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Faecal glucocorticoid metabolite (fGCM) analysis provides a non-invasive, feedback-free approach for monitoring adrenocortical responses to natural and anthropogenic stressors. The use of enzyme-immunoassays (EIAs) to quantify immunoreactive fGCMs has gained popularity in recent years but requires species-specific validation prior to first use. We conducted a pharmacological challenge with adrenocorticotropic hormone (ACTH) to determine whether changes in circulating glucocorticoids are reflected in fGCM, concentrations and therefore to validate excreta as a matrix for monitoring endocrine status in a southern African passerine, the White-browed Sparrow-weaver (Plocepasser mahali) and a columbid, the Laughing Dove (Spilopelia capensis). We tested the suitability of four EIAs to quantify fGCMs in 10 individuals of each species. Two of the EIAs, tetrahydrocorticosterone and 11-Oxoetiocholanolone II, detected significant elevations and were therefore most suitable for quantifying fGCMs in the White-browed Sparrow-weavers. In contrast, the 5α-pregnane-3β, 11β, 21-triol-20-one EIA detected the highest elevations in fGCM concentrations in the Laughing Doves. The lag time between stressor initiation (ACTH injection) and the resulting peak fGCM concentrations was ~2 h in both species. The validations presented here open opportunities for monitoring physiological responses in free-ranging individuals and contribute to our knowledge of the EIAs suitable for non-invasive quantification of avian fGCM concentrations. [ABSTRACT FROM AUTHOR]

Measuring fecal glucocorticoid metabolites (fGCMs) is a widely used, non-invasive method for studies of stress in vertebrates. To study physiological responses in wild Arctic foxes (Vulpes lagopus) to perceived stressors such as fluctuating food availability, occurrence of competitors and predators and disturbance from human activities, a species-specific physiological validation of a method to evaluate adrenocortical activity is needed. Here we used 15 captive Arctic foxes (both males and females and juveniles and adults) to investigate fGCM concentrations following ACTH injection (physiological validation), or handling alone and compared them with their respective baseline concentrations prior to the treatments. A 5α-pregnane-3ß,11ß,21-triol-20-one enzyme immunoassay measured significant fGCM increases following both treatments. The time lags to reach peak fGCM values were 9.3 ± 1.3 h and 12.8 ± 1.7 h for ACTH and handling treatment, respectively. Concentrations of fGCMs varied a lot between individuals, but not attributed to sex nor age of the foxes. However, we found a negative relationship between boldness and fGCM concentrations. Faecal glucocorticoid metabolites concentrations did not change significantly over a period of 48 h in samples kept at temperatures reflecting winter and summer means. This would allow the collection of samples up to two days old in the wild regardless of the season. We conclude that our successfully validated method for measuring fGCMs can be used as a non-invasive tool for studies exploring various stressors both in wild and captive Arctic foxes. [ABSTRACT FROM AUTHOR]

Quantification of glucocorticoids and their metabolites as a central component of the physiological stress response has many potential applications in ornithological research, but require careful species-specific validations. We established a reliable non-invasive method to measure faecal glucocorticoid metabolites (fGCM) in the southern yellow-billed hornbill (Tockus leucomelas). Following an adrenocorticotropic hormone (ACTH) challenge, plasma corticosterone levels increased by 110%, with a tetrahydrocorticosterone enzyme immunoassay (EIA) revealing a ~ fourfold increase in fGCM concentrations approximately 3 h post-injection. These data reveal droppings are a suitable matrix for monitoring stress-associated biomarkers in this species and suggest the tetrahydrocorticosterone EIA may often be appropriate for wild birds. [ABSTRACT FROM AUTHOR]

The non-invasive monitoring of physiological stress can provide conservation and wildlife managers with an invaluable tool for assessing animal welfare and psychological health of captive and free-ranging populations. A significant decrease in free-ranging primate populations globally and an increase in captive-housed primates have led to a need to monitor the stress and general welfare of these animals. We examined the suitability of three enzyme immunoassays (EIAs) for monitoring stress-related physiological responses in the samango monkey, Cercopithecus albogularis erythrarchus. We conducted an adrenocorticotropic hormone (ACTH) challenge on a male and female at the National Zoological Garden, Pretoria, South Africa. Individual faecal samples were collected 8 days pre- and post-ACTH administration and subsequently analysed for faecal glucocorticoid metabolite (fGCM) concentrations. During the study, biological stressors occurred for both the male and female. Two of the three EIAs tested (11-oxoetiocholanolone I and II) were able to reliably monitor fGCM alterations throughout the study period in both sexes. The 11-oxoetiocholanolone I EIA, however, had the lowest mean deviation from the calculated baseline value and was thus chosen as the preferred assay. Both the physiological activation of the stress response and the biological response to a stressor could be monitored with the chosen assay. The successful establishment of a reliable, non-invasive method for monitoring adrenocortical activity in C. albogularis erythrarchus will now allow conservationists, scientific researchers and wildlife managers to evaluate the level of stress experienced, and general welfare, by animals in captivity as well as free-ranging populations.

Faecal hormone monitoring offers a robust tool to non-invasively determine the physiological stress experienced by an individual when faced with natural or human-driven stressors. Although already quantified for several species, the method needs to be validated for each new species to ensure reliable quantification of the respective glucocorticoids. Here we investigated whether measurement of faecal glucocorticoid metabolite (fGCM) provides a feasible and non-invasive way to assess the physiological state of sugar gliders (Petaurus breviceps), an arboreal marsupial native to Australia, by using both a biological and physiological validation. Our analysis confirmed that the cortisol enzyme immunoassay (EIA) was the most appropriate assay for monitoring fGCM concentrations in sugar gliders. Comparing the fGCM response to the physiological and the biological validation, we found that while the administration of ACTH led to a significant increase in fGCM concentration in all individuals, only six of eight individuals showed a considerable fGCM response following the biological validation. Our study identified the most appropriate immunoassay for monitoring fGCM concentrations as an indicator of physiological stress in sugar gliders, but also supports recent suggestions that, if possible, both biological and physiological stressors should be used when testing the suitability of an EIA for a species. Monitoring faecal hormone metabolites offers a non-invasive technique for determining stress responses in wild species. Our study confirms the use of this technique for the marsupial sugar glider (Petaurus breviceps) by validating an appropriate enzyme immunoassay. We show that not only physiological validation, but also separation of individuals from their social group caused a detectable stress response. The validation of an appropriate assay allows the future use of non-invasive monitoring of physiological states in free-ranging sugar glider populations. [ABSTRACT FROM AUTHOR]

