternative to stress studies as the use of stressful handling techniques is no longer needed. However, many factors influence hormone metabolism such as sex, diet, and metabolic rate. Thus, validation should be species- and matrix-specific. To assess stress response in brown howler monkeys Alouatta clamitans, we adopted an ACTH challenge test and parallelism to provide physiological and laboratorial validation. Radioimmunoassay was used to measure fecal levels of corticosterone. All challenged animals presented a peak in fecal glucocorticoids levels the day after the treatment, while control animals did not. There were no significant sex differences, but females with infants had higher levels of corticosterone. Corticosterone levels showed parallelism to the standard curve of the diagnostics kit. Collectively, the data suggest that the method was validated and is useful for monitoring stress, thereby helping in conservation programs both in captivity and in the wild. Transit time information may be coupled with travel distance in seed dispersal studies.

Release from ecological constraint erases sex difference in social ornamentation

Sex differences in animal ornamentation are thought largely genetically fixed due to stronger sexual selection on males of species with conventional sex roles. But, other types of sex differences are not genetically fixed. For example, several differences in human social behavior result instead from sociocultural or economic constraints on women. Since gregarious animals use ornamentation for various social functions, perhaps some of their sex differences are, similarly to human behavior, due to social coercion or ecological constraint (their closest equivalents to human social and economic constraints, respectively). We found sex differences in ornamentation that disappear plastically in a social species with conventional sex roles. The red bill of common waxbills (Estrilda astrild) is on average more saturated in males, but in our experiment female bill color correlated with night temperature, an important energetic stressor, suggesting that sexual dichromatism disappears when ecological conditions are favorable to females. Female ornamentation may be more adversely affected by ecology because of their life history that requires balancing investment in ornamentation with maintaining reproductive condition. Manipulation of stress-related physiology (ACTH challenge) suggests that this effect was not mediated by stress mechanisms. Social coercion appears to not explain sexual dichromatism: males were not more aggressive than females, aggressiveness was not related to bill color, and manipulation of reproductive axis’ physiology (GnRH challenge, which in many species mediates aggressiveness) did not increase bill color. Our results show parallels to the plastic sex differences of humans in social animals and suggest that studying their ecological vs. social causes provides a biological backdrop for understanding the human case as well. Many sex differences in human social behavior result from economic or sociocultural constraints on women, while sex differences in the ornamentation of animals with conventional sex roles are thought largely genetically fixed. We show that a sex difference in ornamentation—the redder bills of male than female common waxbills—disappears plastically in an animal with conventional sex roles due to, in part, changes in female ornamentation. Social coercion did not explain reduced female ornamentation: aggressiveness did not predict bill color, and males were not dominant over females. Instead, female bill color was reduced during colder weather, perhaps because females under energetic stress need to balance investment in social ornamentation vs. maintaining reproductive condition. Similarly to humans, some sex differences of gregarious animals may be due to females requiring appropriate conditions to express their full social potential.

Responses of dairy cows with divergent residual feed intake as calves to metabolic challenges during midlactation and the nonlactating period

