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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.

Context Due to considerable declines in African wildlife populations, most large African mammals are managed inside protected areas. Protected areas come in various sizes, and have different environmental features, climates and management strategies (i.e. ‘hands-on’ or ‘hands-off’) that can influence an animals’ homeostasis. White rhinos (Ceratotherium simum simum) are found almost exclusively within protected areas where population sizes are driven by natural factors and poaching pressures. Aims Our aim was to understand the effect of natural and anthropogenic factors on the adrenocortical response of white rhinos within three protected areas. Specifically, we wanted to understand how poaching pressure, protected area size (

Pangolins are under severe threat of surging poaching rates globally; therefore, there is a demand to ascertain reproductive measures to ensure captive breeding and management strategies. Due to the absence of substantial information on the pangolin, endocrinology and reproductive physiology studies around the globe are merely based on captive observations that have failed to report the chronographs and hormonal cyclicity of the reproductive events. This study attempts to evaluate the annual pattern of reproductive steroids (estradiol-17β and progesterone) and prolactin in 16 wild female Taiwanese pangolins rehabilitated by Pingtung Rescue Center of Endangered Wild Animals, Taiwan. Novel immunoassays, i.e., chemiluminometric assays, have been used to quantify the serum reproductive steroids and contribute to a better understanding of the endocrine correlates of function in the Taiwanese pangolin. The hematological findings were characterized by monthly median concentration. The circulating reproductive hormones demonstrated seasonal reproductive activity by confirming a peak in serum estradiol concentrations in December and considerably higher progesterone concentrations in November/December, and March/April. The rise in prolactin in December and peak values in April suggest participation in the ovulatory process and mating. Collectively, these findings can help maximize the reproductive efficiency of pangolin species in captivity, i.e., by timely pairing and prioritizing the care of the breeding pairs to optimize breeding efforts and, therefore, effectively support conservation breeding programs and restore the natural population in the ecosystems.

The Kruger National Park (KNP) is home to the last genetically viable, minimally managed population of African wild dogs (Lycaon pictus, wild dogs) in South Africa. Until 2004, this population remained stable, but since has been declining. In this study, we aimed to improve our understanding of the ecology of KNP wild dogs by estimating the relative contribution of different prey types to their diet across landscape types. Based on a Bayesian mixing model, we assessed wild dog diet and foraging preferences using stable isotope analysis. We sampled 73 individuals from 40 packs found in six different landscape types. In thickets, packs predominantly prey on small browsing and mixed-feeding species (accounting for ~73% of their diet), but occasionally hunt large grazers (~24%) and large browsers (~3%). In open landscape types where lions (Panthera leo) are more or less absent, such as in the Lowveld sour bushveld, wild dogs prey on large browsers and large grazers (~67%). Our results demonstrate that KNP wild dogs occupy a broader ecological niche than previously thought, with small browsers forming an integral part of their diet. We also present the first data describing differences in wild dog diet–tissue discrimination factors for tail hair and whiskers compared to respective stable nitrogen (δ15N) and carbon (δ13C) values obtained from feces of captive wild dogs, as well as from those of South Africa’s broader managed metapopulation. While these data should be considered preliminary, we suggest that until wild dog diet–tissue discrimination factors are calculated through a controlled feeding study, the discrimination factors calculated for the gray wolf (Canis lupus) should be used for wild dog-related isotope studies, rather than the often cited values for red foxes (Vulpes vulpes).

Funder: University of Zurich; Id: http://dx.doi.org/10.13039/501100006447, Funder: MAVA Foundation, Funder: Mammal Research Institute at the University of Pretoria, The ability of dispersing individuals to adjust their behaviour to changing conditions is instrumental in overcoming challenges and reducing dispersal costs, consequently increasing overall dispersal success. Understanding how dispersers' behaviour and physiology change during the dispersal process, and how they differ from resident individuals, can shed light on the mechanisms by which dispersers increase survival and maximise reproduction. By analysing individual behaviour and concentrations of faecal glucocorticoid metabolites (fGCM), a stress-associated biomarker, we sought to identify the proximate causes behind differences in survival and reproduction between dispersing and resident meerkats Suricata suricatta. We used data collected on 67 dispersing and 108 resident females to investigate (a) which individual, social and environmental factors are correlated to foraging and vigilance, and whether the role of such factors differs among dispersal phases, and between dispersers and residents; (b) how time allocated to either foraging or vigilance correlated to survival in dispersers and residents and (c) the link between aggression and change in fGCM concentration, and their relationship with reproductive rates in dispersing groups and resident groups with either long-established or newly established dominant females. Time allocated to foraging increased across dispersal phases, whereas time allocated to vigilance decreased. Time allocated to foraging and vigilance correlated positively and negatively, respectively, with dispersers' group size. We did not find a group size effect for residents. High proportions of time allocated to foraging correlated with high survival, and more so in dispersers, suggesting that maintaining good physical condition may reduce mortality during dispersal. Furthermore, while subordinate individuals rarely reproduced in resident groups, the conception rate of subordinates in newly formed dispersing groups was equal to that of their dominant individuals. Mirroring conception rates, in resident groups, fGCM concentrations were lower in subordinates than in dominants, whereas in disperser groups, fGCM concentrations did not differ between subordinates and dominants. Our results, which highlight the relationship between behavioural and physiological factors and demographic rates, provide insights into some of the mechanisms that individuals of a cooperative species can use to increase overall dispersal success.

Despite the commonly held belief that reptiles are immune to extreme temperatures, global warming is predicted to result in the loss of 40% of all reptile species by 2080. In order to understand th...

