Non-invasive Monitoring Of Adrenocortical Activity Research Articles

Non-Invasive Monitoring of Adrenocortical Activity in Three Sympatric Desert Gerbil Species.

Simple SummaryIn this era, characterized by remarkable anthropogenic impacts on wildlife, it is crucial to monitor the health of wild animal populations while minimizing the interference to them. To this end, for a better understanding of the eco-physiology of wild animals, the adrenocortical activity can be non-invasively evaluated by measuring glucocorticoid metabolites excreted in feces. However, to ensure that the endocrine information is reliable, the experimental assays should be first validated and the causes for the major variability among individuals should be considered. Here we validated an enzyme immunoassay for measuring fecal corticosterone metabolites (FCM) in three wild gerbil species and emphasized the differences among them. These are endangered species, which play a key role in psammophilic communities, and provide a model system for various aspects in ecology. Thus, this work constitutes the first step toward using the FCMs of these species as indicators for individual and community stress.The study of the endocrine status can be useful to understand wildlife responses to the changing environment. Here, we validated an enzyme immunoassay (EIA) to non-invasively monitor adrenocortical activity by measuring fecal corticosterone metabolites (FCM) in three sympatric gerbil species (Gerbillus andersoni, G. gerbillus and G. pyramidum) from the Northwestern Negev Desert’s sands (Israel). Animals included into treatment groups were injected with adrenocorticotropic hormone (ACTH) to stimulate adrenocortical activity, while control groups received a saline solution. Feces were collected at different intervals and FCM were quantified by an EIA. Basal FCM levels were similar in the three species. The ACTH effect was evidenced, but the time of FCM peak concentrations appearance differed between the species (6–24 h post-injection). Furthermore, FCM peak values were observed sooner in G. andersoni females than in males (6 h and 18 h post-injection, respectively). G. andersoni and G. gerbillus males in control groups also increased FCM levels (18 h and 48 h post-injection, respectively). Despite the small sample sizes, our results confirmed the EIA suitability for analyzing FCM in these species as a reliable indicator of the adrenocortical activity. This study also revealed that close species, and individuals within a species, can respond differently to the same stressor.

ACTH Stimulation Induced Self-Mutilation Behavior in the Golden Conure (Guaruba guarouba).

Simple SummaryPsittacidae are very susceptible to chronic stress and behavioral disorders. We report a successful physiological validation of an enzyme immunoassay for the non-invasive monitoring of adrenocortical activity and, thus, stress in the golden conure. In addition, as an incidental finding, we demonstrate a link between increased glucocorticoid levels and behavioral disorders. Our results are of great relevance for conservation projects and endocrine-behavioral studies of captive golden conures, where the stress evaluation is a fundamental part of animal welfare programs.Psittacidae are very susceptible to chronic stress and behavioral disorders. Information regarding the endocrinology of the golden conure is scarce, especially about adrenocortical activity. Endocrine studies using non-invasive methods are useful, because they allow longitudinal analysis with high numbers of samples without causing additional stress and are viable in the psittacidae management. The objective was to physiologically validate an enzyme immunoassay for measuring glucocorticoid metabolites (GCMs) in this species. Serial droppings were collected from 16 animals. First, one subgroup received adrenocorticotropic hormone (ACTH; Synacthen Depót®) and the other group served as controls. This procedure was reversed afterwards. All birds presented self-mutilation approximately 6 h after the ACTH injection. This behavior disappeared after two days. Peak concentrations (on average nine times higher than baseline values) of GCMs were found 6 (4–8) h after ACTH administration; in all (but one) animals also a second peak was found 14 (10–20) h post injection. GCM levels returned to the baseline after 24 h. We physiologically validated a cortisone enzyme immunoassays to measure adrenocortical activity in the golden conure. Such non-invasive methods are important for studies, which are related to welfare, reproductive, and conservation programs. In addition, we could demonstrate a link between increased glucocorticoid levels and behavioral disorders.

