Non-invasive measurement of faecal glucocorticoid metabolites in Upland Geese <Emphasis Type="Italic">Chloephaga</Emphasis><Emphasis Type="Italic">picta</Emphasis>

Glucocorticoid (GC) hormones rise in response to stressors, including natural events including weather or predator presence, and human activities, such as hunting, scientific research or recreational visits. However, because blood sampling itself causes stress and is dangerous or even impossible in some wildlife species, feedback-free methods for GC determination are needed to assess stress in these animals. Faecal GC analyses have thus gained interest. Here, we validate a non-invasive method to estimate the physiological stress in the Upland goose Chloephaga picta. An adrenocorticotropin hormone (ACTH) challenge was conducted in captive adults (female and male), and droppings were collected before, during and after the experiment. Corticosterone metabolite (CM) secretion in response to the ACTH challenge was measured with several enzyme immunoassays (EIA) to find the most appropriate test. We used CM levels during the periods before and after the experiment as control data. An EIA for 11-oxoetiocholanolone achieved the highest response to the ACTH challenge and also reflected a stress response to unfamiliar environment. Furthermore, CM concentrations of dry samples were highly correlated with the corresponding non-dried (frozen) samples. The data suggest that this method is appropriate to measure the stress in Upland geese, and that samples can be stored either frozen or dry form.     

Validation of an enzyme immunoassay to measure faecal glucocorticoid metabolites from Adélie penguins (<Emphasis Type="Italic">Pygoscelis adeliae</Emphasis>): a non-invasive tool for estimating stress?

To monitor adrenocortical activity in Adélie penguins (Pygoscelis adeliae), we validated an enzyme immunoassay (EIA) for faecal glucocorticoid (GC) metabolites. An adrenocorticotropin hormone (ACTH) challenge was conducted on a paired female and male. The EIA for tetrahydrocorticosterone showed a clear response to the ACTH challenge in both birds. After high-performance liquid chromatography using pooled samples generated from the ACTH challenge, and analysing each individual fraction, three immunoreactive peaks were detected. Both biological and chemical validations strongly suggest that the EIA can be a useful tool for non-invasively measuring GC metabolites in faeces of Adélie penguins.     

A non-invasive method for measuring glucocorticoid metabolites (GCM) in Mountain hares (<Emphasis Type="Italic">Lepus timidus</Emphasis>)

In this study, we evaluated a non-invasive method for measuring glucocorticoid metabolites (GCM) in the Mountain hare (Lepus timidus). An adrenocorticotrophic hormone challenge test was performed in order to select an appropriate enzyme immunoassay (EIA) to measure faecal GCM. Finally, an 11-oxoaetiocholanolone EIA and a 5α-pregnane-3β,11β,21-triol-20-one EIA were chosen. Both assays showed small fluctuations in baseline values and a clear response after stimulation of the adrenocortical activity. To test the stability of faecal metabolites under field conditions, the effects of different storage conditions and periods on GCM concentrations were examined. The assays revealed low fluctuations in metabolite concentrations within the storage period of 12, 24, 48 and 72 h, both at ambient temperatures of 10°C and 25°C, respectively. A washing-out effect of water was found for both assays, which must be taken into account in field studies. The results indicate that this non-invasive method can be used to evaluate glucocorticoid levels of free-ranging Mountain hares.     

Modulation of the hypothalamic-pituitary-adrenal axis of an Arctic-breeding polygynandrous songbird,the Smith's longspur,Calcarius pictus