Animal personality traits (consistent behavioral differences between individuals in their behavior across time and/or situation) affect individual fitness through facets, such as dispersal. In eusocial naked mole-rat (

In nutrient-poor wildlife reserves it has become common-place to provide supplemental mineral resources for wildlife. Yet, the impacts of anthropogenic mineral supplementation on community-wide wildlife nutrition, behaviour and subsequent impact on ecosystem processes remain poorly understood. Here, we examine the contribution of anthropogenic mineral lick provision to wildlife nutrient intake across a community of large mammals (>10kg) in the southern Kalahari Desert. Based on predicted daily nutrient requirements and a faecal nutrient assessment, large herbivores appear deficient in phosphorus (P), sodium (Na) and zinc (Zn). For these nutrients, anthropogenic salt and mineral licks constitute an important (>10%) source of nutrient intake helping to reduce or overcome requirement deficits. Larger-bodied species disproportionately consumed licks (p

Small size in mammals usually restricts long-term, frequent monitoring of endocrine function using plasma as a matrix. Thus, the non-invasive monitoring of hormone metabolite concentrations in excreta may provide an invaluable approach. The aim of the current study was to examine the suitability of enzyme immunoassays (EIAs) for monitoring responses to stressors in the naked mole-rat (Heterocephalus glaber, NMR) using urine and feces as hormone matrices. A saline control administration, and a high- and low-dose adrenocorticotropic hormone (ACTH) challenge were performed on six male and six female disperser morph NMRs. The results revealed that a 5α-pregnane-3β,11β,21-triol-20-one EIA detecting glucocorticoid metabolites (GCMs) with a 5α-3β-11β-diol structure is the most suitable assay for measuring concentrations in male urine samples, whereas an 11-oxoaetiocholanolone EIA detecting GCMs with a 5β-3α-ol-11-one structure appears the most suitable EIA for quantifying GCMs in female urine. An 11-oxoaetiocholanolone EIA detecting 11,17 dioxoandrostanes was the most suitable EIA for quantifying GCMs in the feces of both sexes. There were sex-related differences in response to the high- and low-dose ACTH challenge. We recommend using feces as a more suitable matrix for non-invasive GCM monitoring for NMRs which can be valuable when investigating housing conditions and other welfare aspects.

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.

Little information exists on the sexual behaviours of the roan antelope (Hippotragus equinus; E. Geoffroy Saint-Hilaire, 1803), a species listed vulnerable in southern Africa. To fill this gap, two adult males and 13 adult females from two captive breeding herds, at Lapalala Wilderness, South Africa, were observed three times per day from September 2017 to July 2018. Pre-categorised behaviours exhibited by males and females were recorded and their frequencies quantified. Fifteen pre-defined behaviours were recorded in males with precopulatory behaviours being the most recurrent. Overall, 181 courtships and 73 complete mounts (copulations) were recorded for the two mating periods observed; each mount lasted between 3 and 4 s. In females, eight pre-defined behaviours were observed and homosexual mounts were observed for the first time in females of this species and indeed within the Hippotragini tribe. Receptivity lasted for between 24 and 72 h, with prominent behaviours being copulation, flehmen and urination. Besides hiding, moving away or lying-out, non-receptive females fell on their metacarpal joints when approached by a male, a behaviour not yet reported in hippotragine species. This study showed that courtship is the most prominent component of male mating behaviour and that receptivity in females may vary between 1 and 3 days.

Vertebrate muscle myosin, the motor protein, has evolved over long periods of time. In the present study, we specifically aimed at identifying how this evolution has fine-tuned myosin conformational states to the specific needs of organisms with different body masses. As myosin has two different relaxed states (super-relaxed and disordered-relaxed), we initially hypothesized that a linear positive correlation would exist between the amount of myosin molecules in the super-relaxed conformation and the body weight of the organisms in which they are expressed. To verify this hypothesis, we extracted muscle fibres from multiple vertebrate species with body weights ranging from grams to tons. By using a loaded Mant-ATP chase protocol, we observed that vertebrate species with body weights above 1000 kilograms have significantly higher proportions of super-relaxed myosin proteins in their muscle fibres. As myosin heads in the super-relaxed conformation consume five times less ATP than the ones in the disordered-relaxed state, our finding suggest that heavy vertebrate animals have adapted their motor protein conformations to potentially avoid inappropriate and large energy consumption in their metabolically demanding muscles.

The objective of this study was to determine whether various sources of roughage in nutritionally balanced feedlot diets would evoke a stress response in sheep. A nutritional stress response test was performed on 20 ewes, randomly divided into four treatment groups with 5 ewes per group. Ewes were kept individually in metabolic crates for thirty days. Rations were balanced nutritionally, and various sources of roughage were included; T1: alfalfa hay, T2: maize stover, T3: soya hulls; and T4: Eragrostis tef. Faeces was removed manually from the caudal rectum of each ewe at 05h00 and 19h00. Samples were frozen at -20°C until analyses. A total of 520 faecal samples were analysed to determine the concentrations of faecal glucocorticoid metabolites (fGCM) by enzyme immunoassay (EIA). These concentrations were expressed as mass/g dry weight (ng/g DW). Baseline values were determined, and those greater than the mean plus 2 standard deviations were removed. Baseline values were compared between the groups with one-way ANOVA analysis. The average concentrations of fGCM were 178.77 ± 21.7 in the morning and 183.2 ± 14.4 in the evening. Sheep fed T4 had significantly higher fGCM concentrations in both morning (302.0 ± 86.1 ng/g DW) and evening (237.0 ± 48.1 ng/g DW) compared with the other treatment groups. Thus, fGCM concentrations in sheep were related to the source of roughage, and Eragrostis tef caused a stress response. Keywords: animal welfare, cortisol, dietary fibre, selective grazer, stress response

