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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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (Copyright © 2022. Published by Elsevier Inc.)

It is often hypothesized that organisms exposed to environmental change may experience physiological stress, which could reduce individual quality and make them more susceptible to disease. Amphibians are amongst the most threatened taxa, particularly in the context of disease, but relatively few studies explore links between stress and disease in amphibian species. Here, we use the fungal pathogen Batrachochytrium dendrobatidis (Bd) and amphibians as an example to explore relationships between disease and glucocorticoids (GCs), metabolic hormones that comprise one important component of the stress response. While previous work is limited, it has largely identified positive relationships between GCs and Bd-infection. However, the causality remains unclear and few studies have integrated both baseline (GC release that is related to standard, physiological functioning) and stress-induced (GC release in response to an acute stressor) measures of GCs. Here, we examine salivary corticosterone before and after exposure to a stressor, in both field and captive settings. We present results for Bd-infected and uninfected individuals of three amphibian species with differential susceptibilities to this pathogen (Rana catesbeiana, R. clamitans, and R. sylvatica). We hypothesized that prior to stress, baseline GCs would be higher in Bd-infected animals, particularly in more Bd-susceptible species. We also expected that after exposure to a stressor, stress-induced GCs would be lower in Bd-infected animals. These species exhibited significant interspecific differences in baseline and stress induced corticosterone, though other variables like sex, body size, and day of year were usually not predictive of corticosterone. In contrast to most previous work, we found no relationships between Bd and corticosterone for two species (R. catesbeiana and R. clamitans), and in the least Bd-tolerant species (R. sylvatica) animals exhibited context-dependent differences in relationships between Bd infection and corticosterone: Bd-positive R. sylvatica had significantly lower baseline and stress-induced corticosterone, with this pattern being stronger in the field than in captivity. These results were surprising, as past work in other species has more often found elevated GCs in Bd-positive animals, a pattern that aligns with well-documented relationships between chronically high GCs, reduced individual quality, and immunosuppression. This work highlights the potential relevance of GCs to disease susceptibility in the context of amphibian declines, while underscoring the importance of characterizing these relationships in diverse contexts., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Male reproductive success in most mammals is determined by their success in direct inter-male competition through aggression and conflict, resulting in female-defense mating systems being predominant. This is linked to male testosterone levels and its dynamics. However, in certain environments, a scramble-competition mating system has evolved, where female reproductive behavior takes precedence and male testosterone dynamics are unlikely to be related to inter-male competition. We studied the North American red squirrel (Tamiasciurus hudsonicus), a species with a well-established scramble-competition system. Using an ACTH hormonal challenge protocol as a proxy for competitive interactions, we compared the testosterone dynamics in breeding males in late winter with that in nonbreeding males in late spring in the Yukon. To gain an integrated picture of their physiological state, we also assessed changes in their stress response, body mass, energy mobilization, and indices of immune function. Testosterone levels at the base bleed were high in breeding males (2.72ng/mL) and virtually absent in non-breeding males (0.04ng/mL). Breeding males were in better condition (heavier body mass, higher hematocrit, and higher erythrocytes), had higher indices of immune function (neutrophil:lymphocyte ratio), but a similar ability to mobilize energy (glucose) compared with non-breeding males. Though total cortisol was higher in non-breeding males, free cortisol was twice as high in breeding males as their corticosteroid binding globulin levels were half as high. In response to the ACTH challenge, testosterone levels in breeding males declined 49% over the first hour and increased 36% over the next hour; in non-breeding males levels showed no change. Free cortisol increased only modestly (26% in breeding males; 23% in non-breeding males). Glucose levels changed similarly in breeding and nonbreeding males, declining for the first 30min and then increasing for the next 60min. Thus, testosterone and components of the stress axis function in a profoundly different manner in male red squirrels than in males of mammals with female-defense mating systems. There are four probable interrelated reasons for these adaptations in male red squirrels: the marginal benefits of each mating, the constraints of mate searching away from their own resource-based territories, energy mobilization in a harsh environment, and a long life span., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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: 4h vs 6h) and feces (6h vs 8h). Several metabolites were present in the feces and the tested EIAs reacted with some of them. Time of day had a significant effect on measured fecal steroid metabolites. As expected, males had significantly higher FTM levels than females. ACTH administration significantly increased FCM values; peaks were observed 4-8h after injection. In conclusion, both tested EIAs proved suited for a noninvasive measurement of glucocorticoids and androgens in bank voles., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Land use changes are a significant factor influencing the decline of felid populations. However, additional research is needed to better understand how these factors influence populations in the wild. Hormone analysis can provide valuable information on the basic physiology and overall health of an animal, and enzyme immunoassays (EIA) are generally used for hair hormone analysis but must first be validated for the substrate of choice and species of interest. To date, hormone assays from hair have not been validated for Felidae, despite that the method holds considerable promise for non-invasive sampling of free-ranging animals. We sought to: (1) evaluate whether increased adrenocorticotrophin hormone (ACTH) during the period of hair growth results in elevated hair cortisol; (2) validate the enzyme immunoassay used; and (3) identify any variations in hair cortisol between age, sex and body regions, using Canada lynx. We quantified hair cortisol concentrations in captive animals through an ACTH challenge and collected samples from legally harvested lynx to compare variability between body regions. An EIA was validated for the analysis of hair cortisol. Lynx (n=3) had a qualitative increase in hair cortisol concentration following an ACTH challenge in captive animals (20 IU/kg of body weight weekly for 5 weeks), thereby supporting the use of an EIA to quantify cortisol values in hair. Based on our analysis of sampled lynx pelts, we found that hair cortisol did not vary between age and sex, but varied within the foot/leg region to a greater extent than between individuals. We recommend that future studies identify a standardized location for hair cortisol sampling., (Copyright © 2013 Elsevier Inc. All rights reserved.)