Reliable methods to measure stress-related glucocorticoid responses in free-ranging animals are important for wildlife management and conservation. Such methods are also paramount for our ability to improve our knowledge of the ecological consequences of physiological processes. The brownbear (Ursus arctos) is a large carnivore of ecological and cultural importance and is important for management. Here, we provide a physiological validation for an enzyme immunoassay (EIA) to quantify glucocorticoid metabolites in brown bear feces. We also provide an evaluationof the effects of sample exposure to ambient temperature on measured fecal glucocorticoid metabolite (fGCM) concentrations. We evaluated three EIA systems: a cortisol assay, an 11- oxoetiocholanolone assay, and an 11b-hydroxyetiocholanolone assay. Of these, the cortisol assay provided the best discrimination between peak fGCM concentrations detected 1–4 d after injections of synthetic adrenocorticotrophic hormone and preinjection baseline concentrations in four individual brown bears. The time of exposure to ambient temperature had substantial but variable effects on measured fGCM concentrations, including variation both between samples from the same individual and among samples from different bears. We propose that the validated EIA system for measuring fGCM concentrations in the brown bear could be a useful noninvasive method to monitor stress in this species. However, we highlight that this method requires that fecal samples be frozen immediately after defecation, which could be a limitation in many field situations. [ABSTRACT FROM AUTHOR]

The non-invasive monitoring of physiological stress can provide conservation and wildlife managers with an invaluable tool for assessing animal welfare and psychological health of captive and free-ranging populations. A significant decrease in free-ranging primate populations globally and an increase in captive-housed primates have led to a need to monitor the stress and general welfare of these animals. We examined the suitability of three enzyme immunoassays (EIAs) for monitoring stress-related physiological responses in the samango monkey, Cercopithecus albogularis erythrarchus. We conducted an adrenocorticotropic hormone (ACTH) challenge on a male and female at the National Zoological Garden, Pretoria, South Africa. Individual faecal samples were collected 8 days pre- and post-ACTH administration and subsequently analysed for faecal glucocorticoid metabolite (fGCM) concentrations. During the study, biological stressors occurred for both the male and female. Two of the three EIAs tested (11-oxoetiocholanolone I and II) were able to reliably monitor fGCM alterations throughout the study period in both sexes. The 11-oxoetiocholanolone I EIA, however, had the lowest mean deviation from the calculated baseline value and was thus chosen as the preferred assay. Both the physiological activation of the stress response and the biological response to a stressor could be monitored with the chosen assay. The successful establishment of a reliable, non-invasive method for monitoring adrenocortical activity in C. albogularis erythrarchus will now allow conservationists, scientific researchers and wildlife managers to evaluate the level of stress experienced, and general welfare, by animals in captivity as well as free-ranging populations. [ABSTRACT FROM AUTHOR]

Highlights • Faeces are a suitable matrix for monitoring stress in the southern pied babbler. • Captive babblers have much higher stress levels than wild individuals. • Dominant male babblers are more stressed than subordinate males. • There is no difference in stress levels between dominant and subordinate females. Abstract Using faecal matter to monitor stress levels in animals non-invasively is a powerful technique for elucidating the effects of biotic and abiotic stressors on free-living animals. To validate the use of droppings for measuring stress in southern pied babblers (Turdoides bicolor) we performed an ACTH challenge on captive individuals and determined the effect of temporary separation from their social group on their faecal glucocorticoid metabolite (fGCM) concentration. Additionally, we compared fGCM concentrations of captive babblers to those of wild conspecifics and examined the effects of dominance rank on fGCM concentration. We found droppings to be a suitable matrix for measuring physiological stress in babblers and that individual separation from the group caused an increase in fGCM levels. In addition, babblers temporarily held in captivity had substantially higher fGCM concentrations than wild individuals, indicating that babblers kept in captivity experience high levels of stress. In wild, free-living individuals, dominant males showed the highest levels of stress, suggesting that being the dominant male of a highly territorial social group is stressful. Non-invasive sampling allows field-based researchers to reduce disturbance related to monitoring adrenocortical function, thereby avoiding artificially increasing circulating corticosterone concentration as it is not necessary to physically restrain study animals. [ABSTRACT FROM AUTHOR]

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.

Abstract Entire male pigs display more aggressive and sexual behaviour. This might cause a condition of chronic stress and impair their welfare. In order to assess chronic stress in entire and castrated male pigs, as well as effects of providing grass silage as occupational and feed material on behaviour and health, we carried out a 2 × 2 × 2-factorial experiment with 147 growing-finishing pigs. Factors investigated were castration (entire/castrated), chronic intermittent social stress exposure (yes/no) and access to grass silage (yes/no), as well as their interactions. The stress exposure treatment consisted of repeated short-term confrontations and separations. We recorded different behavioural variables, circadian rhythm of salivary cortisol, response to an ACTH (adrenocorticotropic hormone) challenge test, pathological changes in the gastric mucosa and morphology of the intestinal epithelium. Stress exposure caused a decrease in posture changes and head knocks/bites in the home pen. Reference indicators affected by stress exposure did not differ between entire and castrated male pigs, indicating that there is no permanently increased baseline level of stress in entire male pigs. However, entire males responded more pronouncedly to the stress exposure compared to castrated males in terms of posture changes and play behaviour. Pigs provided with grass silage showed more play behaviour and less manipulative behaviours than pigs not receiving grass silage. Stress treated pigs had more hyperkeratosis in the gastric mucosa and gastric ulcers, while offering grass silage reduced such changes. In conclusion, our results indicate that the increased behavioural stress response of entire male pigs might require some adaptations in housing and management of entire male pigs. Gastric ulceration scoring turned out to be a potential post mortem indicator for chronic stress. Finally, providing roughages like grass silage could be a means to positively affect behaviour and gastric health in pigs. Graphical abstract Unlabelled Image Highlights • We studied effects of castration, chronic intermittent stress and roughage in pigs. • Chronic intermittent social stress exposure impaired gastric health. • Baseline level of stress was not increased in entire male pigs. • Entire males still showed a more pronounced behavioural response to stress exposure. • Providing grass silage improved gastric health and reduced pen mate manipulation. [ABSTRACT FROM AUTHOR]

Dominant females of cooperative breeding species often use aggression to suppress reproduction of subordinate females, resulting in subordinates experiencing stress-related increases in glucocorticoid levels, which may cause reproductive down-regulation. This would suggest a general pattern with higher glucocorticoid levels in subordinate compared to dominant individuals; however, the opposite was found in a number of cooperatively breeding species. Furthermore, breeding females of the cooperatively breeding Damaraland mole-rats ( Fukomys damarensis ) exhibit very high androgen concentrations during the wet season, presumably to support their breeding monopoly. Hormone analysis in Damaraland mole-rats have typically been measured using plasma and urine, but faecal analysis offers additional advantages especially for field studies on this species. The present study examines the suitability of Damaraland mole-rat faecal samples for determining glucocorticoid metabolite (fGCM) and androgen metabolite (fAM) concentrations using enzyme immunoassays. Using these assays, we further evaluated the effects of breeding status on fGCM and fAM concentrations in wild-caught and captive Damaraland mole-rats. Wild-caught breeding and non-breeding males and females exhibited no differences in fAM concentrations. Immunoreactive fGCM concentrations were only high in male breeders and comparatively low in non-breeders and breeding females. Concentrations of fAMs and fGCMs were similar in captive males and females, but fAM concentrations were elevated in captive compared to wild-caught individuals, which may be related to a higher reproductive activity due to removal from the breeding female. The relatively uniform fAM and fGCM concentrations found in wild-caught mole-rats may be explained by a stable colony structure during the dry season during which this study was conducted. Limited dispersal opportunities result in lower aggression and stress levels within a colony and as a result lower fAM and fGCM concentrations. [ABSTRACT FROM AUTHOR]

