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Non-Invasive Assessment of Adrenocortical Activity as a Measure of Stress in Leopards Panthera pardus
Abstract and Figures
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.
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... However, faecal sampling for quantifying GC metabolites presents a number of challenges, such as a possible delay in time between defaecation and collection, and the variability of that time between sampling events (Crossey et al., 2018;Majelantle et al., 2020;Webster et al., 2018). Apart from challenges related to the sampling procedure, variations in sample preparation, including the possible challenge of irregular distribution of hormone metabolites throughout a faecal sample (Millspaugh & Washburn, 2004), and the requirement for specialized and expensive laboratory equipment to prepare samples for analyses (Postiglione et al., 2022) have to be considered. ...
... The opposite trend of declining fGCM concentrations post-defaecation has been shown for brown hyenas (Hyaena brunnea; Hulsman et al., 2011), and banded mongoose (Mungos mungo; Laver et al., 2012), Depending on the assay used, an increase or decrease in fGCM concentrations has also been observed in sheep (Ovis aries; Lexen et al., 2008) and cattle (Bos taurus; Morrow et al., 2002). However, some studies demonstrated little-to-no change in fGCM concentrations over the monitored period, as has been seen in Nile crocodiles (Crocodylus niloticus) (Ganswindt et al., 2014), Jaguars (Panthera onca; Mesa-Cruz et al., 2014) and Leopards (Panthera pardus; Webster et al., 2018). Previous studies have examined potential changes in immunoreactive fGCM concentrations post-defaecation for different species, usually focusing at a single species level. ...
... Three species from each of the three feeding groups investigated were included in our study, four of which have already been investigated, namely; (1) ruminants: (a) impala (Aepyceros melampus), (b) giraffe (Giraffa camelopardalis), (c) blue wildebeest (Connochaetes taurinus) (Wolf et al., 2021); (2) hindgut fermenters: (d) plains zebra (Equus quagga), (e) African elephant (Loxodonta Africana) (Webber et al., 2018), (f) white rhino (Ceratotherium simum) (Nhleko et al., 2022); and (3) carnivores: (g) cheetah (Acinonyx jubatus), (h) spotted hyena (Crocuta crocuta) and (i) leopard (Panthera pardus) (Webster et al., 2018). ...
Quantification of faecal glucocorticoid metabolites (fGCMs) is a popular non‐invasive technique for monitoring wildlife's response to stressors, demanding an understanding of the stability of fGCM concentrations post‐defaecation to ensure comparability of determined fGCM values across samples. To provide species‐specific recommendations for the duration within which sampling can take place, we measured the rate at which the fGCM concentrations of nine different species changed throughout a 7‐day period post‐defaecation. In this study, we explored the temporal dynamics of fGCM concentrations in nine species across three feeding classes (ruminants, hindgut fermenters and carnivores): impala (Aepyceros melampus), giraffe (Giraffa camelopardalis), blue wildebeest (Connochaetes taurinus), plains zebra (Equus quagga), African elephant (Loxodonta africana), white rhino (Ceratotherium simum), cheetah (Acinonyx jubatus), spotted hyena (Crocuta crocuta) and leopard (Panthera pardus). Utilizing enzyme immunoassays already established for each of the focal species, we identified broader feeding class‐specific patterns. All herbivores exhibited a significant decrease in fGCM concentrations over time, starting from 6 h (impala) to 48 h (giraffe, blue wildebeest, white rhino and African elephant) post‐defaecation. For carnivores, concentrations remained fairly comparable for 12–24 h, after which fGCM concentrations either decreased (spotted hyena), increased (leopard) or remained stable (cheetah), with notable variation in triplicate concentrations (cheetah and leopard). These findings offer insights into scheduling faecal sampling for endocrine monitoring, particularly from free‐roaming wildlife, to ensure comparability of determined hormone metabolite concentrations. Furthermore, the species‐specific variation in fGCM concentration post‐defaecation demonstrated in this study underlines the necessity to investigate every new species to ensure accurate and comparable results. Future studies ought to investigate how the mass of collected material, sex and drying methodologies affect the measurement of fGCMs post‐defaecation.