Residual feed intake (RFI) is defined as the difference between the actual and expected feed intake required to support animal maintenance and growth. Thus, a cow with a low RFI can obtain nutrients for maintenance and growth from a reduced amount of feed compared with a cow with a high RFI. Variation in RFI is underpinned by a combination of factors, including genetics, metabolism, thermoregulation and body composition; hypothalamic-pituitary-adrenal (HPA) axis responsiveness is also a possible contributor. Responses to 3 metabolic challenges were measured in lactating and nonlactating dairy cattle. Sixteen Holstein Friesian cows with phenotypic RFI measurements that were obtained during the growth period (188-220 d old) were grouped as either low-calfhood RFI (n = 8) or high-calfhood RFI (n = 8). An ACTH (2 µg/kg of body weight), insulin (0.12 U/kg), and epinephrine (a low dose of 0.1 µg/kg and a high dose of 1.6 µg/kg of epinephrine) challenge were each conducted during both midlactation (122 ± 23.4 d in milk) and the nonlactating period (dry period; approximately 38 d after cessation of milking). Cows were housed in metabolism stalls for the challenges and were fed a diet of alfalfa cubes ad libitum for at least 10 d before the experiment (lactating cows also were offered a total of 6 kg of dry matter/d of crushed wheat grain plus minerals fed as 3 kg of dry matter at each milking) and were fasted for 12 h before the challenges. The efficiency of conversion of feed into milk (the ratio of feed consumed to milk produced over the 7 d before the experiment) during midlactation was better (lower) in low-calfhood RFI cows, although dry matter intake did not differ between RFI groups. Low-calfhood RFI cows exhibited a lower plasma cortisol response to the ACTH challenge than high-calfhood RFI cows, particularly in midlactation (-15%). The low-calfhood RFI cows had a greater plasma insulin-like growth factor-1 response to the insulin challenge and plasma fatty acid response to epinephrine compared with the high-calfhood RFI cows. These data suggest that high-calfhood RFI cows exhibit a more responsive HPA axis. As divergence in RFI measured during growth is retained (although reduced) during lactation, it is possible that energy is used to respond to HPA axis activation at the expense of production in high-calfhood RFI dairy cattle during lactation and contributes to a decrease in overall feed use efficiency.

Development and Validation of an Enzyme Immunoassay for Fecal Dehydroepiandrosterone Sulfate in Japanese Macaques (Macaca fuscata)

Measuring hormonal profiles is important in monitoring stress, physical fitness, and reproductive status in primates. Noninvasive methods have been used to measure several steroid hormones in primates without causing them stress. However, few studies have used feces or urine to measure dehydroepiandrosterone sulfate (DHEAS), an important precursor of sex steroids that has been studied as a biomarker of aging, pregnancy, and stress in humans and nonhuman primates. We developed an enzyme immunoassay to detect DHEAS in the feces of Japanese macaques (Macaca fuscata). Our subjects included eight singly housed Japanese macaques. To validate the assay, we administrated oral DHEA to one male and one female macaque, collected their feces, and measured DHEAS levels over time. Given that DHEAS is related to gonadal steroids and the stress response, we also measured DHEAS concentrations in response to adrenal (adrenocorticotropic hormone [ACTH]) and gonadal (human chorionic gonadotropin [hCG]) stimulation. Our assay successfully detected DHEAS in Japanese macaque feces, and levels of DHEAS were associated with the amount of DHEA ingested. Parallelism and accuracy tests revealed that fecal extracts were reliable measures of DHEAS. Neither ACTH nor hCG challenge appeared to affect DHEAS levels. The method we describe is less expensive than that using the commercially available kits and is applicable to investigations involving aging, stress, and reproduction in Japanese macaques.

Perturbing the Hypothalamic-Pituitary-Adrenal Axis: A Mathematical Model for Interpreting PTSD Assessment Tests.

We use a dynamical systems model of the hypothalamic–pituitary–adrenal (HPA) axis to understand the mechanisms underlying clinical protocols used to probe patient stress response. Specifically, we address dexamethasone (DEX) and ACTH challenge tests, which probe pituitary and adrenal gland responses, respectively. We show that some previously observed features and experimental responses can arise from a bistable mathematical model containing two steady-states, rather than relying on specific and permanent parameter changes due to physiological disruption. Moreover, we show that the timing of a perturbation relative to the intrinsic oscillation of the HPA axis can affect challenge test responses. Conventional mechanistic hypotheses supported and refuted by the challenge tests are reexamined by varying parameters in our mathematical model associated with these hypotheses. We show that (a) adrenal hyposensitivity can give rise to the responses seen in ACTH challenge tests and (b) enhanced cortisol-mediated suppression of the pituitary in subjects with PTSD is not necessary to explain the responses observed in DEX stress tests. We propose a new two-stage DEX/external stressor protocol to more clearly distinguish between the conventional hypothesis of enhanced suppression of the pituitary and bistable dynamics hypothesized in our model.

Free and bound cortisol in plasma and saliva during ACTH challenge in dairy cows and horses.