Leopards Panthera pardus are classified in the IUCN Red List as Vulnerable, primarily due to habitat loss, natural prey base depletion and exploitation caused by various anthropogenic activities. Although protected areas are important for leopard conservation, the majority of suitable leopard habitat lies beyond protected area boundaries exposing individuals to different environmental, physiological and psychosocial stressors. This study aimed to examine the suitability of five different enzyme immunoassays (EIAs) for monitoring adrenocortical function in the leopard based on faecal glucocorticoid metabolite (fGCM) analysis. After performing an adrenocorticotrophic hormone (ACTH) stimulation test and investigating the stability of fGCM post-defaecation, faeces from free-ranging leopards in a peri-urban and a conservation area were collected to investigate the potential impact of habitat variability on glucocorticoid output. An EIA measuring fGCM with a 5-3-11-diol structure performed best, demonstrating a 200%-330% increase in fGCM concentrations approximately 40 h post-ACTH administration. Concentrations of fGCM remained quite stable for up to six days post-defaecation and showed a maximum increase of 8% and a maximum decrease of 9%. Although not significantly different, overall median fGCM concentrations were 68% higher in individuals utilising the peri-urban area compared with leopards utilising the conservation area. The ranges of fGCM concentrations between sites, however, were similar. Individual median fGCM concentrations differed distinctly between free-ranging males and females, possibly linked to female reproductive status. The established method can now assist in addressing some of the issues facing local wildlife managers, conservationists and researchers tackling various aspects related to leopard conservation and management under different land-use practices. [ABSTRACT FROM AUTHOR]

Opportunistic breeding is a strategy used to maximize reproductive success in unpredictable environments. Birds that breed opportunistically are thought to maintain partial activation of the reproductive axis in order to rapidly initiate breeding when environmental conditions become suitable. The physiological mechanisms that modulate reproduction in seasonally breeding birds have been well explored. In contrast, the physiological mechanisms that allow opportunistic breeding birds to maintain a continued state of reproductive readiness has not been well established. Here, we tested the hypothesis that reproductive readiness is modulated through condition-mediated effects on the hypothalamic–pituitaryadrenal (HPA) axis and its downstream effects on corticosterone (CORT) secretion in wild zebra finches ( Taeniopygia guttata ). We examined the variation in body condition, HPA-axis activity (endogenous and adrenocorticotropic hormone (ACTH)-induced responses), and hypothalamic-pituitary-gonadal (HPG) axis activity activity (baseline and gonadotropin-releasing hormone (GnRH) induced testosterone and estradiol levels) in zebra finches across five sites in the Northern Territory in Australia. We found that birds at the sites in the lowest condition had the highest level of baseline and peak CORT. Additionally, males at the sites in the lowest condition had the highest fold increase in testosterone following a GnRH challenge. Across sites, birds with low body condition had high baseline, peak, and ACTH-induced levels of CORT. Our data suggest that reproductive readiness in opportunistically breeding birds is modulated by condition-mediated trade-offs between the HPA- and the HPG-axes. Further work is needed to understand the environmental conditions that influence reproductive activation in opportunistically breeding birds. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• We established enzyme immunoassays (EIAs) to measure fecal hormones in pygmy hippos. • Fecal glucocorticoid metabolites (FGMs) were best assessed by the 5α-3β,11β-diol-CM EIA. • Females experiencing chronic illness and conspecific aggression had higher FGMs. • In males, fecal androgen metabolite concentrations are highest over spring-summer. • Adult males have higher fecal androgen levels than adult females and juvenile males. The pygmy hippopotamus (Choeropsis liberiensis) is an endangered species endemic to the Upper Guinea Forest ecosystem in West Africa. We have limited information concerning the species' reproduction and well-being under managed care. We therefore developed non-invasive methods for characterizing gonadal androgen and adrenal hormone profiles in pygmy hippos using fecal samples collected from 12 males and 12 females housed in North American zoological institutions. We aimed to: 1) identify and validate enzyme immunoassays (EIAs) for measuring metabolites of corticosteroids and testosterone in feces; and 2) test whether gonadal activity is correlated with previous breeding history, season or type of housing. For glucocorticoids, several EIAs for measuring metabolites were investigated. A group-specific EIA exhibiting cross-reactivity with 11,17-dioxoandrostane (DOA) metabolites of cortisol most clearly reflected adrenocortical activity in response to pharmocological challenge with adrenocorticotropic hormone (ACTH) in both males and females. However, day-to-day concentrations of this metabolite in the feces of pygmy hippos that did not undergo ACTH challenge were near the detection limits of the assay, making this EIA impractical for assessing glucocorticoid activity in this species. Another group-specific EIA, exhibiting cross-reactivity with 5α-pregnane-3β,11β,21-triol-20-one, produced biologically relevant data and evidence of an appropriate response to pharmacological challenge with exogenous ACTH. The testosterone metabolite assay C196 (Arbor Assays, Ann Arbor, Michigan, USA) also produced biologically coherent data: adult males exhibited the highest mean androgen metabolite concentrations (477 ng/g), followed by adult females (259 ng/g) and juvenile males (160 ng/g). Proven breeding males had higher, but not significantly different, mean concentrations (472 ng/g) to unproven males (352 ng/g; P = 0.400). Similarly, adult males housed outdoors year-round in subtropical climates exhibited higher, but not statistically different mean concentrations (554 ng/g) to males in temperate climates that were housed indoors at least part of the year (412 ng/g; P = 0.208). There were, however, significant differences in mean concentrations among seasons for adult males, with higher values in spring (546 ng/g) and summer (542 ng/g) than in autumn (426 ng/g) and winter (388 ng/g, P = 0.003). In conclusion, we identified EIAs for the measurement of fecal metabolites of androgens and glucocorticoids that can be used for further studies to monitor gonadal activity in male pygmy hippos and adrenocortical activity in both sexes. We also identified a seasonal trend in male gonadal activity in this species under managed care in North America. Finally, our findings highlight an important consideration when using non-invasive methods for evaluating fecal cortisol metabolites: ACTH used for pharmacological validation of an EIA does not necessarily equate to biological relevance. [ABSTRACT FROM AUTHOR]