... Because several factors (i.e. temperature, bacterial enzyme) can influence fGCM concentrations found in samples, it is important to validate the technique used to monitor fGCM concentration for the species being studied (Touma and Palme 2005;Webster et al. 2018). This is done to ensure samples collected still contain concentrations of biologically relevant target agents (Webster et al. 2018). ...
... temperature, bacterial enzyme) can influence fGCM concentrations found in samples, it is important to validate the technique used to monitor fGCM concentration for the species being studied (Touma and Palme 2005;Webster et al. 2018). This is done to ensure samples collected still contain concentrations of biologically relevant target agents (Webster et al. 2018). As such, we determined the stability of the fGCM concentrations postdefecation. ...
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 (<500 km²), season (wet and dry) and rainfall patterns were responsible for driving adrenocortical activity in white rhino.
Methods
To understand the relationship between rhino adrenocortical responses and different environmental and anthropogenic stressors, we quantified glucocorticoid metabolites in faecal samples (fGCM) collected from four populations within three protected areas (i.e. two small parks, one big park) during the wet and dry seasons.
Key results
We found differences in seasonal fGCM concentrations, with a 42% increase during the dry season, and no differences in fGCM concentrations between the high and low poaching areas. Additionally, we found fGCM concentrations in samples from the small parks were respectively 38% and 42% higher than in samples from the large park during both the dry and wet seasons compared.
Conclusions
Our results suggest that white rhinos may experience physiological stress in smaller parks, especially during the dry season when resources are limited.
Implications
By mitigating stress associated with reduced access to resources and spatial constraints, managers may better promote the viability of large mammals in small protected areas.
... By calculating and comparing the ratio of fAM:fPM concentrations from known sex individuals, distinct thresholds can therefore be used to allocate sex to faecal samples from unknown individuals. Potential alterations in immunoreactive hormone metabolite concentrations post-defecation for each sex can be determined by exposing various sub-sets of a faecal samples to different environmental conditions and subsequently testing samples at defined time intervals [16][17][18][19]. Once validated, this method allows sex determination of free-ranging individuals from faecal samples without observation or the need to capture, restrain, or handle individuals, an approach not yet established for African clawless otters. ...
Method name: Using African clawless otter spraints to quantify reproductive hormones Monitoring reproductive physiology in wildlife can be a useful tool for assessing population dynamics for conservation and management purposes. Utilizing non-invasive approaches for this, such as quantifying reproductive hormone metabolites from faeces, can be challenging when defaecation events are not observed, or when cryptic species like African clawless otters (Aonyx capensis) are involved. Additionally, test systems for quantifying hormone metabolites in a species for the first time must first be reliably validated prior to use. Our results indicate that Epiandros-terone and Progesterone EIAs are most suitable for determining fAM and fPM concentrations in African clawless otter spraints. The fAM:fPM ratio and respective thresholds are more reliable in sex identification compared to the separate use of individual hormone classes. Sex-related hormone metabolite concentrations remained comparable for up to 12hrs post-defaecation in both sexes. • We screened two androgen and two progestagen enzyme-immunoassays (EIAs) for suitabil-ity and reliable quantification of faecal androgen metabolites (fAM) and faecal progestagen metabolites (fPM) in African clawless otters. • We assessed whether the ratio of fAM:fPM concentrations can be used to assign sex to faecal samples from unknown individuals. • We tested the stability of fAM and fPM concentrations post-defaecation to determine the effects of environmental exposure and bacterial metabolism. Specifications table Subject area: Agricultural and Biological Sciences More specific subject area: Zoology; Endocrinology Name of your method: Using African clawless otter spraints to quantify reproductive hormones Name and reference of original method: Original research Resource availability:
... By calculating and comparing the ratio of fAM:fPM concentrations from known sex individuals, distinct thresholds can therefore be used to allocate sex to faecal samples from unknown individuals. Potential alterations in immunoreactive hormone metabolite concentrations post-defecation for each sex can be determined by exposing various sub-sets of a faecal samples to different environmental conditions and subsequently testing samples at defined time intervals [16][17][18][19]. Once validated, this method allows sex determination of free-ranging individuals from faecal samples without observation or the need to capture, restrain, or handle individuals, an approach not yet established for African clawless otters. ...