Non-invasive monitoring of adrenocortical activity in free-ranging Namaqua rock mice Micaelamys namaquensis from South Africa in response to anthropogenic land use and season

Stress in animals has been linked to behavioural and physiological changes in response to environmental, social and anthropogenic stimuli. Hence, stress-related responses in animals, especially in rodents, have been used as biological indicators of ecosystem health. This study aimed to establish an enzyme immunoassay (EIA) for monitoring adrenocortical activity in free-ranging Namaqua rock mice Micaelamys namaquensis (Rodentia: Muridae) using faeces as a prerequisite for assessing the effects of anthropogenic land use and season on faecal glucocorticoid metabolite (fGCM) concentration. Rodents were live-trapped seasonally across four land use types: an agricultural crop farm, an agricultural livestock farm, a human-populated site and a nature reserve; all situated in Limpopo Province, South Africa. Determined fGCM concentrations from capture and recapture events were used for biologically validating an EIA detecting steroids with a 5α-3β-11β-diol structure. Recapturing resulted in a significant overall 40% elevation of individual fGCM concentrations demonstrating the effectiveness of the chosen EIA to reliably detect glucocorticoid output in the study species. Neither land use type nor season affected fGCM concentrations in the species, suggesting that land use and season-related environmental changes do not necessarily act as stressors for M. namaquensis, presumably due to their adaptive and resilient nature. Such species can be used to identify ecosystems affected by human-mediated disturbances and allow insights into the management and restoration of these threatened ecosystems and their associated species.

Validating faecal glucocorticoid metabolite analysis in the Virunga mountain gorilla using a natural biological stressor.

The continued degradation of primate habitat worldwide is forcing many primate populations into small protected forest islands surrounded by high-density human populations. One well-studied example is the critically endangered mountain gorilla (Gorilla beringei beringei). Decades of monitoring and research on Rwanda's mountain gorillas offer a unique opportunity to use non-invasive endocrine analysis to address pressing questions about the conservation of this endangered population. The aims of our study were as follows: (i) to validate field and laboratory methods for assessing stress through faecal glucocorticoid metabolite (FGM) analysis using inter-social unit interactions as a natural stressor; (ii) to determine the excretion lag times between interactions and detectable stress response in faeces; and (iii) to determine whether there are circadian patterns of FGM excretion. We collected ~6000 faecal samples from 127 known gorillas in 10 habituated groups, monitored by the Dian Fossey Gorilla Fund's Karisoke Research Center over 21 months in 2011 and 2012. Extracted FGMs were measured using a cortisol enzyme immunoassay (R4866; C. J. Munro). Results revealed cause–effect relationships between inter-unit interactions and increased FGMs (relative to individual pre-event samples) between 20 and 140 h after interactions, with the peak most often occurring on day 3. There was no evidence of circadian patterns in FGM concentrations, as previously shown in many species with long gut passage times. However, baseline FGM concentrations were lower in adult males than in adult females, and variation was associated with the collection month, indicating possible seasonal variation. This study provides a biologically validated, field-friendly faecal hormone metabolite extraction and laboratory enzyme immunoassay analysis method for non-invasive monitoring of adrenocortical activity in Virunga mountain gorillas. The methods are useful for future evaluation of a variety of environmental and human-induced potential stressors in this critically endangered population.

Non-invasive monitoring of adrenocortical activity in captive African Penguin (Spheniscus demersus) by measuring faecal glucocorticoid metabolites

Measurement of faecal glucocorticoid metabolites (FGMs) has become a useful and widely-accepted method for the non-invasive evaluation of stress in vertebrates. In this study we assessed the adrenocortical activity of five captive African Penguins (Spheniscus demersus) by means of FGM evaluation following a biological stressor, i.e. capture and immobilization. In addition, we detected individual differences in secretion of FGMs during a stage of the normal biological cycle of penguins, namely the breeding period, without any external or induced causes of stress. Our results showed that FGM concentrations peaked 5.5–8h after the induced stress in all birds, and significantly decreased within 30h. As predictable, the highest peak of FGMs (6591ng/g) was reached by the youngest penguin, which was at its first experience with the stressor. This peak was 1.8–2.7-fold higher compared to those of the other animals habituated to the stimulus. For the breeding period, our results revealed that the increase in FGMs compared to ordinary levels, and the peaks of FGMs, varied widely depending on the age and mainly on the reproductive state of the animal. The bird which showed the lowest peak (2518ng/g) was an old male that was not in a reproductive state at the time of the study. Higher FGM increases and peaks were reached by the two birds which were brooding (male: 5552%, 96,631ng/g; female: 1438%, 22,846ng/g) and by the youngest bird (1582%, 39,700ng/g). The impact of the reproductive state on FGM levels was unexpected compared to that produced by the induced stress. The EIA used in this study to measure FGM levels proved to be a reliable tool for assessing individual and biologically-relevant changes in FGM concentrations in African Penguin. Moreover, this method allowed detection of physiological stress during the breeding period, and identification of individual differences in relation to the reproductive status. The increase in FGM levels as a response to capture and immobilization suggests that the measured metabolites are appropriate indicators of adrenal activity in these birds.