Land transformation for anthropogenic use is the leading cause of species decline globally. However, few species are able to succeed in anthropogenically disturbed environments. African clawless otters (Aonyx capensis) occur in a wide variety of habitats, and thus are good model species to investigate animal adaptation to anthropogenic environments. Therefore, we aimed to estimate and compare population densities, group size, and activity time of African clawless otters occurring in a transformed area, Millstream Farm, and two natural areas, Verloren Vallei Nature Reserve and Cobham Nature Reserve. Camera trap arrays, consisting of between 18, 27 and 24 cameras, were placed on all three sites, recording otter presence for a total of 2439 camera days. Using random encounter models, we estimated that the transformed area had the highest density of African clawless otters (8.2 ± 2.3 km−2), compared to otter densities at Verloren Vallei and Cobham Nature Reserve, South Africa, (natural areas) with estimated densities of 0.7 ± 0.2 km−2 and 2.1 ± 0.6 km−2 respectively. We found a significant difference (p = 0.007) between group sizes in the transformed area (detections = 112; group size range = 1–5) and natural areas (detections = 29; group size range = 1–3). Furthermore, there was a significant difference in otter activity time (p = 0.033, activity overlap = 66.5 ± 8.33%) between Verloren Vallei Nature Reserve and the transformed area at Millstream Farm. This study illustrates how African Clawless otters exhibit behavioural plasticity in order to exploit a resource-rich anthropogenic environment.

Elephant interaction programs, specifically ones that provide elephant back riding, have come under public scrutiny, and little information exists to show whether these activities affect animal wel...

In a time of increasing environmental change caused by anthropogenic disturbance, there is a greater need to understand animal adaptations to manmade environments. In this regard, the measurement of stress-related endocrine markers provides a useful tool to examine the impact of environmental challenges and the physiological consequences for wildlife occupying such space. The aims of the present study were to validate fecal glucocorticoid metabolite (fGCM) concentrations as a measure of stress using samples from a male African clawless otter (Aonyx capensis; n = 1) and to compare fGCM concentrations of otters occurring in a transformed and in 2 natural areas in South Africa. From the 5 different enzyme-immunoassays (EIA) tested, a cortisol and oxoetiocholanolone (measuring 11,17 dioxoandrostanes) EIA revealed the highest response (74% and 48% increase, respectively) 30 and 24 hours after a stress event (translocation of a captive individual as part of its rehabilitation prior to release), respectively. For both EIAs, fGCM concentrations were comparable for samples collected up to 3 h post-defecation. Using the cortisol EIA for subsequent analyses, fGCM concentrations of animals from the transformed area (n = 20; mean [± SD]: 0.468 [± 0.539] µg/g dry weight [DW]) were significantly higher (P = 0.013) than those from otters in the natural areas (n = 17; 0.242 [± 0.226] µg/g DW). These preliminary results suggest that African clawless otters may have increased adrenocortical activity that could be due to conditions linked to living in a transformed environment.

Poaching is the primary threat to the survival of rhinoceros’ populations. One frequent consequence of poaching is the creation of orphan calves. If found, orphans are taken into captivity for rehabilitation and subsequent release. However, rehabilitation practices can influence their behavior and welfare, potentially compromising their post-release adaptation and survival. In this study, the effects of hands-off and hands-on rehabilitation methods on the behavior, welfare, and adaptation potential of orphaned white rhinoceros (Certatotherium simum simum) were compared. To achieve these aims, 12 behavioral, one physiological, and four physical indicators of welfare and adaptation potential were measured non-invasively on 25 orphaned rhino at two rehabilitation facilities in South Africa. Results indicated that although orphan welfare was not compromised under either rehabilitation method, the hands-off cohort showed fewer indicators of poor welfare and more indicators of good welfare. Regarding adaptation potential, hands-off rehabilitated rhino showed the species’ natural response to humans, and alert and defense behaviors were part of their behavioral repertoire. The hands-on cohort displayed fewer social interactions than the hands-off cohort, showed habituation to humans, and seldom expressed alert or defense behaviors, which could potentially compromise their survival and social integration after release. Post-release studies are required to confirm whether fitness is compromised in hands-on rehabilitated rhino. Until then, we suggest to minimize anthropogenic exposure during rehabilitation in order to maximize welfare and retain crucial behaviors for post-release adaptation and survival.

South Africa is one of the most diverse countries in the world but the increase in agricultural, industrial and technological development to meet the needs of the growing human population has led to increased amounts of potentially toxic elements (PTEs) and other chemicals in the environment. As regional and global environmental processes influence local conditions to differing degrees, all organisms within a specific environment are exposed to highly complex, ill-defined PTE and chemical mixtures. Differences in feeding strategies within and between vertebrate trophic levels are likely to influence the degree to which individuals may be exposed to and affected by PTE presence. Using vertebrate faeces as a biological matrix, we investigate and compare quantitative differences in PTE concentrations in herbivorous, omnivorous and carnivorous terrestrial vertebrates from two protected areas in South African savannah. Of the eleven PTEs assessed [aluminium (Al), arsenic (As), barium (Ba), cadmium (Cd), chromium (Cr), mercury (Hg), lead (Pb), antimony (Sb), tin (Sn), strontium (Sr), and vanadium (V)], the highest concentrations of Al, As, Cr, Pb, Sn, and V were found in carnivores. General patterns were evident between groups at each site for specific elements, but absolute values for the same elements were site-specific. This is the first study to non-invasively examine and compare PTE concentrations in a variety of free-ranging mammalian wildlife occupying different trophic levels within South African protected areas. Our results confirm that all individuals across trophic levels within these sites are exposed to multiple and varied PTE mixtures on a continuous basis. Whether PTEs at these concentrations cause synergistic or antagonistic disruption of physiological and biological systems alone or in combination in free-ranging African wildlife species is still unclear and requires further investigation.