Cortisol levels reflect hypothalamic-pituitary-adrenocortical (HPA) axis activity. While most plasma cortisol is supposed to be bound to the soluble corticosteroid-binding globulin (CBG), only free cortisol (FC) actively regulates metabolic and immunological processes. We aimed to establish a multispecies suitable method to assess FC in cows and horses which in combination with total cortisol (TC) allows interpreting proportional changes of cortisol in saliva as well as in blood in response to a standardized HPA axis activation via ACTH. We further investigated if the ratios of cortisol fractions as obtained at basal levels in healthy horses (herbivorous and monogastric) and dairy cows (herbivorous and ruminant) change during HPA axis activation, and to which extent saliva cortisol (SC) is representative for alterations in plasma FC and adrenal cortex reactivity. However, it was not the objective of the present study to directly compare the two species. Dosages of ACTH applied in cows and horses were based on published data. Synthetic ACTH was intravenously administered to eight dairy cows (0.16 µg/kg BW) and five horses (1 µg/kg BW). Blood and saliva were collected every 30 min for 3 h from a jugular vein catheter, and analyzed for TC and SC, the ratio of free cortisol (rFC), and the concentration of FC (cFC) in plasma. During the entire sampling period of the ACTH test, plasma TC was paralleled by blood cFC, rFC, and SC in both cows and horses. All cortisol fractions increased within 30 min of ACTH administration compared to basal values (0 min, P < 0.05). Peak TC concentration reached 63.2 ± 9.6 ng/mL and 73.2 ± 11.8 ng/mL in bovine and equine plasma, respectively. Peak values of rFC averaged 17.9 ± 4.5% in cows and 19.2 ± 7.8% in horses. The ratio of SC to cFC in horses remained similar during the ACTH challenge suggesting that SC is recruited from plasma FC. However, SC increased less compared to plasma TC and FC during HPA axis activation in cows. In conclusion, the short-term activation of the HPA axis caused not only an elevation of TC, but also a similar increase of rFC in both species. SC closely reflected changes of FC in horses, but less accurately in cows. The concomitant evaluation of changes among cortisol fractions might give further indications on adaptation mechanisms in glucocorticoid regulation as well as differentiate cortisol-related health disorders.

The effects of kennel size and exercise on the behaviour and stress physiology of individually-housed greyhounds

Greyhounds are routinely bred and managed for racing and are usually housed in individual kennels. To address the question of whether greyhounds would benefit from an increase in the minimum kennel size specified in the Victorian Code of Practice, the effects of kennel size and routine exercise on dog behaviour, stress physiology and injuries were examined. A total of 36 healthy greyhounds (aged 17 months (replicate 1 and 2) and 12 months (replicate 3)) were studied in three time replicates in a 2×2factorial experiment (kennel size of 3m2 and 10m2 of floor space, and two levels of exercise (‘no exercise’ and ‘exercise’(consisting of 2×10min per day and 3 times per week chasing a lure)) over 6 weeks. During the 1st and 5th week of treatment, dog behaviours were recorded for 2days in their kennels using time-lapse video. During the 6th week, six saliva samples were collected to measure basal cortisol concentrations, an ACTH challenge test was performed and injuries were assessed. Intensity of exercise had no significant (p≫>0.05) effect on behaviour, stress physiology and injuries. In the 1st week the adult dogs in large kennels spent more time in the front of the kennels (p≪0.05), less time in the back of the kennels (p≪0.01), less time lying (p≪0.05) and more time standing (p≪0.01) than those adult dogs in the small kennels. In week 5, the younger dogs in the large kennels spent more time at the front of the kennel (p≪0.05), less time in the back (p≪0.01) and spent more of their lying down time at the front and middle of the kennel (p≪0.001) than dogs in the small kennels. No significant differences in any of the measurements were found in the adult dogs in week 5. No abnormal or stereotypic behaviour was observed in Week 1 or 5. No significant differences in injuries, saliva cortisol concentrations and maximum cortisol concentrations in response to ACTH were found. The results of this study do not provide evidence that the smaller kennels and limited exercise increase the risks to dog welfare based on behaviour, stress physiology and injuries. Nevertheless, the observation that dogs tended to spend more time in the front of the large kennels where they were in view of other dogs could indicate that larger kennels may enable better welfare by providing additional social contact.