Non-invasive monitoring of adrenocortical activity in European badgers (Meles meles) and effects of sample collection and storage on faecal cortisol metabolite concentrations

Due to the increasing demand for methods to quantify adrenal activity in response to stressors in wild animals, we evaluated whether stimulation with adrenocorticopic hormone (ACTH) increases faecal cortisol metabolite levels in European badgers. We also conducted several experiments to evaluate the effect of storage time and collection methods on cortisol metabolite levels of faecal samples. Faecal samples were collected from five captive European badgers (Meles meles) and cortisol metabolites were quantified by an enzyme immunoassay. Faecal cortisol metabolite levels increased 1-2 days after ACTH injection and dropped to pre-treatment levels 3 days after ACTH stimulation. We found that cortisol metabolite levels were not affected by the 8-hour time lapse from collection in the field until freezing of faecal samples, environmental exposure (temperature 25.66 ± 2.55°C; range 21.20-32.20) or mean relative humidity 62.77 ± 9.90% (range 42.00-79.00). Cortisol metabolites were not homogenously distributed in the scats. Cortisol metabolite levels were significantly lower when faecal extracts and samples were frozen for long periods of time (14 weeks or more). Thus, the measurement of faecal cortisol metabolites is a suitable method for the non-invasive evaluation of adrenocortical activity in European badgers. However, some aspects related to the collection and storage of faecal samples should be considered in future studies conducted with this species.

Non-invasive monitoring of adrenocortical activity in Greater Rhea (Rhea americana) by measuring fecal glucocorticoid metabolites

The Greater Rhea (Rhea americana) is a near-threatened species. Wild populations are affected by human activities, such as illegal hunting, egg harvesting and conversion of natural habitats to croplands. An indicator of disturbances is the increase of the glucocorticoid corticosterone, a stress hormone that helps to cope with life-threatening situations. Here, we evaluate and characterize adrenocortical function in the Greater Rhea by validating the use of a radioimmunoassay (RIA) to assess fecal glucocorticoid metabolite concentrations, and by comparing the time course of plasma corticosterone and fecal immunoreactive glucocorticoid metabolite excretion. An adrenocorticotropic hormone (ACTH) challenge was performed and serial blood and fecal (cecal and rectal) samples were collected and analyzed by a corticosterone RIA. High-performance liquid chromatography (HPLC) was employed to characterize fecal immunoreactive glucocorticoid metabolites. HPLC analysis revealed the presence of two immunoreactive glucocorticoid metabolites in rectal and cecal Rhea feces. Greater Rheas responded to the ACTH challenge with a 30-fold increase in plasma corticosterone concentration 2 h post-injection, and a 30- to 40-fold increase in concentrations of glucocorticoid metabolites from rectal feces 4–6 h post-injection. In cecal feces, ACTH challenge produced an 11-fold increase. Therefore, collection of rectal feces would be more appropriated to ensure detection of minor increases in adrenocortical activity in this species. The adrenocortical response to ACTH found in Rhea was higher than what is usually observed in other birds and might be associated with their flightless condition and the use of running as an antipredator strategy.

Non-invasive monitoring of adrenocortical activity in free-ranging fallow deer (Dama dama L.)

Measurement of faecal glucocorticoid metabo- lites is increasingly used as a non-invasive tool to examine disturbances in various domestic and wild animals. Because measurements of faecal glucocorticoid metabolites has previously never been reported in fallow deer, we deter- mined 11,17-dioxoandrostanes (11,17-DOA), a group of cortisolmetabolites, in the faecesoffour fallow deeryearlings after an adrenocorticotropic hormone (ACTH) challenge or control salineinjectionbyan11-oxoaetiocholanoloneenzyme immunoassay (EIA), to validate a method. A 2.9- to 4.3- fold increase in measured cortisol metabolites in challenged animals after approximately 22 h demonstrated the suit- ability of this group-specific EIA to monitor adrenocortical activity in respective deer species. To determine faecal cortisol metabolites in fallow deer from a Mediterranean habitat, we collected samples during a 1-year study at Veliki Brijuni Island. The study confirmed seasonal pattern of cortisol release in fallow deer. Higher 11,17-DOA concentrations (median; min-max) were determined for November (99; 50-2,035), March (112; 25-315) and May (92; 40-196 ng/g faeces). Significantly lower concentra- tions were measured during July (30; 10-195 ng/g faeces). This study indicates that the analysis of faecal glucocorti- coid metabolites is a valuable non-invasive technique for monitoring adrenocortical activity in fallow deer. This, together with information about the seasonal pattern of glucocorticoid excretion, could help to improve fallow deer management and welfare, especially in the case of farmed and park animals.