In species where sociality and group cohesion are primarily determined by the maintenance of a reproductive division of labour and cooperative behaviours, the eusocial Damaraland mole-rat (Fukomys damarensis) presents a model which provides behavioural and endocrine distinctions between sex (males and females) and reproductive class (breeders and non-breeders). Although previous studies have demonstrated the endocrine aspects of reproductive suppression and behaviour in Damaraland mole-rats, they have focused on one hormone separately and on different conspecifics and samples across time. Unfortunately, this could introduce extrinsic biases when using these studies to compile complete hormonal profiles for comparisons. This study, therefore, set out to obtain a profile of the reproductive hormones from breeding and non-breeding male and female Damaraland mole-rats at a single point in time, from which circulating plasma prolactin and urinary progesterone, testosterone, and cortisol were measured. As expected, plasma prolactin and urinary cortisol did not differ between the breeders and non-breeders. However, breeders (both male and female) possessed increased urinary testosterone and progesterone concentrations compared to their non-breeding counterparts. These results, in conjunction with the variation in the expression of the respective hormonal receptors within the brains of breeders and non-breeders suggest that elevated testosterone and progesterone in breeders establish a neural dominance phenotype, which ultimately aids in controlling breeding activities. This study has emphasised the need for holistic, comprehensive profiling of reproductive endocrine systems.

Animal personality traits (consistent behavioral differences between individuals in their behavior across time and/or situation) affect individual fitness through facets, such as dispersal. In eusocial naked mole-rat (Heterocephalus glaber) colonies, a disperser morph may arise with distinct morphological, behavioral, and physiological characteristics. This study aimed to quantify the personality traits of a cohort of disperser morphs of naked mole-rat (NMR). Behavioral tests were performed on twelve disperser morphs (six males and six females) in an observation tunnel system that was novel and unfamiliar. Novel stimuli (fresh snakeskin, tissue paper, and conspecific of the same sex) were introduced for fifteen minutes, and the behavioral acts of the individual were recorded. A total of 30 behaviors were noted during the behavioral tests of which eight were used to quantify aggression, boldness, and exploration. The NMR disperser morphs showed consistent individual differences in boldness, and exploration across time and test, indicating a distinct personality. In addition, new naked mole-rat responses including disturbance behaviors; confront, barricade, and stay-away, were recorded. Further investigations into the relationships between animal personality traits and social hierarchy position in entire colonies are needed for more informative results as we further investigate the role of personality in cooperatively breeding societies.

Noninvasive measurement of stress-related alterations in fecal glucocorticoid metabolite (fGCM) concentrations has considerable potential for quantifying physiological responses to very hot weather in free-ranging birds, but practical considerations related to sampling will often make this method feasible only for habituated study populations. Here we evaluate an alternate approach, the use of experimentally manipulated thermal environments for evaluating stress responses to high environmental temperatures in wild-caught birds housed in captivity. Using an enzyme immunoassay utilizing antibodies against 5ß-pregnane-3α,11ß,21-triol-20-one-CMO∶BSA (tetrahydrocorticosterone), we quantified fGCMs in captive individuals of three southern African arid-zone species (southern pied babblers [

In South Africa, blue wildebeest (Connochaetes taurinus) are routinely captured for relocation purposes. To monitor the stress caused by this practise, a non-invasive method assessing adrenocortical function as a measure of stress would minimize disturbance during sample collection. In our study, an adrenocorticotropic hormone challenge, and a mass-capture event were used to examine the suitability of five enzyme immunoassays (EIAs) for monitoring stress-related physiological responses using faeces as a sample matrix. The tested 11-oxoaetiocholanolone I EIA performed best, showing a 2126% increase above baseline after 22 h for a male, and a 474% increase for a female 23 h post-ACTH injection. Baseline faecal glucocorticoid metabolite (fGCM) concentrations did not differ between animals captured on either day 1 or day 2 of the capture event, indicating that the frequent presence of a helicopter during the two days did not influence fGCM concentrations. However, during capture-related restraint for up to 10 h, an overall 1.5-fold elevation in (fGCM) concentrations was found. Storage of faeces at ambient temperature post-defecation indicated a fair stability of fGCMs for up to 8 h. The ability to reliably assess adrenocortical function provides a solid basis to examine endocrine responses to putative stressful circumstances in blue wildebeest.

Information regarding the physiology of African lions is scarce, mainly due to challenges associated with essential routine research procedures. The aim of this experiment was to test the possibility of training six captive lionesses by positive reinforcement conditioning (PRC) to voluntarily allow the collection of vaginal swabs and blood samples. This was done with the final goal of avoiding frequent anesthesia, and potentially stressful management during reproduction research. All lionesses mastered basic clicker and targeting principles within 2 weeks. Routine sampling was possible after 20 weeks of training, enabling the collection of about 750 vaginal swabs and 650 blood samples over 18 months. The animals remained calm and cooperative during all sessions, and demonstrated curiosity in the training. PRC training of captive lionesses proved to be a suitable, minimally invasive method for repeated collection of vaginal swabs and blood. Additionally, PRC may serve as behavioral enrichment for African lions in captive settings. Compared to chemical or physical restraining methods, this noninvasive management approach may reduce distress and physiological negative side effects, thus opening up new avenues for feline research.

Changes in group composition are not exclusive to zoos and conservation breeding centers. The recent increase in poaching of African rhino species has accelerated the arrival of orphan calves at rehabilitation centers. Introducing new members into an existing group is often stressful for many mammal species. However, when young animals are involved, such responses may be reduced or absent. The goal of the present study was to evaluate the effect of introducing orphan calves on the stress responses of young orphan rhino from existing groups. The behavior and fecal glucocorticoid metabolite (fGCM) concentrations of eight orphan southern white rhinoceros (Ceratotherium simum simum) were assessed 1 month before and after the introduction of a new calf. From the 10 variables measured, only the response to humans and submissive behaviors showed significant changes. Stereotypies were not observed during the course of the study, and adrenocortical activity, monitored by means of fGCM concentrations, did not increase after the introductions, showing values within the range observed in free-ranging white rhino. However, strong individual differences were evident in most variables. Our results suggest that the introduction of white rhino calves into an existing group of young rhinos caused minimal stress in existing group members. Although these findings should be treated with caution when generalizing to other captive populations due to the small and heterogeneous sample, our findings may have management implications for rhino orphanages as well as zoos and breeding centers where non-breeding pairs are frequently maintained.