Fecal steroid analysis is a powerful noninvasive tool for behavioral endocrinology, but enzyme immunoassays (EIAs) require experimental validation before they are applied. We conducted a physiological validation of an in-house EIA measuring fecal cortisol metabolites (FCMs) by performing an adrenocorticotrophic hormone (ACTH) challenge, dexamethasone suppression, and control saline solution injection experiment with six male and five female bearded capuchins ( Sapajus libidinosus). We also took advantage of a presumably stressful incident to perform a biological validation for females. In addition, we conducted high-performance liquid chromatography (HPLC) immunograms to characterize the FCMs measured in both sexes of bearded capuchin, and in a closely related species ( S. nigritus, black capuchin monkeys). Male and female S. libidinosus showed FCM peaks after ACTH injections, and females also showed FCM peaks in the biological validation. Three of four individuals (two males and one female) had an FCM peak shortly after injection of dexamethasone and both sexes then showed prolonged low FCM levels after dexamethasone injection. We observed no effects of saline solution injections. The time to peak FCM excretion after ACTH injection or a stressful incident varied 1.5-8.5 h. HPLC results revealed no differences in FCM profiles between sexes or species and suggest that the EIA can also be used in male and female S. nigritus. Our results validate an in-house EIA for both sexes of S. libidinosus but show large individual variation in FCM excretion, which highlights the need for carefully planned feces collection in endocrinologial research. [ABSTRACT FROM AUTHOR]

The development of non-invasive techniques to analyse physiological stress in mammalian species has revolutionised field-based endocrinology. However, careful validation of the methods used to determine faecal glucocorticoid metabolite (fGCM) and other hormone concentrations are required on a species- and sex-specific basis. In this study, we performed an adrenocorticotropic hormone (ACTH) stimulation test on four (two male and two female) captive vervet monkeys ( Chlorocebus pygerythrus ) to determine the most appropriate enzyme immunoassay (EIA) from a suite of available EIAs. Furthermore, we took advantage of a potentially stressful event in our wild vervet population from Samara Private Game Reserve, South Africa, to examine if an alpha–beta female rank reversal increases the physiological stress of those individuals directly involved, as well as other group members. Both our physiological and biological validation studies revealed that a cortisol assay was the most appropriate EIA for monitoring fGCM alterations in vervet monkeys. In addition, we found that the observed rank-reversal had no significant effect on the physiological stress levels of uninvolved group members. Our study highlights that physiological validation is imperative and, where possible, should be conducted in parallel with a carefully considered biologically-relevant test under natural conditions. Overall, our results provide a necessary step for future studies to examine physiological stress of vervet monkeys via fGCM monitoring by validating a suitable EIA for this species. This paves the way for future research into the health and welfare of both captive and wild vervet monkeys, and will allow researchers to assess the behavioural, social and ecological correlates of physiological stress levels of this species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (2.72 ng/mL) and virtually absent in non-breeding males (0.04 ng/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 30 min and then increasing for the next 60 min. 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. [ABSTRACT FROM AUTHOR]

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 3 H-corticosterone and 3 H-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: 4 h vs 6 h) and feces (6 h vs 8 h). 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 4–8 h after injection. In conclusion, both tested EIAs proved suited for a noninvasive measurement of glucocorticoids and androgens in bank voles. [ABSTRACT FROM AUTHOR]

The ocelot (Leopardus pardalis) is included in list of wild felid species protected by CITES and is part of conservation strategies that necessarily involve the use of assisted reproduction techniques, which requires practical and minimally invasive techniques of high reproducibility that permit the study of animal reproductive physiology. The objective of this study was to compare and validate two commercial assays: ImmuChem Double Antibody Corticosterone 125I RIA from ICN Biomedicals, Costa Mesa, CA, USA; and Coat-a-Count Cortisol 125I RIA from DPC, Los Angeles, CA, USA, for assessment of fecal glucocorticoid metabolites in ocelots submitted to ACTH (adrenocorticotropic hormone) challenge. Fecal samples were collected from five ocelots kept at the Brazilian Center of Neotropical Felines, Associação Mata Ciliar, São Paulo, Brazil, and one of the animals was chosen as a negative control. The experiment was conducted over a period of 9 days. On day 0, a total dose of 100 IU ACTH was administered intramuscularly. Immediately after collection the samples were stored at 20C in labeled plastic bags. The hormone metabolites were subsequently extracted and assayed using the two commercial kits. Previously it was performed a trial with the DPC kit to check the best extraction method for hormones metabolites. Data were analyzed with the SAS program for Windows V8 and reported as means ± SEM. The Schwarzenberger extraction method was slightly better when compared with the Wasser extraction method (103,334.56 ± 19,010.37ng/g of wet feces and 59,223.61 ± 12,725.36ng/g of wet feces respectively; P=0,0657). The ICN kit detected an increase in glucocorticoid metabolite concentrations in a more reliable manner. Metabolite concentrations (ng/g wet feces) on day 0 and day 1 were 66,956.28 ± 36,786.93 and 92,991.19 ± 28,555.63 for the DPC kit, and 205,483.32 ± 83,811.32 and 814,578.75 ± 292,150.47 for the ICN kit, respectively. The limit of detection for the ICN kit was 7.7 ng/mL for 100% B/Bo (25ng/mL for 88%B/Bo) and for the DPC kit it was 0.2ug/dL for 90.95% B/Bo (1ug/dL for 81.27% B/Bo). In conclusion it was confirmed that the Schwarzenberger extraction method and the ICN kit are superior for extracting and measuring fecal glucocorticoid metabolites in ocelot fecal samples.A jaguatirica (Leopardus pardalis) é uma das espécies de felídeos selvagens listadas no CITES e integrantes de planos de conservação que necessariamente envolvem o emprego de técnicas de reprodução assistida. Para isso, é fundamental o desenvolvimento de técnicas práticas, de alta repetibilidade e pouco invasivas, que permitam o estudo da fisiologia reprodutiva animal. Objetivou-se neste experimento comparar e validar os ensaios realizados com os conjuntos comerciais ImmuChem Double Antibody Corticosterone 125I RIE da ICN Biomedicals, Costa Mesa, CA, USA e Coat-a-count Cortisol 125I RIE da DPC, Los Angeles, CA, USA, na mensuração dos metabólitos de glicocorticóides das fezes de jaguatiricas por meio do desafio com ACTH. Para este estudo foram utilizadas amostras fecais de cinco indivíduos que se encontravam no Centro Brasileiro de Felinos Neotropicais, Associação Mata Ciliar, Pedreira, SP, Brasil, sendo que um destes animais foi escolhido como controle negativo. O experimento foi conduzido em um período de nove dias. No dia 0 foi administrada uma dose total de 100 UI IM de ACTH (hormônio adrenocorticotrópico). Imediatamente após a colheita, as amostras de fezes foram conservadas a -20ºC em sacos plásticos identificados e posteriormente os metabólitos hormonais foram extraídos e mensurados utilizando-se os dois conjuntos comerciais. Previamente foi realizado com o conjunto comercial da DPC um teste para comparar dois métodos de extração. Os dados foram analisados utilizando o programa The SAS System for Windows V8. A média ± erro padrão dos valores obtidos pela técnica de Schwarzenberger foi levemente superior à média dos valores obtidos pela técnica de Wasser (103.334,56 ± 19.010,37ng/g de fezes úmidas e 59.223,61 ± 12.725,36ng/g de fezes úmidas, respectivamente; P=0.0657). O conjunto comercial da ICN detectou com maior clareza o aumento das concentrações dos metabólitos de glicocorticóides. Os valores do dia 0 e do dia 1 para os conjuntos comerciais DPC e ICN foram respectivamente 66.956,28 ± 36.786,93ng/g de fezes úmidas e 92.991,19 ± 28.555,63ng/g de fezes úmidas; 205.483,32 ± 83.811,32ng/g de fezes úmidas e 814.578,75 ± 292.150,47ng/g fezes úmidas. Para o conjunto comercial da ICN a sensibilidade foi de 7,7ng/mL para 100% B/ Bo (25 ng/mL para 88%B/Bo) e para o conjunto comercial da DPC foi de 0,2ug/dL para 90,95% B/Bo (1ug/dL para 81,27% B/Bo). Confirmou-se, portanto, que a melhor técnica de extração foi a descrita por Schwarzenberger e que o conjunto comercial da ICN Biomedicals teve um melhor desempenho para a quantificação de metabólitos de glicocorticóides em fezes de jaguatiricas.