Noninvasive monitoring of adrenocortical activity in carnivores by fecal glucocorticoid analyses

Noninvasive monitoring of adrenocortical activity in carnivores by fecal glucocorticoid analyses

Noninvasive monitoring of adrenocortical activity in carnivores by fecal glucocorticoid analyses

Measurement of glucocorticoid metabolites in feces has become an accepted method for the noninvasive evaluation of adrenocortical activity. The objective of this study was to determine if a simple cortisol enzyme immunoassay (EIA) was suitable for monitoring adrenocortical activity in a variety of carnivore species. Performance of the cortisol EIA was gauged by comparison to a corticosterone radioimmunoassay (RIA) that has been used for measuring glucocorticoid metabolites in feces of numerous species. Tests for parallelism and extraction efficiency were used to compare the cortisol EIA and corticosterone RIA across eight species of carnivores (Himalayan black bear, sloth bear, domestic cat, cheetah, clouded leopard, black-footed ferret, slender-tailed meerkat, and red wolf). The biological relevance of immunoreactive glucocorticoid metabolites in feces was established for at least one species of each Carnivora family studied with an adrenocorticotropic hormone (ACTH) challenge. High performance liquid chromatography (HPLC) analysis of fecal extracts for each species revealed (1) the presence of multiple immunoreactive glucocorticoid metabolites in feces, but (2) the two immunoassays measured different metabolites, and (3) there were differences across species in the number and polarities of metabolites identified between assay systems. ACTH challenge studies revealed increases in fecal metabolite concentrations measured by the cortisol EIA and corticosterone RIA of ∼228–1145% and ∼231–4150% above pre-treatment baseline, respectively, within 1–2 days of injection. Concentrations of fecal glucocorticoid metabolites measured by the cortisol EIA and corticosterone RIA during longitudinal evaluation (i.e., >50 days) of several species were significantly correlated ( P<0.0025, correlation coefficient range 0.383–0.975). Adrenocortical responses to physical and psychological stressors during longitudinal evaluations varied with the type of stimulus, between episodes of the same stimulus, and among species. Significant elevations of glucocorticoid metabolites were observed following some potentially stressful situations [anesthesia (2 of 3 subjects), restraint and saline injection (2 of 2 subjects), restraint and blood sampling (2 of 6 episodes), medical treatment (1 of 1 subject)], but not in all cases [e.g., gonadotropin injection ( n=4), physical restraint only ( n=1), mate introduction/breeding ( n=1), social tension ( n=1), construction ( n=2) or relocation ( n=1)]. Results reinforced the importance of an adequate baseline period of fecal sampling and frequent collections to assess adrenocortical status. The corticosterone RIA detected greater adrenocortical responses to exogenous ACTH and stressful exogenous stimuli in the Himalayan black bear, domestic cat (female), cheetah, clouded leopard, slender-tailed meerkat, and red wolf, whereas the cortisol EIA proved superior to resolving adrenocortical responses in the black-footed ferret and domestic cat (male). Overall results suggest the cortisol EIA tested in this study offers a practical method for laboratories restricted in the usage of radioisotopes (e.g., zoological institutions and field facilities) to integrate noninvasive monitoring of adrenocortical activity into studies of carnivore behavior and physiology.

Social, state-dependent and environmental modulation of faecal corticosteroid levels in free-ranging female spotted hyenas.

Little is known about to what extent the sensitivity of the hypothalamic-pituitary-adrenal (HPA) axis may be state dependent and vary in the same species between environments. Here we tested whether the faecal corticosteroid concentrations of matrilineal adult female spotted hyenas are influenced by social and reproductive status in adjacent ecosystems and whether they vary between periods with and without social stress. Females in the Serengeti National Park frequently become socially subordinate intruders in other hyena territories by undertaking long-distance foraging trips to migratory herds, whereas in the Ngorongoro Crater they usually forage inside their own small territories on resident prey. The faecal corticosteroid concentrations in Serengeti females were significantly higher than in Ngorongoro females. Energy expenditure by lactation is exceptionally high in spotted hyenas and this may be reflected in their corticosteroid levels. The faecal corticosteroid levels in both populations were higher in lactating than in non-lactating females. During periods of social stability, faecal corticosteroid concentrations increased in non-lactating females but not in lactating females as social status declined. Lactating Serengeti females had significantly higher faecal corticosteroid concentrations during periods with acute severe social stress than during periods without, indicating that the HPA axis is sensitive to social stimuli even in lactating females. So far few studies have used non-invasive monitoring methods for assessing social stress in freeranging animals. This study demonstrates for the first time, to the authors' knowledge, that corticosteroid concentrations may differ between periods with and without social stress for a free-ranging female mammal and that the modulating effect of social status may depend on reproductive status.