Males in female-philopatric social groupings leave their natal groups to pursue successive reproductive opportunities in one or more other groups. In vervet monkeys, Chlorocebus pygerythrus , adult males coexist and physical eviction is not a driver of male movement. Migratory decisions are expected to turn on an evaluation of future reproductive opportunity, as indexed principally by local operational sex ratio and relative competitive ability. Although vervet males' reproductive success is correlated with dominance, they are distinctive in that the attainment of rank is contingent on integration into female sociospatial networks and we expect decisions about continued residency to reflect this. We used 8 years' data from three groups to confirm that male dispersal between groups is seasonal in our population, with a peak that is coterminous with androgen levels and precedes peak mating and conception by 4 weeks. The average length of completed residency was 459 days, with an increase in the logged rate of departure after 1428 days, which is 150 days longer than the estimated modal age at first conception by putative daughters. There were positive correlations between a male's initial and highest rank, and between his highest rank and the length of time to reach it. We found that a male's residency was positively and independently associated with his highest achieved rank and both his grooming centrality and proximity degree. Additionally, increasing rank and proximity degree also had positive effects on residency length subsequent to the attainment of his highest rank. The probability of emigration was associated negatively with both female number and grooming centrality scores. We conclude that emigration from a group is linked to male rank attainment and mediated by a male's integration into female sociospatial networks. We found no evidence that emigration preceded the sexual maturity of putative daughters.

Although sickness behaviour in response to non-lethal parasites has been documented in wild animals, it remains unclear how social and environmental stress might also shape an animal’s behavioural response to parasitism, nor do we know whether simultaneous infection with more than one parasite changes the way animals respond. Here, we combine physiological, environmental, behavioural and parasite measures to investigate behavioural responses to infection in wild vervet monkeys (Chlorocebus pygerythrus) living in a semi-arid region of South Africa. We quantified both activity budget and behavioural predictability to investigate the occurrence of sickness behaviour and infection with two non-lethal gastrointestinal parasite genera. Higher parasite load was linked to an increase in the time spent resting. However, the nature of the relationship with other behaviours was contingent on both the parasite genus in question, and how parasite species interacted, highlighting the importance of considering co-infection. Overall, food availability was the dominant predictor of behavioural change suggesting that, for monkeys living in a more extreme environment, coping with ecological stress may override the ability to modulate behaviour in response to other physiological stressors. Our findings provide insight into how animals living in harsh environments find ways to cope with parasite infection, avoidance, and transmission.

Relatively little effort has been directed towards elucidating the role of physiological stress pathways in mediating avian responses to global heating. For free-ranging southern pied babblers, Turdoides bicolor, daily maximum air temperatures (Tmax) between ∼35 and ∼40°C result in reduced foraging efficiency, loss of body mass and compromised breeding success. We tested the hypothesis that very hot days are experienced as stressors by quantifying relationships between Tmax and faecal glucocorticoid metabolite (fGCM) levels in naturally excreted droppings. On days when Tmax38°C, however, fGCM levels increased linearly with Tmax and averaged 190.79±70.13 ng g−1 dry mass. The effects of Tmax on fGCM levels did not carry over to the following morning, suggesting that very hot days are experienced as acute stressors.

Populations of roan antelope (Hippotragus equinus) in southern Africa have experienced a drastic decline over the past few decades and this situation has led to the development of intensive breeding programmes to support conservation efforts. However, little is known about related welfare aspects, including stress-related physiological biomarkers. The present study set out to establish a non-invasive method to monitor faecal glucocorticoid metabolite (fGCM) concentrations as a measure of stress and determine fGCM concentrations in relation to male reproductive activity and female reproductive status in the roan antelope. An adrenocorticotrophic hormone challenge was performed using two adult roan antelope (one male and one female) at Lapalala Wilderness Nature Reserve, South Africa, to determine the suitability of five enzyme immunoassays (EIA) for monitoring adrenocortical function in roan antelope. An 11-oxoaetiocholanolone I EIA detecting 11,17 dioxoandrostanes performed best showing 17-20 folds increases in fGCM concentrations after 12 h-17 h post-injection. The identified EIA was then used to monitor fGCM concentrations during active and non-active reproductive periods in males (n = 3), and during periods of cyclicity, gestation, and postpartum in females (n = 10). Males showed an overall 80% increase in fGCM concentrations when reproductively active and females showed a progressively significant increase in fGCM levels throughout pregnancy, with overall fGCM concentrations being 1.5 to 2.6-fold higher than the respective fGCM concentrations during periods of postpartum and cyclicity, respectively. Furthermore, fGCM concentrations remained above baseline for up to 21 days post-partum. A correlation between ecological parameters (rainfall and temperature) and fGCM concentrations revealed elevated fGCM concentrations during the dry season for males, but not females. The non-invasive method validated in this study provides a valuable tool to quantify stress-related biomarkers in roan antelope, and findings can be used to support management decisions in conservation breeding facilities.

Endocrine studies using faeces as hormone matrix have become increasingly popular to examine adrenocortical activity in wildlife. A prerequisite for this approach is to minimize alteration of faecal glucocorticoid metabolite (fGCM) composition post-defecation. This is done by freezing the collected material as soon as possible after collection, and removing moisture from the frozen faecal samples afterwards (usually by freeze-drying). In remote areas, freeze-drying opportunities are often limited, and in the case of the African wild dog (Lycaon pictus), established assays revealed that fGCM concentrations remain comparable for only ∼24h post-defaecation. In the present study, three cost-effective drying treatments (exposure to sunlight, placement in a solar oven, and use of a food dehydrator) were investigated as alternatives to the golden standard of freeze-drying faeces.•In comparison to freeze-dried material, African wild dog faecal samples dried through sunlight exposure, a solar oven, and use of a food dehydrator revealed no significant differences in respective fGCM concentrations measured.•A food dehydrator would be the preferable option to dry African wild dog faeces if limited electrical supply is available. This technique dries faeces the fastest, and negates any reliance on weather conditions., Graphical abstract Image, graphical abstract