To successfully reproduce in the Arctic, birds must modulate their neuroendocrine and behavioural systems. These adjustments include an attenuation of the stress responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis to external stimuli and a behavioural insensitivity to high corticosterone (B) levels. The HPA axis was examined in free-living territorial polygynandrous Smith's longspurs (Calcarius pictus) that migrate to breed on the Arctic tundra. Basal and stress-induced B levels were measured through the breeding season and were found to be significantly lower in females compared with males. This was not a consequence of adrenal insensitivity, because intrajugular injections of adrenocorticotrophin hormone (ACTH) enhanced B release in incubating females. In males the adrenocortical response to stress was significantly attenuated during the parental phase compared with arrival at the breeding ground. In contrast to temperate passerines, there was no significant decrease in male territorial aggressive behaviour when B was experimentally elevated, suggesting a behavioural insensitivity to glucocorticoids. This mechanism is hypothesized to increase reproductive success by preventing interruptions to parental care during transient deleterious environmental perturbations, which are often experienced in the short Arctic breeding season. Modulation of the HPA axis in this species in relation to life-history stage, lifetime reproductive success and the polygynandrous mating system is discussed.     

Non-invasive monitoring of glucocorticoid metabolites in brown hyaena (Hyaena brunnea) feces

The brown hyaena (Hyaena brunnea) is the least known of the large predators of southern Africa. The current IUCN status of the brown hyaena is “Near Threatened”, and there are conservation concerns related to a general lack of biological knowledge of the species. For instance, a better knowledge of the responses to environmental and social stressors would improve our abilities to sustainably manage brown hyaena populations in both captive and free‐ranging environments. We conducted adrenocorticotrophic hormone (ACTH) challenges in one female and one male adult brown hyaena at Lion Park Zoo, South Africa, to validate measurements of glucocorticoid metabolites (GCM) in brown hyaena feces via an enzyme immunoassay (EIA). We also measured gastrointestinal transit times (GIT times) and the GCM degradation in feces left in ambient temperature for up to 32 hr to more reliably assess the use of this assay as a tool for non‐invasive glucocorticoid measurements. Intramuscular injections of synthetic ACTH yielded GCM levels of 388% (female) and 2,682% (male) above baseline with peak increases occurring 25‐ to 40‐hr after injection. The time delay of fecal GCM excretion approximately corresponded with food transit time in the brown hyaenas. Fecal GCM levels declined significantly over time since defecation. Our results provided a good validation that fecal GCMs accurately reflect circulating glucocorticoid stress hormones in brown hyaenas, but we highlight that samples have to be frozen immediately after defecation to avoid bias in the measurements as a result of bacterial degredation. Zoo Biol 30:451–458, 2011. © 2010 Wiley‐Liss, Inc.     

Comparison of plasma ACTH and corticosterone levels after embryo bursectomy

Plasma levels of both adrenocorticotropic hormone (ACTH) and corticosterone (B) were determined in embryos (day 15 of incubation), chicks (day 3 after hatch) and young chickens (8 weeks). Experimental animals were bursectomized at 80 hr of incubation, i.e., before any anlage of the bursa of Fabricius could develop. Bursectomized (BFX) animals were compared to sham-operated controls (T), in basal, resting condition and 7 (ACTH) or 14 min (B) after ether stress was delivered for 30 sec. Basal B and ACTH levels seemed not to be significantly modified in BFX embryos, chicks and chickens. Hypophysial and adrenocortical response to stress appeared more precociously in BFX embryos (day 15 of incubation) than in intact ones (day 19). The non stress-responsive period that was observed for one week after hatch of T birds did not appear in 3-day-old BFX chicks whose both B and ACTH stress-induced levels were as high as in intact adults. In contrast, adrenocortical and pituitary corticotropic responses to stress were markedly impaired (by 50%) in adult BFX chickens as compared to intact controls.     

Validation of noninvasive monitoring of adrenocortical endocrine activity in ground-feeding aardwolves (Proteles cristata): exemplifying the influence of consumption of inorganic material for fecal steroid analysis