Monitoring reproductive physiology in wildlife can be a useful tool for assessing population dynamics for conservation and management purposes. Utilizing non-invasive approaches for this, such as quantifying reproductive hormone metabolites from faeces, can be challenging when defaecation events are not observed, or when cryptic species like African clawless otters (Aonyx capensis) are involved. Additionally, test systems for quantifying hormone metabolites in a species for the first time must first be reliably validated prior to use. Our results indicate that Epiandrosterone and Progesterone EIAs are most suitable for determining fAM and fPM concentrations in African clawless otter spraints. The fAM:fPM ratio and respective thresholds are more reliable in sex identification compared to the separate use of individual hormone classes. Sex-related hormone metabolite concentrations remained comparable for up to 12hrs post-defaecation in both sexes.•We screened two androgen and two progestagen enzyme-immunoassays (EIAs) for suitability and reliable quantification of faecal androgen metabolites (fAM) and faecal progestagen metabolites (fPM) in African clawless otters.
•We assessed whether the ratio of fAM:fPM concentrations can be used to assign sex to faecal samples from unknown individuals.
•We tested the stability of fAM and fPM concentrations post-defaecation to determine the effects of environmental exposure and bacterial metabolism.
... By calculating and comparing the ratio of fAM:fPM concentrations from known sex individuals, distinct thresholds can therefore be used to allocate sex to faecal samples from unknown individuals. Potential alterations in immunoreactive hormone metabolite concentrations post-defecation for each sex can be determined by exposing various sub-sets of a faecal samples to different environmental conditions and subsequently testing samples at defined time intervals [16][17][18][19]. Once validated, this method allows sex determination of free-ranging individuals from faecal samples without observation or the need to capture, restrain, or handle individuals, an approach not yet established for African clawless otters. ...
... (voided within the previous 24 h) were deposited into a double-layer Ziploc plastic bag. To minimize microbial alteration of steroid metabolite composition, all samples were placed in a field cooler box and transported to the laboratory within 30 min where they were immediately frozen at − 20 • C. Frozen fecal samples were lyophilized, pulverized, sieved through a wire-mesh filter, and finally extracted with 80 % ethanol as described by Webster et al. [16]. Fecal extracts were stored in Eppendorf vials at − 20 • C until analyses. ...
... Bacterial enzymes can cause the fGCM concentration in feces to continue to rise or fall after a certain time [14,88]. In White-tailed Sea Eagles, the stability of urine GCM concentration lasted for 9 days [43], in the Nile Crocodile the fGCM concentration in feces remained stable for 72 h at ambient temperature [89], while in Grey Mouse Lemurs it remained stable for at least 10 h [64], in Cheetahs for 22 h at 0-4 • C [90], in Blue Wildebeest for 8 h [71], and in Leopards even up to 6 days post-defecation [91]. In the present study, glucocorticoid concentrations remained relatively stable with a variation of up to 18% within the first 4 h. ...