As global non-human primate populations show dramatic declines due to climate change, land transformation and other anthropogenic stressors, it has become imperative to study physiological responses to environmental change in order to understand primate adaptability and enhance species conservation strategies. We examined the effects of seasonality on faecal glucocorticoid metabolite (fGCM) concentrations of free-ranging male and female thick-tailed greater galagos (Otolemur crassicaudatus) in an Afromontane habitat. To do so, we established an enzyme immunoassay (EIA) for monitoring fGCM concentrations in the species using a biological validation. Following this, faecal samples were collected each month over the course of a year from free-ranging males and females situated in the Soutpansberg Mountains, Limpopo, South Africa. Multivariate analyses revealed lactation period was a driver of fGCM levels, whereas sex and food availability mostly influenced seasonal fGCM concentrations in the total population. Thus far, the results of this study show that drivers of fGCM levels, an indication of increased adrenocortical activity, in O. crassicaudatus are numerous and complex within the natural environment. The species may be adapted to such conditions and an extreme change to any one component may result in elevated fGCM levels. This increases our understanding of strepsirrhine primate physiology and offers initial insights into species adaptability to a challenging environment.

Despite the importance of ecotourism in species conservation, little is known about the industry’s effects on wildlife. In South Africa, some African penguin (Spheniscus demersus) colonies have become tourist attractions. The species is globally endangered, with population sizes decreasing over the past 40 years. As African penguin chicks are altricial and unable to move away from anthropogenic stressors, it is important to evaluate the effect of tourist activities on baseline glucocorticoid levels as a measure of potential disturbance. Chicks at three study sites within two breeding colonies (Robben Island, Stony Point), with varying levels of exposure to tourism (low/moderate/high) were monitored. Urofaecal samples were collected to determine urofaecal glucocorticoid metabolite (ufGCM) concentrations as an indication of baseline stress physiology. Morphometric measurements were taken to compare body condition between sites. Penguin chicks experiencing low, infrequent human presence had significantly higher mean (± standard deviation) ufGCM levels [1.34 ± 1.70 μg/g dry weight (DW)] compared to chicks experiencing both medium (0.50 ± 0.40 μg/g DW, P = 0.001) and high levels of human presence (0.57 ± 0.47 μg/g DW, P = 0.003). There was no difference in chick body condition across sites. These results suggest that exposure to frequent human activity may induce habituation/desensitization in African penguin chicks. Acute, infrequent human presence was likely an important driver for comparatively higher ufGCM levels in chicks, though several other environmental stressors may also play an important role in driving adrenocortical activity. Nevertheless, as unhabituated chicks experiencing infrequent anthropogenic presence showed significantly higher ufGCM levels, managers and legislation should attempt to minimize all forms of activity around important breeding colonies that are not already exposed to regular tourism. Although the results of this study are crucial for developing enhanced conservation and management protocols, additional research on the long-term effect of anthropogenic activities on African penguin physiology is required.

Repeatability of hormone concentrations is of great interest for studies investigating the evolution of hormonal traits. Particularly the repeatability of glucocorticoids (GCs) in response to a stressor is frequently investigated, but often only point (initial and/or response value), or single measures are used. A new method takes into account the entire individual hormone profile and generates an individual profile repeatability (PR) score. The method was developed for response profiles, but it may also be valuable for baseline values in species with diurnal changes in hormone concentrations. GCs are determined in a variety of matrices, and repeatability can vary considerably depending on the matrix. We investigated the repeatability of baseline GC metabolite (GCM) concentrations measured in urine (uGCM) and faeces (fGCM) of captive eastern rock sengis (Elephantulus myurus) using the more traditional linear mixed model approach and the PR method. GCMs were assessed over 24 h and measurements were repeated twice with two weeks between replicates. A diurnal rhythm in GCM concentrations associated with the activity period of the sengis was found in urine, but not in the faeces. Urinary GCM concentrations exhibited a moderate repeatability, whereas the repeatability of fGCM concentrations was low. Urinary GCM concentrations and their repeatability differed between the sexes; with higher concentrations and lower PR scores in females. No such sex differences were apparent for fGCM concentrations and the PR score was not able to characterise repeatability of fGCM concentrations, which were lacking a distinct profile. The PR score enabled a successful quantification of the repeatability of the diurnal uGCM profiles. Hormone profile, sex and sample matrix can affect hormonal traits considerably and the results may be obscured if these factors are not carefully considered.

Pangolins are the most trafficked wild mammals, with their scales in high demand. Scales are often the only part of the animal confiscated from the trade, but they represent accessible material for forensic investigations, including for sexing. This study aimed to develop a sexing tool for Temminck’s pangolin, using scales for hormone quantification. Scales from males and females were liquidised using keratinase and the resulting suspension analysed for progestagen and androgen metabolite (scPM and scAM) concentrations. Scale PM and scAM concentrations were compared between sexes, while overall median values for scPM and scAM, as well as a ratio of scPM to scAM (P/A) were used as boundary values for sex identification. Neither scPM nor scAM concentrations were significantly different between the sexes and concentrations of a juvenile and sub-adult male overlapped with females, possibly indicating later sexual maturity in males. Boundary values for scAM concentrations and the P/A ratio predicted sex with 100% accuracy for females and 78% for males, while the accuracies for the scPM boundary value were lower. When only adult individuals are considered, scAM and P/A ratio boundaries are 100% accurate for both sexes. Therefore, scale hormone ratios show promise as a sex identification tool for Temminck’s pangolin, particularly applicable in forensic investigations on the pangolin trade.