Biologically inert material in feces may confound interpretations of noninvasive fecal endocrine data, because it may induce variance related to differences in foraging behavior rather than to differences in endocrine activity. We evaluated two different enzyme immunoassays (EIAs) for the noninvasive evaluation of adrenocortical activity in ground-feeding aardwolves (Proteles cristata) and tested the influence of soil content in aardwolf feces on the interpretation of fecal glucocorticoid metabolite data. Using adrenocorticotropic hormone (ACTH) challenges for validation, we successfully identified a cortisol EIA suitable for assessing adrenocortical activity in aardwolves. An alternatively tested 11-oxoetiocholanolone EIA failed to detect a biologically relevant signal after ACTH administration. Although the proportion of inorganic content in aardwolf feces did not alter qualitative conclusions from the endocrine data, the data related to mass of organic content had a larger amount of variance attributed to relevant biological contrasts and a lower amount of variance attributed to individual variation, compared with data related to total dry mass of extracted material. Compared with data expressed as dry mass of extracted material, data expressed as mass of organic content may provide a more refined and statistically powerful measure of endocrine activity in species that ingest large amounts of indigestible material.     

Plasma cortisol and corticosterone concentrations in the golden hamster, (Mesocricetus auratus)

Because some recent studies of hamster adrenocortical function have depended on older studies that may have been inadequate or misinterpreted, the present study re-examined plasma corticosterone and cortisol concentrations in hamsters under several conditions to determine which plasma glucocorticoid predominated in this animal. Sensitive radioimmunoassays were used to measure separately the two glucocorticoids in the basal condition, after adrenocorticotropin (ACTH) treatment, after acute stress, and after chronic stress. In the basal condition, corticosterone concentrations were 3-4 times higher than those of cortisol. After stimulation, this difference disappeared, but rarely were any hamster's cortisol levels higher than their corticosterone levels. Both ACTH and acute stress elevated plasma corticosterone and cortisol concentrations, but only plasma cortisol concentrations were elevated following chronic stress. The dissociation between cortisol and corticosterone concentrations after chronic stress suggests that the two glucocorticoid hormones in the hamster may be regulated independently. The data also indicate that both corticosterone and cortisol should be measured when assessing adrenocortical function in the hamster.     

Exposure to chronic stress downregulates corticosterone responses to acute stressors

We used captive European starlings (Sturnus vulgaris) to test whether corticosterone responses differed in birds held under normal laboratory conditions or conditions of chronic stress. Surprisingly, both basal corticosterone concentrations and corticosterone responses to acute stress were significantly reduced when birds were chronically stressed. To determine the mechanism underlying this reduced response, animals under both conditions were injected with lactated Ringer's solution (control), adrenocorticotropin (ACTH), arginine vasotocin (AVT), or dexamethasone (DEX). ACTH increased corticosterone concentrations above stress-induced levels in both cases, although maximum responses were lower in chronically stressed birds. AVT did not augment the corticosterone response under nonchronically stressed conditions, but it did under chronically stressed conditions. DEX reduced maximal corticosterone concentrations in both cases. Neither ovine nor rat corticotropin-releasing factor (CRF) altered normal stress responses. These data indicate that changes in responsiveness of the hypothalamic-pituitary-adrenal axis to ACTH and AVT serve to downregulate corticosterone responses during chronic stress. Furthermore, these data lead to the following hypothesis: ACTH output from the pituitary limits maximum corticosterone concentrations under normal conditions, but reduced AVT release from the hypothalamus regulates lower corticosterone concentrations under chronic stress conditions.     

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.     

Modulation of the adrenocortical response to acute stress with respect to brood value, reproductive success and survival in the Eurasian hoopoe

Reproducing parents face the difficult challenge of trading-off investment in current reproduction against presumed future survival and reproduction. Glucocorticoids are supposed to mediate this trade-off because the adrenocortical response to stress disrupts normal reproductive behaviour in favour of self-maintenance and own survival. According to the brood-value hypothesis, individuals with a low survival probability until the next reproductive season have to invest in current reproduction, a process driven by a down-regulation of their adrenocortical response. If the adrenocortical response to stress effectively mediates the trade-off between current reproduction versus future survival and reproduction, we expect a negative relationship with reproductive success and a positive correlation of the adrenocortical stress response with survival. We studied the relationship between corticosterone secretion in parents and their current brood value, reproductive success and survival in a short-lived multi-brooded bird, the Eurasian hoopoe Upupa epops. The adrenocortical response to acute handling stress was correlated with the brood value within the individual (first and second broods of the year) and between individuals. Birds breeding late in the season mounted a lower total corticosterone response to acute stress than birds breeding earlier, while females showed lower levels than males. We observed a negative relationship between the adrenocortical stress response and rearing success or fledging success in females, as predicted by the brood-value hypothesis. However, we could not evidence a clear link between the adrenocortical stress response and survival. Future research testing the brood-value hypothesis and trade-offs between current reproduction and future survival should also measure free corticosterone and carefully differentiate between cross-sectional (i.e. between-individual) and individual-based experimental studies.     