Simple Summary
The measurement of stress in wildlife, especially using non-invasive methods, is an important tool when monitoring wild animals taken into human care. However, the methods used to measure stress hormone metabolites from feces must be validated for each species. Eight Common Buzzards habituated to humans were placed into prepared aviaries and their feces were collected over a period of seven days. For biological validation, handling and restraint were used as a stress event. Hormone metabolites were analyzed using three different enzyme immunoassays to find the most suitable one. In addition, a degradation experiment was conducted to find out how long the fecal glucocorticoid metabolites (fGCMs) remained stable in the feces at room temperature. The cortisone enzyme immunoassay detected a distinctive peak of excreted glucocorticoid metabolites in response to the stress event. We found no significant differences between the sexes, but a diurnal variation in the stress hormone metabolites. Immunoreactive metabolite concentrations showed a significant change eight hours after defecation, indicating degradation processes. Our study successfully validated the non-invasive measurement of fGCMs as a stress indicator in the Common Buzzard and could lay the foundation for future studies providing new insights for animal welfare research in this species.
Abstract
For wild animals, being in captivity in wildlife centers can cause considerable stress. Therefore, it is necessary to establish and validate non-invasive tools to measure chronic stress during rehabilitation. Eight Common Buzzards which lived in permanent husbandry were placed individually into prepared aviaries and their feces were collected before, during and after a stress event for biological validation over a period of seven days. The extracted fecal glucocorticoid metabolites (fGCMs) were analyzed with three different enzyme immune assays (EIA) to find the most suitable one. Additionally, we aimed to investigate the stability of fGCM levels after defecation because further metabolization by bacterial enzymes can lead to changed results. The Cortisone-EIA performed best in males and females and showed that the stress event led to an fGCM increase of 629% (557% in females and 702% in males) in relation to basal values. We found no significant differences between the sexes, but observed significant differences between different times of day. FGCM concentration significantly changed after eight hours at room temperature. Our study successfully validated the non-invasive measurement of fGCM as a stress indicator in Common Buzzards and could therefore lay the foundation for future studies providing new insights for animal welfare research in Buzzards.
... En comparación con estudios realizados en leopardos indios (Panthera pardus fuska), los valores obtenidos son mucho menores a los de un estudio efectuado en dos zoos de India (10400 ±3870 ng cortisol/g heces) (Panchal, Desai, & Ghosal, 2021) y algo menores que los obtenidos en leopardos de seis zoos de India (270 ± 13 ng cortisol/g heces) (Vaz, Narayan, Kumar, Thenmozhi, Thiyagesan, & Baskaran 2017) y en leopardos en áreas de conservación (290-390 ng cortisol/g heces) (Ganswind, Webster, Burroughs, & Laver, P., 2018). ...
Conditions for wild animals in captivity in zoological parks have been in constant change throughout the years, to improve animal welfare. One of the proposed methods was the introduction of sensorial enrichment in these centres trying to make them resemble as much as possible the animals’ lifestyle in their natural habitat. In this study, a sensorial enrichment has been tested in big felines from the zoological centre Terra Natura Murcia (6 lions, 2 leopards and 2 lynx) using scents. For this purpose, two products were tested, being one a synthetic product commercialised as Feliway® (synthetic analogue of the feline pheromone F3) and the other a natural concentrated lavender essence with the intention of surveying their effects on the animals’ behaviour and their possible stress levels measuring faecal cortisol levels. Both the ethograms and the faecal sample collection took place before and after the introduction of the tested scents. The ethogram’s results indicated an increase in the behaviours such as smelling and rubbing against each other as well as the appearance of new behaviours such as attacking/playing or jumping/climbing up. The results of the measurements of faecal cortisol could not prove a decrease in stress levels due to lack of sample individualisation and limited number of samples. Despite the need of further studies, this study shows that the use of the tested products could improve animal welfare in Terra Natura Murcia.