Subspecies-specific response to ACTH challenge test in the house mouse (Mus musculus)

Long term changes in hormonal levels of small and rare animal species can be effectively monitored by non-invasive methods such as immunoenzymatic analysis. These methods generally analyze metabolites instead of the hormone itself and thus do not invade the organism. However, they can be influenced by many factors, so before they are used, they need to be validated. For this purpose we used the “ACTH challenge” test based on stimulating the animal’s adrenocortical activity and measuring the consequent increase of the level of its glucocorticoid (“stress”) hormones. Specifically, we measured concentrations of fecal glucocorticoid metabolites of two house mouse subspecies, Mus musculus musculus and M. m. domesticus. Using polyclonal antibody we investigated the mice’s naturally occurring circadian fluctuation and their reaction to the adrenal stimulation. This study confirmed that the selected method is suitable for analysis of fecal corticosterone metabolites in the wild house mouse. More importantly, we revealed a subspecies-specific stress response at the level of corticosterone production: while the significant effect of ACTH was proved in both subspecies, a notable adrenocortical reaction was also elicited by injecting the saline solution in M. m. domesticus. Our results thus highlight the importance of considering potential cryptic variation within the species under study when designing projects on adrenal stress hormone assessments.

When the ball is in the female’s court: How the scramble-competition mating system of the North American red squirrel has shaped male physiology and testosterone dynamics

Male reproductive success in most mammals is determined by their success in direct inter-male competition through aggression and conflict, resulting in female-defense mating systems being predominant. This is linked to male testosterone levels and its dynamics. However, in certain environments, a scramble-competition mating system has evolved, where female reproductive behavior takes precedence and male testosterone dynamics are unlikely to be related to inter-male competition. We studied the North American red squirrel (Tamiasciurus hudsonicus), a species with a well-established scramble-competition system. Using an ACTH hormonal challenge protocol as a proxy for competitive interactions, we compared the testosterone dynamics in breeding males in late winter with that in nonbreeding males in late spring in the Yukon. To gain an integrated picture of their physiological state, we also assessed changes in their stress response, body mass, energy mobilization, and indices of immune function. Testosterone levels at the base bleed were high in breeding males (2.72ng/mL) and virtually absent in non-breeding males (0.04ng/mL). Breeding males were in better condition (heavier body mass, higher hematocrit, and higher erythrocytes), had higher indices of immune function (neutrophil:lymphocyte ratio), but a similar ability to mobilize energy (glucose) compared with non-breeding males. Though total cortisol was higher in non-breeding males, free cortisol was twice as high in breeding males as their corticosteroid binding globulin levels were half as high. In response to the ACTH challenge, testosterone levels in breeding males declined 49% over the first hour and increased 36% over the next hour; in non-breeding males levels showed no change. Free cortisol increased only modestly (26% in breeding males; 23% in non-breeding males). Glucose levels changed similarly in breeding and nonbreeding males, declining for the first 30min and then increasing for the next 60min. Thus, testosterone and components of the stress axis function in a profoundly different manner in male red squirrels than in males of mammals with female-defense mating systems. There are four probable interrelated reasons for these adaptations in male red squirrels: the marginal benefits of each mating, the constraints of mate searching away from their own resource-based territories, energy mobilization in a harsh environment, and a long life span.

Adrenal cortex reactivity in dairy cows differs between lactational stages and between different feeding levels