Sex differences in the excretion of fecal glucocorticoid metabolites in the Syrian hamster

We verified the relevance of measuring fecal glucocorticoid metabolites (FGM) to assess the stress response of the Syrian hamster. Male and female hamsters (n = 10 each) were submitted to an adrenocorticotropic hormone (ACTH) challenge test, whereas animals in the control group received 0.5 mL of sterile isotonic saline solution. All feces voided by each animal were collected at 4 h intervals from 24 h before (baseline) until 48 h after injections. FGM were quantified using an 11-oxoetiocholanolone enzyme immunoassay (EIA). Basal concentrations of FGM were almost four times higher in males than in females. Following ACTH administration, FGM levels started rising from 8 h onwards, reaching peak concentrations 20 or 28 h post injection in males and females, respectively. Despite the much higher absolute concentrations present in males, the relative increase (500%) in response to the ACTH stimulation was similar in both sexes. Sex differences in FGM levels are in accordance with results reported by others regarding the hamster adrenal physiology. The comparison of the adrenocortical response of males and females to an ACTH challenge provided new information about the amplitude and the timing of such a response and the excretion of glucocorticoids in both sexes. We demonstrated for the first time in the Syrian hamster that adrenocortical activity can be monitored in fecal samples in a noninvasive way. Our study provides a humane, practical, and noninvasive alternative to blood removal and therefore a powerful tool for stress-related studies in a species frequently used as an animal model in medical research.     

Prolonged kallikrein inhibition does not affect the basal growth and secretory capacity of rat adrenal cortex, but enhances mineralo- and glucocorticoid response to ACTH and handling stress

The effects on the pituitary-adrenocortical functions of the prolonged (7-day) blockade of endogenous bradykinin (BK) synthesis, obtained by the administration of the kallikrein inhibitor (K-I) cyclohexylacetyl-Phe-Arg-Ser-Val-Gln amide, were investigated in the rat. K-I treatment did not cause significant changes in the (i) body and adrenal weights; (ii) basal plasma levels of ACTH, aldosterone and corticosterone; and (iii) average volume of adrenocortical cells and their basal secretory capacity. Conversely, K-I administration induced a significant magnification of the in vivo mineralo- and glucocorticoid responses to the intraperitoneal (i.p.) bolus injection of ACTH. Moreover, K-I-treated rats, but not control ones, displayed a moderate and short-term adrenal secretory response to the mild stress evoked by the placebo i.p. injection. Collectively, these findings rule out the possibility that endogenous BK plays a relevant role in the control of adrenocortical function under basal conditions. However, they suggest that endogenous BK may be involved in quenching exceedingly high adrenocortical responses to ACTH and stresses.     

Behavioural and Hormonal Stress Responses to Social Separation in Ravens,Corvus corax