Las condiciones de los animales salvajes en cautividad en los parques zoológicos han ido cambiando a lo largo de los años, buscando fundamentalmente mejorar el bienestar de los animales. Los primeros cambios, que afectaban a el diseño de alojamientos y espacio por animal, han sido implementados con otras medidas que consisten en la introducción de enriquecimiento ambiental de modo que las condiciones de vida en estos centros se asemejen en cierta medida al estilo de vida de cada una de las especies de animales en el medio natural En el presente estudio, se ha ensayado un enriquecimiento de tipo sensorial en grandes felinos del centro zoológico Terra Natura Murcia (6 leones, 2 leopardos y 2 linces) a través de olores. Para ello se han utilizado dos productos, uno sintético comercializado con el nombre de Feliway® (análogo sintético de la feromona F3 felina) y otro natural, esencia de lavanda concentrada con el fin de valorar el efecto que producen tanto en el comportamiento de los animales, mediante la realización de etogramas como en los posibles niveles de estrés a partir de la medición de cortisol en heces. Las observaciones del comportamiento y su recogida en etogramas, así como la recogida de muestras fecales fueron realizadas antes y después de la aplicación de los estímulos sensoriales. Los resultados del etograma mostraron aumento de la actividad de los animales con comportamientos como olfateo y restregarse además de la aparición de nuevos comportamientos como atacar/jugar o saltar/trepar. Los resultados de cortisol en heces no permitieron concluir que se produzca una disminución del estrés en los animales, sin embargo, esto puede ser debido a la falta de individualización de las muestras y el limitado número de las mismas. Por lo tanto, y, aunque son necesarios más estudios, el uso de estos productos podría mejorar el bienestar de los animales presentes en el Parque Terra Natura Murcia.
Noninvasive faecal hormone analyses can provide valuable information on the physiological state of wild animals and how they respond to ecological changes or anthropogenic disturbances. However, preservation techniques to prevent hormone alteration can be problematic, and not all are field friendly. We compared five processing methodologies to preserve samples for faecal glucocorticoid, progestagen and thyroid hormone metabolites. Samples were collected from adult zoo Africa savanna elephants (Loxodonta africana) (one male, four females) immediately after defecation. Subsamples were then subjected to five preservation methods: lyophilisation (LYO) (considered the gold standard), dehydration, solid-phase extraction (SPE) and two ethanol extraction methods—with and without being immediately dried down. Faecal glucocorticoid, progestagen and thyroid hormone metabolites were quantified by validated enzyme immunoassays. After 7 days at room temperature (to emulate shipping conditions), faecal glucocorticoid metabolite concentrations were lower for all methods compared to LYO. For thyroid hormone metabolite concentrations, the dehydration process resulted in higher concentrations compared to LYO, whereas with SPE, concentrations were lower. For faecal progestagen metabolite concentrations, there were no discernible differences across methods. Based on these results, we recommend ethanol extraction followed by immediate sample desiccation, a method that combines technical simplicity with the advantage of ambient temperature sample storage and transportation. Nevertheless, each investigator should consider the best method for the research question, field conditions, budget, equipment accessibility and shipping requirements, especially as results can vary by species and assay used. With growing interest in assessing animal welfare, validating field methods for noninvasive hormone monitoring is essential.
Monitoring stress-related faecal glucocorticoid metabolite (fGCM) concentrations is a reliable, popular and established approach for understanding wildlife responses to perceived stressors. To maintain fGCM integrity post-defaecation, faecal material must be promptly stored frozen, or dried to prevent continued suspected bacterial enzyme activity. We compare the effectiveness of freeze-drying with other field-friendly drying techniques (food dehydrator and homemade solar oven). We collected 10 fresh faecal samples each from nine species (giraffe, impala, blue wildebeest, plains zebra, African elephant, white rhino, cheetah, spotted hyena, and leopard) and monitored alterations in fGCM concentrations over time utilizing these different drying techniques. Our findings indicate that a homemade solar oven is as effective as freeze-drying faecal samples. A food dehydrator is also a suitable method for drying faecal samples for the carnivores monitored. Our findings provide field-friendly methods for researchers dealing with logistical constraints in remote field sites.•For all species examined, a homemade solar oven offers a practical and affordable alternative to freeze-drying faeces for fGCM quantification.
•A food dehydrator provides an affordable alternative to freeze-drying faeces for fGCM analysis when monitoring carnivores.