Changes in ACTH challenge test characteristics in dairy cows changing their physiological status at different lactational stages and different feeding levels were not investigated in terms of repeatability yet. In 23 multiparous Holstein cows (10 cows fed a sole fresh herbage diet without concentrate, 13 cows fed with concentrate), three ACTH challenge tests were performed: once during pregnancy shortly prior to drying off (T1), and in week 3 (T2) and 8 (T3) after parturition. Test characteristics were correlated to performance and metabolic parameters: DMI, BW, energy balance (EB), plasma concentrations of free fatty acids (NEFA) and beta-hydroxybutyrate (BHB). Basal plasma cortisol concentrations were higher at T1 compared with T2 and T3 (p<.05). The adrenal cortex sensitivity (expressed as total AUC (AUCt ) of cortisol response after ACTH application) was lowest at T2 compared with T1 and T3 (p<.05). Ranking of the individual animals' responses was not repeatable between time points of the ACTH tests. Enhancing the energy deficiency during early lactation by omission of concentrate did not affect baseline cortisol concentrations in plasma, but decreased peak height at T2 (p<.05). Baseline plasma cortisol concentrations were positively correlated with cortisol peak values after ACTH application, previous lactation performance, milk yield and BW (p<.05). The AUCt was positively correlated with baseline cortisol concentrations, EB and DMI. Cortisol release after ACTH injection was lower in animals with high plasma concentrations of NEFA, BHB and with higher contents of fat and free fatty acids in milk (p<.05). Cortisol peak height after ACTH administration was higher in cows with a more positive EB, higher DMI and lower plasma concentrations of NEFA and BHB. In summary, cortisol responses to ACTH challenges in this study were not repeatable in dairy cows changing their physiological status.

Measuring fecal testosterone metabolites in spotted hyenas: Choosing the wrong assay may lead to erroneous results

ABSTRACTEnzyme-immunoassays (EIA) that detect fecal testosterone metabolites (fTM) are powerful tools to monitor gonadal activity non-invasively. However, a challenge with testosterone EIAs might be their potential for cross-reactivities with structurally similar glucocorticoid metabolites. Therefore, we aimed to verify the capability of four different testosterone EIAs to monitor fTM without reflecting changes in adrenocortical activity in spotted hyenas by analyzing fecal samples following testosterone and ACTH challenge tests. We demonstrated that none of the testosterone EIAs is appropriate to measure fTM as all of them showed substantial cross-reactivities to unknown metabolites. Our study underlines the importance of validating androgen EIAs.

Excretion and measurement of corticosterone and testosterone metabolites in bank voles (Myodes glareolus)

The bank vole is a commonly used model species in behavioral and ecophysiological studies. Thus, presenting a validated method for noninvasive monitoring of corticosterone and testosterone secretion is of high relevance. Here, we evaluated the effect of time of day and an ACTH challenge test on measured fecal corticosterone (FCM) and testosterone (FTM) metabolites in both sexes. Furthermore, we performed radiometabolism experiments for both steroids and sexes to study metabolism and excretion of 3H-corticosterone and 3H-testosterone. FCM and FTM were analysed with a 5α-pregnane-3β,11β,21-triol-20-one enzyme immunoassay (EIA) and a testosterone (measuring 17β-hydroxyandrostanes) EIA, respectively. Males had significantly higher FCM levels than females and their main excretion route was via the feces (∼72%), whereas females excreted nearly equal portions in both feces and urine. For testosterone the main excretion route was via the feces in both sexes (∼80%). The time course of excretion was similar in both sexes, but for the first time a significant difference between injected steroids was found: Corticosterone was excreted faster than testosterone, both in urine (median of peak levels: 4h vs 6h) and feces (6h vs 8h). Several metabolites were present in the feces and the tested EIAs reacted with some of them. Time of day had a significant effect on measured fecal steroid metabolites. As expected, males had significantly higher FTM levels than females. ACTH administration significantly increased FCM values; peaks were observed 4–8h after injection. In conclusion, both tested EIAs proved suited for a noninvasive measurement of glucocorticoids and androgens in bank voles.

A comparison between the equine and bovine hypothalamus-pituitary-adrenocortical axis

In this review, we address the function of the hypothalamus-pituitary-adrenocortical (HPA) axis with special emphasis on the comparison between the bovine and equine species. The pars intermedia of the pituitary gland is particularly well developed in horses and cattle. However, its function is not well appreciated in cattle yet. The Wulzen's cone of the adenohypophysis is a special feature of ruminants. Total basal cortisol concentration is much higher in horses than that in cows with similar free cortisol fractions. Corticotropin-releasing factor (CRF) concentrations in equine pituitary venous blood are lower compared with other species, whereas plasma ACTH concentrations in cows are higher than those in horses. A CRF challenge test induced a more pronounced cortisol response in horses compared with cattle, whereas regarding ACTH challenge testing, the opposite seems true. Based on data from literature, the bovine species is characterized by relatively high basal blood CRF and ACTH and low cortisol and glucose concentrations. Obviously, further lowering of blood cortisol in cattle is easily prevented by the high sensitivity to ACTH, and as a consequence, subsequent increased gluconeogenesis prevents imminent hypoglycemia. Hypoglycemia is less likely in horses given their high muscle glycogen content and their relatively high cortisol concentration. When assessing HPA axis reactivity, response patterns to exogenous ACTH or CRH might be used as a reliable indicator of animal welfare status in cows and horses, respectively, although it is emphasized that considerable caution should be exercised in using measures of HPA activity solely to assess animal welfare.