In current research guidelines, much focus is placed on ethical management of animals and the application of principles of reduction, refinement and replacement. Of these refinements through environmental enrichment is an important aspect when housing primate to prevent behavioural problems. In this study, we investigated the co-housing of domestic cats and vervet monkeys as a novel method of enrichment based on the cohabitation and stress alleviation effect of horses housed with goats and from seeing cats cohabitating with vervet monkeys in an animal sanctuary. The study used a habituation method whereby the cats were stepwise introduced to the monkeys by sight and smell but with physical separation. Assessment included changes in behaviour, weight and faecal glucocorticoid metabolite (fGCM) concentrations over time. On the first day of housing, the vervets whilst inquisitive kept their distance. The vervets housed in cages that were closest to the cats were the most active and during the first minute of introduction made more alarm calls, which stopped a few days later. The fGCMs were non-significantly different. The results of this study provide evidence that vervet monkeys and domestic cats could potentially be housed together without overt aggression. We thus suggest further observations to ascertain if the co-housing could have long-term benefits for vervet monkeys, from the companionship that would be offered by the cats.

The savannah biome supports unique biodiversity and provides a multitude of ecosystem services. Defining background concentrations for trace elements in the environment is beneficial for the determination of nutrient deficiencies/hotspots and for the management of pollution. Sediment and corresponding vegetation samples were collected around 48 surface water points in two savannah wildlife areas for assessment and comparison of 20 trace elements using ICP-MS. Site-specific and matrix-specific differences were evident for essential B, Co, Cu, Fe, Mn, Mo, Ni, Se and Zn, potentially toxic As, Cd, Cr, Hg, Pb and V and additional elements Al, Ba, Sb, Sn and Sr analysed. Sediment and vegetation from all sampled locations at both sites contained single or multiple potentially toxic elements at various concentrations. Although the presence of all elements can be linked to underlying geology and geochemistry specific to each site, evidence of anthropogenic cause was also evident at both sites. This paper covers the widest range of trace elements assessed in protected terrestrial wildlife reserves in the South African savannah biome to date and highlights the potential for deleterious consequences of trace element contamination of the environment.

Anthropogenic activity is a major driver of seabird injury and mortality in the 21st century. Although most seabirds perish within the natural environment as a result of human activities, some are rescued and admitted to rehabilitation centres. Despite the considerable number of admissions, little is known regarding the physiological response seabirds have to specific admission reasons and the rehabilitation process. In this study, we aimed to determine the effect of injury, emaciation, oiling, individual removal from the natural environment and the rehabilitation process on the physiological stress response of the African penguin (Spheniscus demersus). Urofaecal samples were collected from African penguins throughout a three-stage rehabilitation process and quantified for glucocorticoid metabolites (ufGCM). The three stages included an initial ICU crate stage (Stage 1), an ICU pen stage (Stage 2) and a rehabilitation stage (Stage 3). Data were analysed using a generalised linear model in order to determine the effect of admission reason, age and rehabilitation stage (Stage 1, 2 and 3) on ufGCM levels. Although the model indicated that only Stage 1 was a significant driver of adrenocortical activity in the study population, ufGCM levels of injured and emaciated animals within Stage 1 were considerably higher than those of birds with any other admission reason across all stages. This is the first study examining the causes and effect of rehabilitation on the physiological stress response in African penguins. Enhanced care and attention should be given to rescued individuals, especially during the first stage of rehabilitation, to reduce perception of additional stressors and thus increase the chance of full recovery.

The southern ground-hornbill (SGH) is a cooperatively breeding bird endemic to eastern and southern Africa, but is endangered in its southern distributional range. The national conservation restoration program harvests redundant chicks for captive breeding and reintroduction; with sexing and social grouping of the species evaluated by throat-skin coloration, with adult males displaying a completely red color compared to dark blue within the red observed in adult females. However, recent findings indicate that dominant and subordinate adult males exhibit patches of blue throat-skin. To optimize SGH management practices, it is vital to determine the role of red and blue coloration, as well as the possible drivers thereof. As a prerequisite, an enzyme immunoassay for monitoring fecal androgen metabolite (fAM) concentrations in SGH was established. Following this, fresh fecal samples were collected from 78 SGH, of various demographics and origin, across 12 captive institutions, to determine whether fAM concentrations differ between blue (B), partially blue (sB), and fully red (R) throat-skin colored SGH. Furthermore, fAM concentrations were compared between males housed in different social groups of different age and sex classes. Individual median fAM concentrations of B, sB, and R adult males did not differ significantly but were considerably higher in B and sB males compared to R males. Social dynamics within captivity, for example, dominance, played no role as a driver of male gonadal activity or throat skin coloration. The results of the study indicate that androgens and apparent social dynamics are not primary determinants of throat coloration in male SGH.

The social environment of animals can have profound implications on their behaviour and physiology. Naked mole-rats (Heterocephalus glaber) are highly social with complex dominance hierarchies that influence both stress- and reproduction-related hormones. Homeostasis may be affected by aggressive interactions, colony instability and social isolation. Furthermore, naked mole-rat colonies are characterised by a marked reproductive skew; a single female and few males are reproductively active while other colony members are reproductively suppressed. Thus, there are distinct differences in related hormone concentrations between reproductively active and non-active animals; however, this changes when non-reproductive individuals are removed from the colony. We investigated the effects of social isolation and colony disruption on plasma cortisol and progesterone concentrations in non-breeding naked mole-rats. During colony disruption, we found a significant increase in cortisol concentrations in females removed from the colony for social isolation (experimental) as well as in females that remained in the colony (control). Cortisol concentrations were reduced in both groups after experimental animals were paired up. No changes in cortisol concentrations were observed in control or experimental males after removal from the colony or pairing. This suggests that the females, but not the males, found colony disruption and social isolation stressful. Upon removal from the colony, both control and experimental females showed a small increase in progesterone, which returned to basal levels again in the control animals. Experimental females showed a dramatic spike in progesterone when they were paired with males, indicating reproductive activation. The sex difference in the stress responses may be due to the stronger reproductive suppression imposed on females, or the increased likelihood of dispersal for males. It is clear that the social environment reflects on the endocrine correlates of animals living in a colony, and that the colony structure may affect the sensitivity of the animals to changes in their environment.