Social life is profitable, but it facilitates conflicts over resources and creates interdependence between individuals. Separating highly social animals triggers intense reactions aimed at re‐establishing lost connections. Less is known, however, about behavioural and physiological responses to separation in socially facultative species, where individuals temporarily form groups and may subsequently leave them. Non‐breeding common ravens (Corvus corax) gather in large numbers at feeding and roosting sites, but otherwise spend time seemingly solitary or in small subgroups. We here studied how ravens cope with being socially isolated, and investigated the life characteristics that might explain potential individual differences. For this, we individually separated captive subadult ravens (n = 25) and housed them in physical and visual isolation from their group members across 4 d. During the separation period, we collected behavioural data and measured the amount of immunoreactive corticosterone metabolites from bird droppings to assess the ravens’ physiological stress response. We found behavioural indicators of stress at the start of the separation, when ravens showed higher levels of tension than of comfort – a pattern that reversed at the end of the separation. Furthermore, we found that the upbringing of ravens affected their behaviour during separation. Hand‐raised birds produced more vocalisations in the beginning of the separation, and were less active at the end, while the reverse pattern occurred with parent‐raised ravens. Contrary to our predictions, we did not find differences in hormonal responses between the beginning and end of the separation period or any link between hormonal responses and behaviours. Ravens’ behavioural responses to social separation stress seem to be dependent on their arousal states, although possible links with hormonal reactions remain unclear. Our results show that behavioural reactions are not always linked with hormonal responses to stress, and further emphasise the importance of investigating effects of early‐life experiences.     

Body condition is associated with adrenocortical response in the barn swallow (Hirundo rustica L.) during early stages of autumn migration

Migration is an energy-demanding life-history period and also a significant population-limiting factor of long-distance migratory birds. It is important to understand how corticosterone, the main energy regulating hormone in birds, is associated with behavioural and physiological changes during migration. According to the migration modulation hypothesis (MMH), individual birds may express elevated levels of baseline corticosterone to facilitate fuelling, but down-regulate the adrenocortical response in order to protect skeletal muscles from the catabolic effects of the hormone. We measured the baseline and stress-induced levels of corticosterone in barn swallows (Hirundo rustica L.) during early stages of autumn migration. Here, we show that, while barn swallows clearly responded to the capture and handling stress by increasing the corticosterone level, the strength of this acute response was related to their energetic condition: birds with high body mass responded more rapidly and had lower peak values of corticosterone than lighter birds. Further, the baseline levels of corticosterone correlated negatively with the magnitude of the adrenocortical response. Barn swallows did not show elevated baseline levels of corticosterone in the course of autumn, which suggests that, instead of fuelling, the birds were actively migrating. Our results indicate that MMH also applies to aerial feeders, whose foraging habits differ from model birds of previous studies.     

Chronic melatonin treatment and the hypothalamo-pituitary-adrenal axis in the rat: Attenuation of the secretory response to stress and effects on hypothalamic neuropeptide content and release

The pituitary-adrenal secretory response to acute and chronic stress, suppressibility of adrenocortical secretions by exogenous glucocorticoids, and hypothalamic content and in vitro release of the two major peptidergic activators of the hypothalamo-pituitary-adrenal (HPA) axis, corticotropinreleasing hormone (CRH) and arginine-vasopressin (AVP), were examined in rats receiving daily melatonin (MEL) injections coincident with the circadian increment of endogenous pineal and adrenocortical secretory activity. After 7 days of MEL administration, the rats displayed a significant attenuation of the adrenocortical secretory response to acute and chronic stress. Chronic MEL treatment also prevented the decline in adrenocorticotropic hormone (ACTH) release resulting from chronic stress exposure. Hypothalamic CRH content was significantly lower in rats receiving MEL treatment, while AVP remained largely unaltered; however, MEL administration counteracted the chronic stress-induced decrease in hypothalamic AVP content and in vitro release. When exposed to dexamethasone in vitro, hypothalamic explants from MEL-treated rats responded with a stronger suppression of CRH and AVP release than those originating from vehicle-injected animals. These observations indicate that MEL attenuates the adrenocortical response to stress and influences the biosynthesis, release and glucocorticoid responsiveness of hypothalamic ACTH secretagogues.     