Background: Numbers of giraffes are declining rapidly in their native habitat. As giraffe research and conservation efforts increase, the demand for more complete measures of the impact of conservation interventions and the effects of captive environments on animal health and welfare have risen. We compared the ability of six different enzyme immunoassays to quantify changes in fecal glucocorticoid metabolites (FGM) resulting from three sources: adrenocorticotropic hormone stimulation test, transport, and time of day that samples were collected. Results: Two male giraffes underwent ACTH injections; all six assays detected FGM increases following injection for Giraffe 1, while only three assays detected FGM increases following injection for Giraffe 2. Consistent with other ruminant species, the two 11-oxoetiocholanolone assays (one for 11,17-dioxoandrostanes and the other for 3α,11-oxo metabolites) measured the most pronounced and prolonged elevation of FGM, while an assay for 3β,11β-diol detected peaks of smaller magnitude and duration. Both of the 11-oxoetiocholanolone assays detected significant FGM increases after transport in Giraffes 3-7, and preliminary data suggest FGM detected by the assay for 11,17-dioxoandrostanes may differ across time of day. Conclusions: We conclude the assay for 11,17-dioxoandrostanes is the most sensitive assay tested for FGM in giraffes and the assay for FGM with a 5β-3α-ol-11-one structure is also effective. 11-oxoetiocholanolone enzyme immunoassays have now been demonstrated to be successful in a wide variety of ruminant species, providing indirect evidence that 5β-reduction may be a common metabolic pathway for glucocorticoids in ruminants. As FGM peaks were detected in at least some giraffes using all assays tested, giraffes appear to excrete a wide variety of different FGM. The assays validated here will provide a valuable tool for research on the health, welfare, and conservation of giraffes. [ABSTRACT FROM AUTHOR]

• ACTH administration results in an increase in plasma GC and FGM levels. • A non-invasive method for quantifying echidna FGMs has been validated. • Echidna adrenocortical responses seem not less involved than in other mammals. The monotreme adrenocortical response to stress may not rely as heavily on the hypothalamic–pituitaryadrenal (HPA) axis compared to other mammals. This study aimed to validate a technique in which glucocorticoid metabolites could be quantified non-invasively in short-beaked echidna faeces by examining the secretion of glucocorticoids (GC) using an adrenocorticotrophic hormone (ACTH) challenge on sexually mature captive echidnas. Echidnas were housed individually for 15 days, with the ACTH challenge occurring on day five. Blood samples were collected on day five during the challenge and faecal samples were collected each morning for the 15 days. Both sample types were analysed for glucocorticoids (GC) or its metabolites. Plasma corticosterone concentrations increased significantly after 30 min and 60 min relative to time 0, whilst plasma cortisol concentrations increased significantly after 60 min. The ACTH challenge also resulted in an increase in glucocorticoid metabolite concentration in faecal samples from four of the six echidnas detected one to two days post ACTH injection, thereby validating a non-invasive method to assess adrenal response in the echidna. These results confirm that echidnas respond to a synthetic ACTH challenge in a similar manner to that of eutherian species indicating that echidnas appear to use the HPA axis in their stress response. [ABSTRACT FROM AUTHOR]

Animals in captivity and in the wild face numerous challenges, including the risk of enduring acute or chronic stress. In captivity, facilities attempt to alleviate the risk of chronic stress by providing environmental enrichment, shown to minimize behavioral disorders and stress in several species. One potential form of enrichment in zoos is training animals to provide rides for guests, however, the effect of this activity on the welfare of individual animals has never been examined. We validated the use of saliva for assessing stress in dromedary camels ( Camelus dromedarius ), an animal commonly used for rides. We then measured variation in salivary cortisol in four male camels while providing rides of differing frequency for guests at the Toronto Zoo. The camels were sampled during the ride season (June to September) using four treatments: (1) in their pasture, (2) at the ride area when not performing rides, (3) while providing a low number of rides ( n = 50/day) and (4) while providing a high number of rides ( n = 150/day). Furthermore, samples were taken before and after the ride season for comparison. There was a significant difference between the post-ride season treatment and the three treatments involving guest presence during the ride season (ride area, low rides, high rides). In general, cortisol concentrations were lower during the ride season and higher during the non-ride season. Based on the metrics we used, performing rides is not a stressful experience for these dromedary camels and suggests that rides may be a form of enrichment. [ABSTRACT FROM AUTHOR]

Highlights: [•] Validated EIA to quantify hair cortisol concentrations in Canada lynx. [•] Demonstrated hair cortisol concentration increases after ACTH challenge in captive lynx. [•] Showed hair cortisol concentration does not vary with age and sex, but does vary with body region. [•] Suggested hair cortisol concentration samples be collected from a standardized region. [ABSTRACT FROM AUTHOR]

