Method development: Assigning sex to African clawless otter spraints and assessing stability of faecal androgen and progestagen metabolites post-defaecation.

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.

A frog in hot water: the effect of temperature elevation on the adrenal stress response of an African amphibian.

Amphibians, with their unique physiology and habitat requirements, are especially vulnerable to changes in environmental temperatures. While the activation of the physiological stress response can help to mitigate the impact of such habitat alteration, chronic production of elevated glucocorticoid levels can be deleterious in nature. There is no empirical evidence indicating the physiological response of African amphibians to temperature changes, where individuals are unable to emigrate away from potential stressors. To rectify this, we used the edible bullfrog (Pyxicephalus edulis) as a model species to determine the effect of elevated temperature on the adrenocortical response of the species using a recently established matrix. While a control group was kept at a constant temperature (25 °C) throughout the study period, an experimental group was exposed to control (25 °C) and elevated temperatures (30 °C). Mucous swabs were collected throughout the study period to determine dermal glucocorticoid (dGC) concentrations, as a proxy for physiological stress. In addition to this, individual body mass measurements were collected. The results showed that individuals within the experimental group who experienced increased temperatures had significantly elevated dGC levels compared to the control animals. Furthermore, there was a significant difference in the percentage mass change between experimental and control animals . These findings indicate the physiological sensitivity of the edible bullfrog to a thermal stressor in captivity. While this study shows the importance of proper amphibian management within the captive environment, it also highlights the coming danger of global climate change to this and similar amphibian species.

An International Investigation of the Prevalence of Negative Visitor Behaviour in the Zoo.

Negative visitor behaviour is an understudied area of zoo research, even though negative actions can have serious implications for animal welfare. This research project investigated the prevalence of negative visitor behaviours at four different zoos. It included observations of visitors at seven different taxa exhibits and three different types of enclosures. A modified version of behaviour sampling was used to record visitor behaviour and the activity of the animals, while a negative binomial regression was conducted to test the significance of several predictor variables against the number of negative behaviours observed. Negative visitor behaviour was relatively common, occurring in 57% of observations. Banging was the most commonly observed negative action. Negative behaviours were influenced by zoo (p < 0.001), species (p < 0.001) and the number of visitors present (p < 0.001). The charismatic species were the most harassed animals included in the study, while children were the most likely to engage in negative behaviour. Negative visitor behaviours occurred more frequently when animals were active and in close proximity to visitors. It is imperative for zoos to understand visitors' behaviour so that they can effectively communicate with their visitors to minimise negative actions and promote better animal welfare.

Seasonal drivers of faecal glucocorticoid metabolite concentrations in an African strepsirrhine primate, the thick-tailed greater galago (Otolemur crassicaudatus).

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.

Urofaecal glucocorticoid metabolite concentrations in African penguin (Spheniscus demersus) chick populations experiencing different levels of human disturbance.

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.

Degradation of Temminck's pangolin (Smutsia temminckii) scales with a keratinase for extraction of reproductive steroid hormones.

Hormone monitoring in keratinous tissues has become increasingly popular. The insoluble keratin materials are generally pulverised before hormone extraction; however, this is difficult for thicker keratin structures like baleen plates or hooves. A new method, involving the use of keratinase, allows enzymatic digestion of keratin and hormone analysis in the resulting suspension. Pangolins are unique mammals covered in keratinous scales, which are one of the reasons these animals are extensively trafficked. This study aimed to investigate the suitability of Temminck's pangolin scales as hormone matrix for quantifying reproductive steroids. A protocol was developed to digest scales with a keratinase before measuring hormone concentrations. This method can be used to investigate the reproductive endocrinology of Temminck's pangolins but may also be extended to the other extant pangolin species.•Keratinase digests Temminck's pangolin scales and reproductive steroid metabolite concentrations are measurable in the resulting suspension.•Isopropanol is an ideal washing solvent for scales to remove surface contaminants and scale sample mass should be standardised to allow comparisons.•Any section of a scale and scales from any pangolin body region can be used as samples for hormone quantification.

Non-invasive monitoring of glucocorticoid metabolite concentrations in native Indian, as well as captive and re-wilded tigers in South Africa.

Over the last century, wild tiger (Panthera tigris) numbers have declined from over 100 000 individuals to fewer than 4 000, with animals now confined to less than 5% of their historic range due to habitat loss, persecution, inadequate management, and poaching. In contrast, 15 000-20 000 tigers are estimated to be housed in captivity, experiencing conditions vastly different than their wild counterparts. A total of 280 tigers are currently held at 44 different facilities within South Africa, including zoos, semi-captive 're-wilded' populations, and pets; these animals provide a unique opportunity to measure the impact of extrinsic factors, found in exotic habitats, on the adrenocortical activity of tigers. By monitoring and comparing stress-related faecal glucocorticoid metabolite (fGCM) concentrations of tigers housed at different locations, and free ranging tigers in natural tiger reserves, this project aimed to get a better understanding of the impact of extrinsic factors on adrenocortical function as a measure of stress. The results of this study showed no significant difference in fGCM concentrations between captive, re-wilded, and free-ranging tigers with the exception of one site. Furthermore, factors such as sex and season were not significant drivers of fGCM concentrations. One study group had elevated fGCM concentrations, showing population variation in the stress response. This indicates that populations are able to cope with exotic environments, however, as population-specific differences in the stress response exist, we suggest management protocols be created for each population. This study offered the unique opportunity to see how well tigers are faring outside of their native range and if having re-wilded tigers in exotic locations is a potential welfare-acceptable management option for tiger conservation globally.