•Different faecal sample drying techniques should not be utilized within a single study to ensure comparable analyses of fGCM values.
Data on the population dynamics and threats to large carnivores are vital to conservation efforts, but these are hampered by a paucity of studies. For some species, such as the leopard (Panthera pardus), there is such uncertainty in population trends that leopard trophy hunting has been banned in South Africa since 2016 while further data on leopard abundance are collected. We present one of the first assessments of leopard population dynamics, and identify the key threats to a population of leopards outside of protected areas in South Africa. We conducted a long-term trap survey between 2012 and 2016 in the Soutpansberg Mountains, and drew on a previous estimate of leopard population density for the region from 2008. In 24 sampling periods, we estimated the population density and assessed population structure. We fitted eight leopards with GPS collars to assess threats to the population. Leopard population density declined by 66%, from 10.73 to 3.65 leopards per 100 km² in 2008 and 2016, respectively. Collared leopards had a high mortality rate, which appeared to be due to illegal human activity. While improving the management of trophy hunting is important, we suggest that mitigating human–wildlife conflict could have a bigger impact on carnivore conservation.
Little is known about the levels of stress experienced by African buffalos affected by injury, disease, or socio-ecological and anthropogenic factors. To be able to start filling this gap, we examined the suitability of two 11-oxoaetiocholanolone enzyme-immunoassays (EIAs) detecting 11,17 dioxoandrostanes (11,17-DOA) as well as faecal glucocorticoid metabolites(FGMs) with a 5β-3α-ol-11-one structure (3α,11oxo-CM), respectively, for monitoring stress-related physiological responses in African buffalo. An adrenocorticotrophic hormone (ACTH) challenge in one male and one female housed at Mokopane Biodiversity Conservation Centre, South Africa, showed a threefold increase in circulating cortisol levels in a sample taken 40 min post-injection. Corresponding 11,17-DOA levels increased tenfold (female) and 15-fold (male) above baseline, and 3α,11oxo-CM concentrations increased ninefold (female) and 12-fold (male) above pre-injection levels, indicating that both EIAs are suitable for measuring FGMs in African buffalo.In addition, 11,17-DOA levels monitored during the adaptation process of individual housing revealed an up to 14-fold elevation in FGMs. Storage of faeces at ambient temperature for up to 16 h post-defecation resulted in an significant increase in 11,17-DOA levels 2 h after defecation. Finally, higher individual baseline 11,17-DOA concentrations were found in samples defecated overnight, indicating a possible diurnal effect in excretion of FGMs in African buffalo.
The leopard’s (Panthera pardus) broad geographic range, remarkable adaptability, and secretive nature have contributed to a misconception that this species might not be severely threatened across its range. We find that not only are several subspecies and regional populations critically endangered but also the overall range loss is greater than the average for terrestrial large carnivores. To assess the leopard’s status, we compile 6,000 records at 2,500 locations from over 1,300 sources on its historic (post 1750) and current distribution. We map the species across Africa and Asia, delineating areas where the species is confirmed present, is possibly present, is possibly extinct or is almost certainly extinct. The leopard now occupies 25-37% of its historic range, but this obscures important differences between subspecies. Of the nine recognized subspecies, three (P. p. pardus, fusca, and saxicolor) account for 97% of the leopard’s extant range while another three (P. p. orientalis, nimr, and japonensis) have each lost as much as 98% of their historic range. Isolation, small patch sizes, and few remaining patches further threaten the six subspecies that each have less than 100,000 km2 of extant range. Approximately 17% of extant leopard range is protected, although some endangered subspecies have far less. We found that while leopard research was increasing, research effort was primarily on the subspecies with the most remaining range whereas subspecies that are most in need of urgent attention were neglected.