Epizootiology of a nuclear-polyhedrosis virus in European spruce sawfly,Gilpinia hercyniae: Birds as dispersal agents of the virus during winter

In Welsh spruce forest the relationship of birds to a nuclear polyhdedrosis virus (NPV) was studied outside the larval period of its sawfly host, Gilpinia hercyniae. Birds passed feces containing infective NPV throughout the nonlarval period (November–June). It is thought that birds acquired inoculum by feeding on the corpses of NPV-killed larvae adhering to spruce trees. The proportion of infective droppings declined in the 2 months (May and June) prior to the new larval period. Infective droppings were collected from all six species of birds netted in January. The infectivity of droppings of goldcrest was less and that of longtailed tit greater in January than in the previous September. Experimental evidence suggests that birds may carry NPV for at least 6 km.     

Correction by natural adaptogens of hormonal-metabolic status disorders in rats during the development of adaptation syndrome using functional tests with dexamethasone and ACTH]

Rats exposed to stress by fixation develop a complex of hormonal metabolic homeostasis disturbances (as evidenced by changed levels of ACTH, insulin, 11-HOCS, urea, glucose). One of the major mechanisms of these disorders in reduced hypothalamus sensitivity to regulatory signals and exhausted adrenocortical functional activity, developing at the stages of anxiety and exhaustion of the adaptation syndrome, respectively, and detectable by functional tests with dexamethasone and ACTH. Administration of natural adaptogens (Scutellaria baicalensis extract and its active principle, baikalin flavonoid) was conducive to normalization of the majority of the examined parameters whatever the direction of changes.     

Immunoreactive Corticotrophin Levels in Adrenocortical Insufficiency

Plasma concentrations of immunoreactive corticotrophin (ACTH) have been determined in 14 patients with untreated Addison''s disease and in 42 patients with secondary adrenocortical insufficiency. Basal morning plasma ACTH levels were markedly raised in those with Addison''s disease but were either in the normal range or undetectable in the group with secondary adrenocortical insufficiency. In the group with Addison''s disease circulating ACTH values showed a definite nyctohemeral rhythm, a pronounced rise in response to insulin-induced hypoglycaemia, and an immediate fall following the intravenous injection of corticosteroids, with a half-life of between 13·5 and 44·2 minutes. When assays were performed with antisera directed against the portion of the ACTH molecule responsible for corticosteroidogenesis (the N-terminal portion) the apparent ACTH concentrations were lower than with antisera directed against the non-steroidogenic (C-terminal) portion of the molecule. This emphasizes that different antisera may give different apparent hormone concentrations, and that the ranges of values obtained in normal and abnormal states must be established for each antiserum.     

Hair cortisol: a parameter of chronic stress? Insights from a radiometabolism study in guinea pigs

Measurement of hair cortisol has become popular in the evaluation of chronic stress in various species. However, a sound validation is still missing. Therefore, deposition of radioactivity in hair and excretion into feces and urine after repeated injection of (3)H-cortisol was studied in guinea pigs (n?=?8). Each animal was given intraperitoneally 243.6?kBq (3)H-cortisol/day on 3 successive days. After the first injection, all voided excreta were collected for 3?days. After the second injection, hair was shaved off the animals' back and newly grown hair was obtained on day 7. Following methanol extraction, radiolabeled and unlabeled glucocorticoid metabolites (GCM) in fecal and hair samples were characterized by high-performance liquid chromatography (HPLC) and enzyme immunoassays (EIA). In feces, maximum radioactivity was reached 8?h (median) post each injection, whereas maxima in urine were detected in the first samples (median 2.5?h). Metabolites excreted into feces (13.3?%?±?3.7) or urine (86.7?%) returned nearly to background levels. HPLC of fecal extracts showed minor variation between individuals and sexes. In hair, small amounts of radioactivity were present. However, two EIAs detected large amounts of unlabeled GCM, including high levels at the position of the cortisol standard; radioactivity was absent in this fraction, demonstrating that (3)H-cortisol was metabolized. Furthermore, large amounts of immunoreactivity coinciding with a radioactive peak at the elution position of cortisone were found. These results show for the first time that only small amounts of systemically administered radioactive glucocorticoids are deposited in hair of guinea pigs, while measurement of large amounts of unlabeled GCM strongly suggests local production of glucocorticoids in hair follicles.