Scales of our lives: Sex identification of Temminck's pangolin (Smutsia temminckii) using scales retrieved out of the illegal wildlife trade.

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.

Endocrine correlates of gender and throat coloration in the southern ground-hornbill (Bucorvus leadbeateri).

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.

Validating a non-invasive technique for monitoring physiological stress in the samango monkey.

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

Validating the use of dermal secretion as a matrix for monitoring glucocorticoid concentrations in African amphibian species.

The complex interaction between factors leading to amphibian declines is responsible for the inability to develop robust, standardized conservation action plans. Monitoring physiological stress responses in amphibians may provide an ideal tool to assist conservationists in this regard. This study aimed to validate dermal secretions as a robust matrix for monitoring glucocorticoid alterations in an African amphibian, the edible bullfrog (Pyxicephalus edulis). Both a biological (handling) and physiological (ACTH challenge) validation were conducted to determine which of five available enzyme immunoassays (EIAs) tested is most suited for monitoring alterations in dermal glucocorticoid (dGC) concentrations. Additionally, the most optimal body region for monitoring dGC concentrations in P. edulis was identified. To confirm the activation of the hypothalamic-pituitary-interrenal (HPI) axis following ACTH administration, urinary glucocorticoid metabolites (uGCM) were also quantified. The tested corticosterone EIA was the only assay able to monitor alterations in dGC concentrations following the handling event in P. edulis. Further validation during the ACTH challenge supported the corticosterone EIA in this regard. Dermal secretions collected from both the ventral and dorsal regions were suitable for monitoring dGC concentrations in both sexes. A considerable increase in uGCM concentrations following ACTH administration was found, suggesting that the dGC concentrations observed were derived from the HPI axis. This study offers further support for the use of dermal secretions as an important matrix for monitoring physiological stress, and thus general health, in amphibian species.

Non-invasive monitoring of glucocorticoid metabolite concentrations in urine and faeces of the Sungazer (Smaug giganteus).

Developing non-invasive techniques for monitoring physiological stress responses has been conducted in a number of mammal and bird species, revolutionizing field-based endocrinology and conservation practices. However, studies validating and monitoring glucocorticoid concentrations in reptiles are still limited. The aim of the study was to validate a method for monitoring glucocorticoid metabolite concentrations in urine (uGCM) and faeces (fGCM) of the cordylid lizard, the Sungazer (Smaug giganteus). An adrenocorticotropic hormone (ACTH) challenge was conducted on one male and two females with both urine and faecal material being collected during baseline and post-injection periods. Steroid extracts were analysed with four enzyme immunoassays (EIAs)namely: 11-oxoaetiocholanolone, 5α-pregnane-3ß-11ß-21-triol-20-one, tetrahydrocorticosterone, and corticosterone. A considerable response in fGCM and uGCM concentrations following ACTH administration was observed in all subjects, with the 5α-pregnane-3ß-11ß-21-triol-20-one and tetrahydrocorticosterone EIAs appearing to be the most suited for monitoring alterations in glucocorticoid metabolite concentrations in S. giganteus using faeces or urine as hormone matrix. Both EIAs showed a significantly higher concentration of glucocorticoid metabolites in faeces compared to urine for both sexes. Collectively, the findings of this study confirmed that both urine and faeces can be used to non-invasively assess adrenocortical function in S. giganteus.

Female reproductive activity and its endocrine correlates in the African lesser bushbaby, Galago moholi.

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.

The hustle and bustle of city life: monitoring the effects of urbanisation in the African lesser bushbaby.

Urbanisation has become a severe threat to pristine natural areas, causing habitat loss and affecting indigenous animals. Species occurring within an urban fragmented landscape must cope with changes in vegetation type as well as high degrees of anthropogenic disturbance, both of which are possible key mechanisms contributing to behavioural changes and perceived stressors. We attempted to elucidate the effects of urbanisation on the African lesser bushbaby, Galago moholi, by (1) recording activity budgets and body condition (body mass index, BMI) of individuals of urban and rural populations and (2) further determining adrenocortical activity in both populations as a measure of stress via faecal glucocorticoid metabolite (fGCM) levels, following successful validation of an appropriate enzyme immunoassay test system (adrenocorticotropic hormone (ACTH) challenge test). We found that both sexes of the urban population had significantly higher BMIs than their rural counterparts, while urban females had significantly higher fGCM concentrations than rural females. While individuals in the urban population fed mainly on provisioned anthropogenic food sources and spent comparatively more time resting and engaging in aggressive interactions, rural individuals fed almost exclusively on tree exudates and spent more time moving between food sources. Although interactions with humans are likely to be lower in nocturnal than in diurnal species, our findings show that the impact of urbanisation on nocturnal species is still considerable, affecting a range of ecological and physiological aspects.

Spicing up the menu: evidence of fruit feeding in Galago moholi.

The African lesser bushbaby, Galago moholi, is described as a food specialist, feeding exclusively on small arthropods and gum primarily from Acacia karroo trees. We studied a population of G. moholi in a highly fragmented habitat in the southernmost part of its natural distributional range in South Africa. In this habitat, we opportunistically observed bushbabies feeding on fruits of the winter fruiting tree, Pappea capensis. Plot counts of tree composition revealed that although the dominant tree species in the area belonged to the genus Acacia, A. karroo trees were widely absent and gum could only be found in small quantities on other Acacia species. The analysis of P. capensis fruits showed high levels of protein, fat, and energy content, making the fruits a potentially important food source for G. moholi during winter when insect availability is low. Our observation is the first documented case of fruit feeding in G. moholi, suggesting that the species is not a food specialist as previously reported but can supplement its diet with fruit when available.