Steroid hormones play an important role in female reproductive physiology and behaviour and are often used to monitor important female reproductive events. However, such studies are often attempted on captive populations alone, delivering limited data. One such example is the African lesser bushbaby, Galago moholi, for which contradicting observational data exist between captive and free-ranging populations, while hormonal analyses have only been obtained from a single captive population. To extend and rectify the limited information, we monitored faecal progestagen and oestrogen metabolite levels across various important life history stages of both captive and free-ranging G. moholi. We additionally recorded changes in vaginal state as well as the occurrence of reproductive and aggressive behaviour throughout the study. Data from our captive population revealed an ovarian cycle length of 33.44 ± 0.59 days (mean ± SD), with follicular and luteal phases of 14.2 ± 1.0 and 19.1 ± 1.5 days, respectively, and an average pregnancy length of 128 ± 3.3 days. The initiation of female reproductive activity was closely linked to an oestrus-related increase in faecal oestrogen metabolite levels. Four of the seven captive females monitored in our study conceived during the May mating period, with one additional female fertilised in September, supporting the idea that the September mating period functions as a back-up for female G. moholi. Identified benchmark faecal progestagen metabolite levels (non-pregnant: >1 µg/g dry weight (DW), pregnant: >9 µg/g DW) should help researchers to determine pregnancy status of randomly wild-caught females in even a cross-sectional study setup.
Rhinoceros populations in Africa are under severe threat as a result of surging poaching rates and risk-mitigation strategies are continuously adapted in an attempt to ensure the survival of the species. This study compared faecal glucocorticoid metabolite (fGCM) levels of two age classes of limited free-ranging female white rhinos with fGCM levels of adult free-ranging female white rhinos. Subsequently, fGCM alterations in the limited free-ranging animals were monitored following routine dehorning as a measure of the animals’ short-term physiological stress response. Baseline fGCM levels differed significantly between tested groups, with both free-ranging and limited free-ranging adult animals showing significantly higher fGCM levels compared with limited free-ranging juvenile females. In contrast, baseline fGCM levels did not differ significantly between limited free-ranging and free-ranging adult individuals. Routine dehorning procedures resulted in a short-term stress response expressed by a significant increase in fGCM levels 48 h post-dehorning, with stress steroid levels returning to pre-dehorning concentrations 72 h after the procedure.
Measuring physiological stress reactions through the quantification of plasma cortisol often involves physical restraint, which acts as a stressor itself. Here, we present the validation of a non-invasive method for assessing adrenocortical activity as an indicator of stress in the bat-eared fox (Otocyon megalotis). By conducting an adrenocorticotropic hormone (ACTH) challenge, we examined the suitability of three enzyme immunoassays (EIAs) detecting 11,17 dioxoandrostanes (11,17-DOA) as well as faecal glucocorticoid metabolites (fGCM) with a 5β-3α-ol-11-one (3α,11oxo-CM), or 11,17,21-trihydroxy-4-ene-20-one structure (cortisol), respectively, for monitoring stress-related physiological responses in male and female bat-eared foxes. Our results suggest that the cortisol EIA seems most suitable for measuring fGCMs in this myrmecophageous mammal. Using the cortisol EIA, we compared fGCM concentrations of three populations of foxes. Only one population experienced a sudden change in social environment and the assay appeared to effectively detect the expected resulting increase in stress hormone levels. Therefore, the identified EIA is effective at detecting intraspecific variation in fGCM levels and hence is a useful tool to evaluate physiological stress responses in this species.
Corticoids are a group of vital hormones. Measuring the corticoid concentration in plasma, saliva, hair or feathers, and the amount of glucocorticoids or their metabolites in urine or faeces offers valuable information for clinical diagnosis, livestock management, animal welfare, and conservation biology. Disturbances of animals influence the endogenous glucocorticoid production, but a careful selection of the sample matrix can overcome some of the sampling difficulties. Some matrices also offer new possibilities, especially post hoc information about the adrenal status. Since most glucocorticoids are excreted as metabolites, the biological activity of these substances is covered in this respect.