There are few stressful factors which have been investigated to affect adrenocortical function in the African wild dog (AWD) Lycaon pictus. Understanding what animals perceive as stressors is important for not only the implementation of management practices promoting general animal welfare in captivity, but also because a prolonged stress perception is known to disrupt reproduction, immune function and ultimately pose a threat to survival. In this regard, faecal glucocorticoid metabolite (fGCM) measurements are commonly used as a non-invasive approach to assess the impact of factors which may be acting as perceived stressors in wildlife. This study was aimed at determining if there are significant differences in AWD fGCM concentrations as a result of sex, presence of absence of an injury, age-class, body condition, dietary provisioning (through stable nitrogen (δ15N) and carbon (δ13C) isotope analysis), hierarchal status, and setting (i.e. captive or free-ranging). A total of 47 faecal samples were collected immediately post-defaecation from 39 AWDs across four free-ranging sites (Hluhluwe-iMfolozi Park, Madikwe Game Reserve, Kruger National Park and the Waterberg), two permanently captive sites (Johannesburg and Pretoria Zoo), and four temporary captive holding facilities (Hoedspruit Endangered Species Centre and Maremani, Tembe and Zimanga bomas) in South Africa. Captive AWDs had distinctively higher fGCM levels than their free-ranging counterparts, regardless of sex, body condition, hierarchal status, age-class or dietary provisioning. The present study is the first to assess physiological stress responses across permanently captive, temporarily captive and free-ranging AWDs within the South African managed metapopulation, while incorporating the use of stable isotope analysis to quantify differences in dietary isotopic profiles between these different settings. Ultimately this demonstrates the usefulness of fGCM analysis as a tool for assessing animal welfare in both captive and free-ranging AWDs, and underpins the importance of understanding factors perceived as stressors for the management of the species. Keywords: animal welfare, body condition scoring, faecal glucocorticoid metabolite monitoring, managed metapopulation, stress, wildlife management.

Non-invasive methods for measuring glucocorticoids and their metabolites are frequently used in ecological, behavioural and physiological studies of mammals. Using faeces, urine and other matrices for such a measurement has considerable advantages in comparison to more traditional methods, but also requires thorough validation of the methods used. Eastern rock sengis (Elephantulus myurus) are fascinating African mammals and the non-invasive monitoring of the adrenocortical activity opens up new opportunities to study their biology. We were able to validate two assays for measuring urinary (uGCM) and faecal glucocorticoid metabolite (fGCM) concentrations in this species using a dose-dependent challenge with adrenocorticotropic hormone (ACTH). A higher concentration of ACTH elicited higher uGCM and fGCM concentrations in both males and females. Interestingly, uGCM and fGCM concentrations and the responses to ACTH were higher in females than in males and small changes in faecal glucocorticoid metabolites could not be reliably detected in males. In contrast to ACTH, a saline injection did not result in an increase in uGCM or fGCM concentrations. The study also provided insight into when responses to a stressor are likely to be detected in the urine and faeces of sengis and opens up new opportunities to study the stress physiology of this and other sengi species. It further emphasises the importance of thoroughly validating non-invasive methods for measuring hormones in both sexes of a species and for incorporating dose-dependent approaches.

Veterinary schools rely on live animals to assist in teaching veterinary students clinical procedures. The aims of the study were to (1) quantify a potential physiological stress response of teaching mares during veterinary student transrectal palpation of their reproductive tracts using fecal glucocorticoid metabolites (fGCM) and salivary glucocorticoid concentrations (sGC) and (2) to determine the relationship between the glucocorticoid concentrations and both the ages and palpation experience of the study animals. Twenty-nine, adult, nonpregnant Nooitgedacht mares, which were accustomed to the routine procedure of palpation, were subjected to a palpation of 20 minutes per mare by a single student. Median sGC concentrations before palpation (collected in paddocks) were not significantly different from those determined at 10, 40, and 70 minutes after palpation (χ 2 = 1.69, df = 3, P = .64). Median fGCM concentrations before palpation (start of palpation) were not significantly different from those determined at 26 hours after palpation (W = −89.0; P = .34). Neither prepalpation sGC nor postpalpation fGCM concentrations correlated with either age of the mares (Pearson's r = −0.02, n = 28, P = .91; Pearson's r = 0.30, n = 29, P = .11) or practical experience (number of transrectal palpations conducted before the study; Pearson's r = −0.008, n = 28, P = .97; Pearson's r = 0.26, n = 29, P = .18) of the mares, respectively. This study suggested that student transrectal palpations of the reproductive tract in habituated mares did not activate the hypothalamic-pituitary-adrenal axis.

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.

Reports of the vaginal flora of wild cats such as lions or leopards are scarce. The micro-organisms most commonly found in the vagina of clinically healthy cats are aerobic bacteria such as coagulase-negative Staphylococcus, Streptococcus canis, and Escherichia coli. Simonsiella spp are large Gram-negative bacteria belonging to the Neisseriaceae family, typically found in the oral cavity and upper respiratory tract of many species. To date, there are no reports of the detection of Simonsiella spp in the vaginal flora of any felid. For a period of six months, daily behaviour monitoring was performed on six captive lionesses at a South African conservation centre, in parallel with the collection of vaginal swabs and interpretation of the resultant vaginal cytologies every other day. Oestrus was identified by typical female reproductive behaviours, as well as by enlarged and separated vulvar lips, and a predominant proportion of superficial cornified cells, clearing of the background, and high bacterial presence in the vaginal smear. Simonsiella spp were identified by their characteristic morphology in 58% (60 of 103) of the vaginal samples collected during oestrus. They were also found in oral swabs of three out of three lions tested. Additionally, Simonsiella spp were opportunistically found in a vaginal smear from a zoo housed female Sri Lankan leopard in oestrus, during a routine reproduction assessment. The finding of Simonsiella spp may be more common than previously suspected, transitory, and without detectable clinical relevance. A connection between occurrence of these bacteria and oestrus was apparent.

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 5α-3β-11β-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.