Environmental changes result in physiological responses of organisms, which can adversely affect population dynamics and reduce resistance to disease. These changes are expressed in chronic levels of stress. The measurement of glucocorticoid (GC) concentrations in faeces is a non-invasive method for monitoring stress in wildlife. The metabolism and excretion of steroids differ significantly between species and, as a consequence, non-invasive methods must be physiologically validated for each species. Koalas (Phascolarctos cinereus) are declining in numbers through much of their range. The role of chronic stress in koala populations has not been identified. Prior to the assessment of faecal GC concentrations in wild koala populations, the excretion timing and concentrations of GCs need to be determined. In this study, we assessed a method for identifying and measuring the concentrations of GC metabolites in faecal pellets of captive koalas following ACTH treatment. The results show that an elevation of plasma cortisol concentrations, using sustained release of ACTH, results in elevated concentrations of faecal cortisol/cortisol metabolites. Taking into account the excretion time lag, an increase in faecal cortisol metabolite concentrations corresponds to the release of GCs from the adrenal cortex as early as 36 h before faecal pellet collection. The calculations of steroid partitioning of plasma cortisol showed that the ACTH-stimulated values were significantly different from the control values for the concentrations of free, corticosteroid-binding globulin-bound and albumin-bound cortisol. This study validates the use of faecal cortisol analysis to assess the activity of the hypothalamo-pituitary-adrenocortical axis in freshly collected koala faecal pellets and indicates that the method should be suitable to assess the adrenocortical status of koalas in wild populations. [ABSTRACT FROM AUTHOR]

Analysis of fecal glucocorticoid (GC) metabolites has recently become the standard method to monitor adrenocortical activity in primates noninvasively. However, given variation in the production, metabolism, and excretion of GCs across species and even between sexes, there are no standard methods that are universally applicable. In particular, it is important to validate assays intended to measure GC production, test extraction and storage procedures, and consider the time course of GC metabolite excretion relative to the production and circulation of the native hormones. This study examines these four methodological aspects of fecal GC metabolite analysis in tufted capuchins ( Cebus apella). Specifically, we conducted an adrenocorticotrophic hormone (ACTH) challenge on one male and one female capuchin to test the validity of four GC enzyme immunoassays (EIAs) and document the time course characterizing GC metabolite excretion in this species. In addition, we compare a common field-friendly technique for extracting fecal GC metabolites to an established laboratory extraction methodology and test for effects of storing 'field extracts' for up to 1 yr. Results suggest that a corticosterone EIA is most sensitive to changes in GC production, provides reliable measures when extracted according to the field method, and measures GC metabolites which remain highly stable after even 12 mo of storage. Further, the time course of GC metabolite excretion is shorter than that described yet for any primate taxa. These results provide guidelines for studies of GCs in tufted capuchins, and underscore the importance of validating methods for fecal hormone analysis for each species of interest. [ABSTRACT FROM AUTHOR]

Abstract: The non-invasive measurement of adrenocortical function in cheetahs is an important tool to assess stress in captive and free-ranging individuals, because stress has been suggested to be one of the causes of poor reproductive performance of captive cheetahs. We tested four enzyme immunoassays (EIA) in two captive cheetahs in Germany using adrenocorticotropic hormone (ACTH) challenges and identified the corticosterone-3-CMO EIA to be most sensitive to the increase in faecal glucocorticoid metabolite (fGCM) concentrations after the ACTH challenge. This EIA performed also well in five captive cheetahs in South Africa. The fGCM concentrations across all seven cheetahs increased within 24h by 681% compared to the baseline levels prior to ACTH. Storage of faecal samples at 0–4°C did not strongly affect fGCM concentrations within 24h, simplifying sample collection when immediate storage at −20°C is not feasible. The two cheetahs in Germany also received an injection of [3H]cortisol to characterise fGCMs in faecal extracts using high-performance liquid chromatography (HPLC) immunograms. HPLC fractions were measured for their radioactivity and immunoreactive fGCM concentrations with the corticosterone-3-CMO EIA, respectively. The results revealed a polar peak of radiolabelled cortisol metabolites co-eluting with the major peak of immunoreactive fGCMs. Thus, our EIA measured substantial amounts of fGCMs corresponding to the radioactive peaks. The peaks were of higher polarity than native cortisol and corticosterone, suggesting that the metabolites were conjugated, which was confirmed by solvolysis of the HPLC fractions. Our results show that the corticosterone-3-CMO EIA is a reliable tool to assess fGCMs in cheetahs. [Copyright &y& Elsevier]

The budgerigar ( Melopsittacus undulatus) is a small parrot native to Australia that is commonly held in zoos, laboratories, and private homes. Assessment of budgerigar stress levels would aid welfare monitoring and improve our understanding of their biology. Analyzing fecal glucocorticoid metabolites provides a noninvasive method to measure stress levels in birds. For this method to be reliable, the antibody to be used in an immunoassay must be carefully selected for each species, and validation must be performed. A common limitation in many existing assays is the inability to accurately detect variable fecal glucocorticoid metabolites in minute quantities of feces, requiring small samples to be combined. We have developed a double antibody radioimmunoassay protocol based on a commercially available 125I-corticosterone radioimmunoassay kit for use in detecting fecal glucocorticoid metabolites in small quantities (<20 mg) of budgerigar droppings. The assay was validated pharmacologically with an adrenocorticotropic hormone challenge and with oral administration of corticosterone. Our validation has demonstrated our assay is both sensitive and a reliable approach to noninvasive monitoring of stress in budgerigars. Zoo Biol. 32:112-116, 2013. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: The objectives of this study were to investigate whether; 1) temperament or 2) behavioral responsiveness to humans, can affect the behavior, physiology and productivity of dairy cows being milked in a familiar and novel milking environment. Temperament of multi-parous cows was defined based on exit time from a restraint device, as High Responders (HR; n=10), Medium Responders (MR; n=10) or Low Responders (LR; n=10). The behavioral response of cows to humans was assessed using four tests: restraint, exit speed, avoidance distance test and a voluntary approach test. Cows were milked according to their established routines in a rotary (familiar) milking parlor and behavioral, physiological and production data were collected over five consecutive days, including heart rate, cortisol and oxytocin concentrations and milk yield. The following week, cows were milked in a novel environment (herringbone parlor within the same farm facility) over five consecutive days, and the data and sample collection program was repeated. Cows were then given an exogenous adrenocorticotropic hormone (ACTH) challenge to measure adrenal responsiveness. Exit time was negatively correlated with the behavioral responses of cows to restraint and human avoidance distance (HAD) in the paddock and arena. The behavioral response of cows to the milking process was greater in MR than LR and HR cows in the familiar and novel milking environments. Milk yields were greater in LR than HR cows in the novel but not the familiar milking parlor. Oxytocin concentrations increased during milking in the novel environment, regardless of cow temperament. In the familiar and novel environments, heart rates were higher in HR than LR cows before and during milking and rMSSD was lower in HR cows during milking in a novel environment. There was no difference in cortisol concentrations between LR and HR cows in response to an ACTH challenge, but HR cows had higher baseline cortisol levels than LR cows. The number of leg lifts cows performed when restrained in the crush was associated with several physiological and milk production measures. These results indicate that cows with different temperaments vary in their basal physiology as well as their behavioral and physiological responses to milking and stress associated with being milked in a novel environment. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: Improving the husbandry in the southern three-banded armadillo (Tolypeutes matacus) through gaining knowledge of its stress physiology is imperative to maintaining a healthy, zoo-housed population. Our objectives were to: 1) validate the use of fecal hormone analysis for monitoring adrenocortical activity using both an adrenocorticotropic hormone (ACTH) challenge and biological events; and 2) characterize longitudinal adrenocortical activity in male and female southern three-banded armadillos. An ACTH injection was given intra-muscularly to one male (4IU/kg; 5.6IU total) and one female (5.5IU/kg; 8IU total) southern three-banded armadillo. Fecal samples were collected 1day pre- and continued 5days post-ACTH to capture the physiological response measured by elevated fecal glucocorticoid metabolites (FGM) to validate these techniques. Additionally, natural and routine events, including pairing individuals for breeding and veterinary procedures/handling, were used to biologically validate these techniques. To characterize adrenocortical activity, fecal samples (∼3025 total; n =275/animal/yr) were collected from 11 (5 males; 6 females) southern three-banded armadillos 5–7 times a week for 1year at Lincoln Park Zoo (Chicago, IL). A cortisol enzyme immunoassay was used for FGM analysis. The ACTH challenge in the male resulted in a twofold increase of FGM (1123.2±36.2ng/g dry feces) above baseline (675.7±10.0ng/g dry feces) at approximately 54–94h post- injection. The female exhibited a twofold increase (1635.4ng/g dry feces) over baseline FGMs (608.5±12.3ng/g dry feces) approximately 30h post-injection. Reproductive behaviors and veterinary procedures resulted in elevated FGM concentrations from all individuals except for one male. The longitudinal characterization demonstrated that sex and season did not influence (P <0.05) FGM concentrations. Individuals were highly variable with mean FGM concentration of 2010.1±862.4ng/g dry feces (range, 816.3–7889.1ng/g dry feces). Mean FGM baseline concentration was 878.5±201.8ng/g dry feces (range, 475.2–1955.5ng/g dry feces) with a mean elevated FGM concentrations of 2694.3±1111.4ng/g dry feces (range, 1110.3–10,683.3ng/g dry feces). This study provides the foundation for future research on how the environment directly affects the adrenocortical activity in this species of armadillo. [Copyright &y& Elsevier]