Loner or socializer? Ravens' adrenocortical response to individual separation depends on social integration

Non-breeding common ravens (Corvus corax) live in complex social groups with a high degree of fission–fusion dynamics. They form valuable relationships and alliances with some conspecifics, while taking coordinated action against others. In ravens, affiliates reconcile their conflicts, console each other after conflicts with a third party, and provide each other with social support — all behaviors that presumably reduce corticosterone levels and alleviate stress. However, how well an individual is socially integrated in a (sub)group might vary substantially. This raises the question whether the social integration of a raven affects its stress responses to fission–fusion dynamics. The present study aims to investigate this effect experimentally by separating single ravens (n=16) individually from their group for four days and subsequently reintroducing them. To determine stress response patterns in the separated individuals we measured the amounts of immunoreactive corticosterone metabolites (CM) in droppings. We compared two enzyme immunoassays, which we validated by conducting an ACTH challenge, and finally decided to apply an 11-oxoetiocholanolone enzyme immunoassay. Additionally, we determined levels of social integration using focal observations. Our findings suggest that a strong social integration is related to low CM levels when the individuals are within the group and high levels during separations, implying that separation leads to stress in these birds. In contrast, poorly socially integrated ravens seem to exhibit the opposite pattern, indicating that to them group living is more stressful than being temporarily separated. We, therefore, conclude that the birds' adrenocortical activity is modulated by their social integration.

Associations between temperament and gene polymorphisms in the brain dopaminergic system and the adrenal gland of sheep

Sheep of calm or nervous temperament differ in their physiological (cortisol secretion) and behavioural (motor activity) responses to stressors, perhaps due to variation in genes that regulate glucocorticoid synthesis or brain dopamine activity. Using ewes that had been selected over 20 generations for nervous (n=58) or calm (n=59) temperament, we confirmed the presence of a polymorphism in a gene specifically involved in cortisol production (CYP17), and identified polymorphisms in three genes specifically associated with personality and behavioural traits: dopamine receptors 2 and 4 (DRD2, DRD4), and monoamine oxidase A (MAOA). The calm and nervous lines differed in their frequencies of CYP17 SNP628 (single nucleotide A-G mutation at position 628) and DRD2 SNP939 (single nucleotide T–C mutation at position 939), but not for other SNPs detected in DRD2 or MAOA. In a second experiment, we then genotyped a large, non-selected flock of ewes for DRD2 SNP939 and CYP17 SNP628. Responses to the ‘arena’ and ‘isolation box’ challenges were associated with the DRD2 SNP939 genotype and the response to ACTH challenge was associated with the CYP17 SNP628 genotype. We conclude that, for sheep, a combination of the DRD2 SNP939 C allele and the CYP17 SNP628 A/A genotype could be used as a genetic marker for nervous temperament, and that a combination of DRD2 SNP939 T/T and CYP17 SNP628 G/G could be used as a genetic marker for calm temperament.