Noninvasive Monitoring of Adrenocortical Activity in Roe Deer (Capreolus capreolus) by Measurement of Fecal Cortisol Metabolites

A method for measuring glucocorticoids noninvasively in feces of roe deer was established and validated. The enzyme immunoassay (EIA) measures 11,17-dioxoandrostanes (11,17-DOA), a group of cortisol metabolites. Such measurement avoids blood sampling and reflects a dampened pattern of diurnal glucocorticoid secretion, providing an integrated measure of adrenocortical activity. After high-performance liquid chromatography, the presence of at least three different immunoreactive 11,17-DOA in the feces of roe deer was demonstrated. The physiological relevance of these fecal cortisol metabolites to adrenocortical activity was evaluated with an adrenocorticotropic hormone challenge test: cortisol metabolite concentrations exceeded pretreatment levels (31–78 ng/g) up to 13-fold (183–944 ng/g) within 8–23 h. Starting from basal levels between 13 and 71 ng/g, a suppression of adrenocortical activity after dexamethasone administration, indicated by metabolite levels close to the detection limit, was obtained 36–81 h after treatment, whereas unmetabolized dexamethasone was detectable in feces 12 h after its injection. Fecal glucocorticoid metabolite assessment via EIA is therefore of use in the monitoring of adrenocortical activity in roe deer. In a second experiment, capture, veterinary treatment, and transportation of animals were used as experimental stresses. This resulted in a 7.5-fold increase of fecal metabolites (1200 ± 880 ng/g, mean ± SD) compared to baseline concentrations. The administration of a long-acting tranquilizer (LAT), designed to minimize the physiological stress response, 2 days prior to a similar stress event led to a reduced stress response, resulting in only a 4-fold increase of fecal metabolites (650 ± 280 ng/g; mean ± SD). Therefore, LATs should be further investigated for their effectiveness in reducing stress responses in zoo and wild animals, e.g., when translocations are necessary.

Excretion of corticosteroids in urine and faeces of hares ( Lepus europaeus )

Increased production of glucocorticoids by the adrenal cortex is found in mammals under stress. As cortisol itself is absent in the faeces, an enzyme immunoassay (11-oxoaetiocholanolone) measuring 11,17-dioxoandrostanes has already been established to measure faecal cortisol metabolites in ruminants for non-invasive monitoring of adrenocortical activity. The aim of this study was to establish route and delay of excretion of glucocorticoids in hares and to determine whether a cortisol-, corticosterone- or this new enzyme immunoassay is best suited to detect faecal glucocorticoid metabolites. In the first experiment radioactive-labelled glucocorticoids (14C-cortisol and 3H-corticosterone) were administered intravenously to two groups of three hares in metabolic cages. All voided urine and faecal samples were collected for 4 days. Metabolites of both steroids were found predominantly in the urine (91 +/- 4%). Peak concentrations were observed in the first urinary sample following infusion (13 +/- 6 h) and in the faeces with a delay of about 1 day (23 +/- 7 h). Most of the radioactivity was not extractable with diethylether, indicating that the metabolites excreted in urine and faeces are mainly conjugated or polar unconjugated ones. This was confirmed by reverse-phase high-performance liquid chromatography separations of the metabolites, which also revealed marked differences concerning the metabolism of the two glucocorticoids injected. Compared with the cortisol and the corticosterone enzyme immunoassay, only the group-specific enzyme immunoassay for 11,17-dioxoandrostanes detected high quantities of immunoreactive metabolites. In a second experiment hares (n = 20) were stressed by rousing them three times (5 min, 10 min and another 5 min) with a 20-min break in-between. Faecal samples were collected 2 days before until 4 days after stress and analysed using the 11-oxoaetiocholanolone enzyme immunoassay. After stress significantly (P < 0.001) increased 11,17-dioxoandrostane concentrations were found. Based on these results, measuring 11,17-dioxoandrostanes in faeces enables non-invasive monitoring of disturbances in hares and thus provides a tool for field investigations elucidating the role of stress in hare populations.