Jaguars are threatened with extinction throughout their range. A sustainable captive population can serve as a hedge against extinction, but only if they are healthy and reproduce. Understanding how jaguars respond to stressors may help improve the captive environment and enhance their wellbeing. Thus, our objectives were to: (1) conduct an adrenocorticotrophic hormone (ACTH) challenge to validate a cortisol radioimmunoassay (RIA) for noninvasive monitoring of adrenocortical function in jaguars; (2) investigate the relationship between fecal corticoid (FCM) and androgen metabolite (FAM) concentrations in males during the ACTH challenge; and (3) establish a range of physiological concentrations of FCMs for the proposed protocol. Seven jaguars (3 M, 4 F) received 500 IU/animal of ACTH. Pre- and post-ACTH fecal samples were assayed for corticoid (M and F) and androgen metabolites (M) by RIA. Concentrations of FCMs increased ( P80.01) after ACTH injection (pre-ACTH: 0.90 ± 0.12 µg/g dry feces; post-ACTH: 2.55 ± 0.25 µg/g). Considering pre- and post-ACTH samples, FCM concentrations were higher ( P80.01) in males (2.15 ± 0.20 µg/g) than in females (1.30 ± 0.20 µg/g), but the magnitude of the response to ACTH was comparable ( P>0.05) between genders. After ACTH injection, FAMs increased in two (of 3) males; in one male, FCMs and FAMs were positively correlated (0.60; P80.01). Excretion of FCMs was assessed in 16 jaguars (7 M, 9 F) and found to be highly variable (range, 80.11-1.56 µg/g). In conclusion, this study presents a cortisol RIA for monitoring adrenocortical function in jaguars noninvasively. Zoo Biol 31:426-441, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The article presents a study which validates the use of fecal glucocorticoid metabolite (FGM) assay to assess endogenous adrenal activity in collared peccary (Pecari tajacu) through adrenocorticotropic hormone (ACTH). The feces samples were free-dried and extracted using an ethanol vortex method and assayed using an enzyme immunoassay. It compares FGM concentrations using repeated measures analysis of variance (ANOVA). Results demonstrate that fecal glucocorticoid metabolite assay can be an effective tool for noninvasive stress monitoring in peccaries.

Abstract: The response of animals to handling by humans has been extensively evaluated in domesticated livestock, but rarely examined in wildlife species. Twelve captive wombats (Lasiorhinus latifrons) were subjected to two treatments in a replicated design: (1) daily handling, involving 15min of tactile contact 5 d/wk for 12 wk and (2) no-handling, involving no contact apart from that received during routine husbandry. The effect of handling was assessed via overt responses to human approach and touch, a stressor, and a novel stimulus. Daily handling reduced the wombat''s flight distance in response to human approach; more in the first handling replicate (−0.16±0.02m/wk) than in the second (−0.06±0.02m/wk). A behavioural reactivity score also declined faster in the first than second handling replicate. Synthetic ACTH was used to validate the measurement of faecal cortisol metabolites in L. latifrons by EIA. Faecal cortisol metabolite secretion consistently increased in reaction to a handling procedure involving forced human contact (indicating a lack of habituation) but the magnitude of this response was not reduced by regular handling. Regular handling therefore changed the human–wombat relationship by lowering reactivity to and avoidance of the human handler, but did reduce the stress response, suggesting that the wombats entered into a state of learned helplessness. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Glucocorticoids or their metabolites can be measured in several body fluids or excreta, including plasma, saliva, urine and faeces. In recent years the measurement of glucocorticoid metabolites (GCMs) in faeces has gained increasing attention, because of its suitability for wild populations. In horses, however, the group-specific enzyme immunoassay described so far has a limited practicability due to its complex extraction procedure. Therefore, we tested the applicability of other enzyme immunoassays for glucocorticoid metabolites. The present study clearly proved that an enzyme immunoassay (EIA) for 11-oxoaetiocholanolone using 11-oxoaetiocholanolone-17-CMO: BSA (3α,11-oxo-A EIA) as antigen showed high amounts of immunoreactive substances. Therefore it was possible to use just a small amount of the supernatant of a methanolic suspension of faeces. The results correlated well with the already described method for measuring GCMs in horse faeces, i.e. analysing the samples with an EIA after a two step clean up procedure of the samples (Merl et al. 2000). In addition, the 3α,11-oxo-A EIA has the advantage of providing a bigger difference between baseline values and peak values after ACTH stimulation. The new assay increased the accuracy of the test, lowered the expenses per sample, and storing samples at room temperature after collection was less critical than with other assays investigated in our study. This is a big advantage both in the field of wildlife management of equids and in the field of equestrian sports and it shows the importance of choosing an assay which is in good accordance with the metabolites excreted in a given species. [ABSTRACT FROM AUTHOR]