The physiological consequences of crib-biting in horses in response to an ACTH challenge test

Stereotypies are repetitive and relatively invariant patterns of behavior, which are observed in a wide range of species in captivity. Stereotypic behavior occurs when environmental demands produce a physiological response that, if sustained for an extended period, exceeds the natural physiological regulatory capacity of the organism, particularly in situations that include unpredictability and uncontrollability. One hypothesis is that stereotypic behavior functions to cope with stressful environments, but the existing evidence is contradictory. To address the coping hypothesis of stereotypies, we triggered physiological reactions in 22 horses affected by stereotypic behavior (crib-biters) and 21 non-crib-biters (controls), using an ACTH challenge test. Following administration of an ACTH injection, we measured saliva cortisol every 30min and heart rate (HR) continuously for a period of 3h. We did not find any differences in HR or HR variability between the two groups, but crib-biters (Group CB) had significantly higher cortisol responses than controls (Group C; mean±SD: CB, 5.84±2.62ng/ml, C, 4.76±3.04ng/ml). Moreover, crib-biters that did not perform the stereotypic behavior during the 3-hour test period (Group B) had significantly higher cortisol levels than controls, which was not the case of crib-biters showing stereotypic behavior (Group A) (B, 6.44±2.38ng/ml A, 5.58±2.69ng/ml). Our results suggest that crib-biting is a coping strategy that helps stereotypic individuals to reduce cortisol levels caused by stressful situations. We conclude that preventing stereotypic horses from crib-biting could be an inappropriate strategy to control this abnormal behavior, as it prevents individuals from coping with situations that they perceive as stressful.

Validation and use of hair cortisol as a measure of chronic stress in eastern chipmunks (Tamias striatus).

Stress levels of individuals are documented using glucocorticoid concentrations (including cortisol) in blood, saliva, urine or faeces, which provide information about stress hormones during a short period of time (minutes to days). In mammals, use of hair cortisol analysis allows for the assessment of prolonged stress over weeks and months and provides information on chronic stress levels without bias associated with handling. Here, we validate hair cortisol analysis in wild rodents using exogenous adrenocorticotrophic hormone (ACTH challenge) and apply the technique to evaluate stress in eastern chipmunks inhabiting logged and natural sites. Chipmunks were subjected to a mark-recapture study and injected weekly with ACTH (Synacthen Depot) or saline, with hair being collected at the conclusion of the challenge. Subsequently, faecal and hair samples were collected from chipmunks occupying logged and natural sites to assess the utility of hair cortisol in comparison with faecal cortisol metabolites. Following extraction, cortisol concentrations were quantified in hair and faecal extracts by enzyme immunoassay. Hair cortisol concentrations were significantly elevated in samples from ACTH-injected chipmunks compared with saline-injected control animals (five times higher). Chipmunks inhabiting logged sites had increased faecal cortisol metabolite concentrations compared with those in natural sites, but no differences were observed in hair cortisol concentrations. Faecal cortisol metabolite levels were positively correlated with hair cortisol levels in chipmunks. Hair cortisol levels reflect changes in circulating cortisol levels and can be used to evaluate the adrenal stress response, and thus stress, in natural populations. Nonetheless, because of the differences in the temporal scale of stress that hair and faeces represent, we caution the use of hair cortisol for detecting differences in physiological stress when comparing individuals within populations and suggest that it is best suited to examining population-level differences.

Dosagem de metabólitos de glucocorticoides e progesterona em fezes de papagaio-verdadeiro (Amazona aestiva)

Os objetivos do presente trabalho foram avaliar as concentrações fecais de metabólitos de glicocorticoides, mensurados por enzimaimunoensaio, empregando-se anticorpos contra cortisol, e por radioimunoensaio, empregando-se anticorpo contra corticosterona, e dos metabólitos da progesterona, mensurados por raioimunoensaio empregando-se anticorpo contra progesterona, em papagaios-verdadeiros (Amazona aestiva) após desafio com ACTH. A estimulação da adrenal com 25 UI/animal de ACTH resultou na elevação das concentrações de metabólitos de glicocorticoides, mas não modificou a dos metabólitos da progesterona fecal em papagaio-verdadeiro. Embora não tenha sido observada a sincronização dos picos de excreção fecal dos metabólitos de glicocorticoides mensurados por enzimaimunoensaio e radioimunoensaio, houve dois picos de excreção, um entre 2 e 4 horas e outro entre 8 e 10 horas. Apesar dos picos, não foram detectados efeitos de Grupos (Tratamento x Controle), momento (horas de coleta) ou sexo (macho x fêmea) nos resultados observados nas concentrações fecais de metabólitos de glicocorticoides e de progesterona, com os métodos empregados.