Abstract: Our knowledge of glucocorticoid actions in vertebrates comes primarily from laboratory studies, which are often conducted with little consideration of how animals experience changes in glucocorticoid secretion in natural contexts. Typically, free-living animals are exposed to acute perturbations of the environment, ranging from a few minutes to a few hours duration, with varying frequency. The cumulative effects of these perturbations and their resultant glucocorticoid surges are not well known. To investigate the possible cumulative effects of repeated, acute surges in glucocorticoid secretion, we developed an ecologically relevant methodology for treating captive white-crowned sparrows (Zonotrichia leucophrys gambelii) with corticosterone (CORT). We dissolved CORT in dimethylsulfoxide (DMSO) and administered this cocktail directly on the skin. Treatments resulted in small elevations of CORT within the physiological range. In our first experiment at the end of the breeding life stage, birds were treated three times a day (3×). Two control groups were used: one treated with DMSO 3× and one not handled nor treated. In a second study at the beginning of the breeding life stage, one group was treated once a day and a second group 3×. A DMSO-control group was used for each dosage regime. Repeated, acute administration of CORT resulted in higher baseline CORT levels and a down-regulation of the endogenous adrenocortical response to a standardized stress. Maximum CORT and plasma corticosterone binding globulin levels increased in response to the CORT treatments only at the end of the breeding season. CORT treatment did not alter adrenal size, adrenal response to ACTH, or hepatic CORT metabolism. [Copyright &y& Elsevier]

Abstract: Hypothalamic–pituitary–adrenal (HPA) axis function, in Brahman heifers of differing temperament, was evaluated using separate challenges with CRH and ACTH. Exit velocity (EV) measurement was used to classify heifer temperament as calm [C; consisted of 6 slowest heifers (EV=1.05±0.05 m/s)] or temperamental [T; 6 fastest heifers (EV=3.14±0.22 m/s)]. During the 6 h prior to CRH challenge, areas under the ACTH (P =0.025) and cortisol (P <0.001) curves were greater in the temperamental heifers. Baseline cortisol (P <0.001) but not ACTH (P =0.10) differed between temperament groups. Following CRH challenge, areas under the ACTH (P =0.057) and cortisol (P <0.01) response curves were greater in the calm animals. The same animals were subjected to an ACTH challenge 14 d following their utilization in the CRH stimulation experiment. Prior to ACTH challenge, baseline cortisol concentrations were higher (P <0.001) in the temperamental heifers (T=18±2.6, C=4.3±0.6 ng/mL). Following ACTH administration, area under the cortisol response curve was greater (P =0.07) in the calm heifers. After declining below baseline concentrations during the post-challenge recovery period, cortisol in temperamental animals was again greater (P =0.02) than in the calm heifers. These data demonstrate that cattle with an excitable temperament exhibit increased stress responsiveness to handling, increased baseline adrenal function but not increased basal pituitary function, and a muted responsiveness to pharmacological stimulus. Thus, functional characteristics of the HPA axis vary with animal temperament. [Copyright &y& Elsevier]

Abstract: Studies in anthropoid primates and other mammals suggest that reproductive season, rank, reproductive skew, aggression received, and social support are the major factors influencing glucocorticoid output. In which way these are also affecting adrenal function in lemurid primates has been studied rarely. Here, we examine the influence of reproductive season and rank on glucocorticoid output in male sifakas (Propithecus verreauxi), a species characterized by high breeding seasonality, a hierarchy among males and extreme reproductive skew towards dominant males. We established a fecal assay for non-invasively monitoring adrenal activity and collected 315 fecal samples during the reproductive and birth season from 10 male sifakas living in 5 groups in Western Madagascar. We found a significant effect of season on glucocorticoid output, with males exhibiting higher fecal glucocorticoid levels during the reproductive compared to the birth season in conjunction with an increase in overall aggression rates during the former period. Moreover, our data indicate a significant effect of rank on adrenocortical activity with dominant males exhibiting higher glucocorticoid levels than subordinate males in the reproductive season. However, dominant males did not differ significantly in rates of initiated or received aggression and rates of affiliative behavior from subordinates but showed significantly lower rates of submission. Given their highly formalized dominance relationships, we conclude that higher glucocorticoid output in dominant males during the 4-month reproductive season is likely related to higher energetic demands necessary to cope with the challenges of male reproduction rather than to physical demands of increased fighting frequency to maintain dominance status. High rank in sifakas may thus carry high costs, which, however, may be outweighed by monopolization of almost all paternities. In sum, our data generally support the findings on the relationship between environmental and social factors and glucocorticoid output found in non-lemurid primates. [Copyright &y& Elsevier]

Abstract: Exposure to prenatal stress can impair the behavioural and hormonal development in mammals. However, the consequences for the immune system are rarely investigated and there is only limited evidence that naturalistic prenatal stressors do also have the potential to affect the offspring. Thus, by using a social conflict model in female Long-Evans rats, we investigated the effects of prenatal social stress on several behavioural, hormonal and immunological parameters. Offspring from stressed and non-stressed pregnant females were housed in pairs after weaning, and tested at an age of 4–6 months. Prenatally stressed (PS) males were more active in the elevated plus-maze test as indicated by significantly more frequent entries into the open arms compared to prenatal control males (PC). In addition, PS males had significantly lower serum corticosterone concentrations under basal conditions as well as after ACTH-challenge. The basal number of total leukocytes was significantly lower in the PS group due to significantly lower lymphocyte counts. In particular, the CD4+ T-helper cell subset was affected. The lymphocyte proliferation to pokeweed mitogen was lower in PS males. Because some of the present findings do not correspond to previous studies using conventional stressors, we assume that the nature of the stressor plays an important role for pregnancy outcome and behaviour and physiology of the offspring in later life. [Copyright &y& Elsevier]

Presents abstracts of research on animal production, management and the environment. "Minimum Temperature and Maximum Humidity: Predictors for Conception of Crossbred Holstein Cows in Thailand," by V. Punyapornwithaya et al; "Can Acceptable Pregnancy Rates be Achieved Using Exclusive Timed Artificial Insemination for First Service?," by M. B. Capel, D. V. Nydam and R. Saltman; "Effect of Time of Feed Delivery on the Feeding and Lying Behavior of Lactating Dairy Cows," by T. J. DeVries and M. A. G. von Keyserlingk.

Presents abstracts of research on animal production, management and the environment. "Minimum Temperature and Maximum Humidity: Predictors for Conception of Crossbred Holstein Cows in Thailand," by V. Punyapornwithaya et al; "Can Acceptable Pregnancy Rates be Achieved Using Exclusive Timed Artificial Insemination for First Service?," by M. B. Capel, D. V. Nydam and R. Saltman; "Effect of Time of Feed Delivery on the Feeding and Lying Behavior of Lactating Dairy Cows," by T. J. DeVries and M. A. G. von Keyserlingk.