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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.

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.

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 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.

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.

Amphibians are experiencing drastic population declines. New semi-invasive conservation methods are required to protect threatened species. The collection of mucus, from dorsal and ventral body regions, is an ideal matrix for monitoring stress hormones, and thus physiological stress, in African amphibians. This technique will assist in amphibian conservation in Africa., 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.

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., Graphical abstract Image, graphical abstract

Due to considerable global decline in wildlife population numbers and species diversity, because of various anthropogenic activities, conservationists increasingly rely on captive and managed populations as important reservoirs to ensure the survival of endangered and vulnerable species. However, very few of these facilities implement robust, non-invasive monitoring techniques to confirm the effectiveness of their management practices to address animal welfare challenges. This study assessed adrenocortical activity as an indication of environmental stress by investigating the effects of both natural (climate, life-history stages) and anthropogenic (visitor presence) factors on captive-housed African penguins. Seven male-female African penguin breeding pairs were housed in a large, naturalistic outside enclosure at the National Zoological Garden (NZG), South Africa. Weekly urofaecal samples were collected from all individuals over one-year to measure urofaecal glucocorticoid metabolite (ufGCM) concentrations. General linear mixed model analysis determined that visitor presence (for males) and rainfall (for females) were the two factors which best explained the variation in ufGCM concentrations of the study population; however, none of the environmental and anthropogenic factors monitored were found to be significant. A posthoc graphical analysis showed considerable individual variation in terms of ufGCM concentrations within and between sexes when comparing life-history stages. This study confirms that non-invasive steroid monitoring can be an effective tool set for defining and assessing environmental stressors for African penguins and potentially other captive seabirds. However, conservationists and wildlife managers should also consider that individual-, sex-, and population-specific differences in the response to environmental stressors can exist. As such, a generalized management protocol for a specific species may not be sufficient and should be customized according to the specific captive population and/or individual.

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-3β-11β-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-3β-11β-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.

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 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.

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.

Faecal hormone monitoring offers a robust tool to non-invasively determine the physiological stress experienced by an individual when faced with natural or human-driven stressors. Although already quantified for several species, the method needs to be validated for each new species to ensure reliable quantification of the respective glucocorticoids. Here we investigated whether measurement of faecal glucocorticoid metabolite (fGCM) provides a feasible and non-invasive way to assess the physiological state of sugar gliders (Petaurus breviceps), an arboreal marsupial native to Australia, by using both a biological and physiological validation. Our analysis confirmed that the cortisol enzyme immunoassay (EIA) was the most appropriate assay for monitoring fGCM concentrations in sugar gliders. Comparing the fGCM response to the physiological and the biological validation, we found that while the administration of ACTH led to a significant increase in fGCM concentration in all individuals, only six of eight individuals showed a considerable fGCM response following the biological validation. Our study identified the most appropriate immunoassay for monitoring fGCM concentrations as an indicator of physiological stress in sugar gliders, but also supports recent suggestions that, if possible, both biological and physiological stressors should be used when testing the suitability of an EIA for a species.

Urbanisation is an important factor driving species and biodiversity decline. Although habitat alterations can be detrimental for species, studies have shown that many diurnal primates are able to adapt to novel environments. Little is known about the ability of nocturnal primates to survive within an urban environment. To increase our understanding on this topic, we present ad libitum observations on group movement and sociality from urban and rural populations of the African lesser bushbaby (Galago moholi) in South Africa from 2014-2018. Our data show considerable changes in the social dynamics within urban bushbaby populations. In contrast to rural individuals, which spent the majority of their activity period solitarily or in pairs, urban individuals displayed a larger degree of sociality throughout their active period, forming groups of up to ten individuals. Furthermore, urban individuals spent less time moving around, while increasing social (communication/pair-grooming), foraging and feeding behaviour. Urban individuals fed on a range of different anthropogenic and natural food sources (insects/tree gum/nectar) compared to their rural counterparts. In summary, urban bushbabies showed a large degree of behavioural plasticity, with changes in social dynamics and structure frequently observed. Such alterations in sociality, along with the ability to utilise different feeding resources, may explain the ability of the species to survive within a highly altered environment.

Periods of reproduction are linked to changes in male behaviour, physiology and physical parameters. Although high androgen concentrations hold numerous advantages, especially during reproductive periods, chronically elevated androgen concentrations over long periods may be costly and thus need to be regulated. As such seasonal breeders will display temporary elevated androgen concentrations, increased testis levels and mating behaviour only during important reproductive periods. We studied a captive as well as a free‐ranging population of the polygamous strepsirrhine primate, the African lesser bushbaby Galago moholi, to clarify the link between androgen concentration, reproductive behaviour and testis size and the importance of the two mating periods observed in the species. To monitor androgen patterns, we used faecal sampling and quantification of faecal androgen metabolites (fAM). We additionally collected testicular measurements and behavioural data. G. moholi displayed a strong degree of reproductive seasonality, with maximum fAM concentration, testicular volume (TV) and behavioural activity focused on the mating periods. In contrast to other studies, TV increased prior to fAM, with reproductive activity being initiated only when fAM concentrations reached high levels. Changes in TV and fAM concentrations were not significantly different between both mating periods. Based on the absence of a significant difference between mating seasons, it is likely that male G. moholi attempt to maximize their reproductive success by utilizing both mating periods equally. This study is the first to describe the reproductive endocrine pattern linked to physical changes and mating behaviour in any galago species, increasing our understanding of the reproductive biology of nocturnal, polygamous primates.

In seasonal breeders, periods of reproductive activity often coincide with high levels of glucocorticoids. We studied seven male and seven female African lesser bushbabies (Galago moholi A. Smith, 1836) over two mating periods via noninvasive faecal hormone metabolite monitoring to investigate the relationship between reproductive and adrenocortical hormone activity. We used linear mixed-effect models to investigate the effect of physiological (endocrine) variables on faecal glucocorticoid metabolite concentrations. Our results indicate faecal androgen (males) and progestagen metabolite (females) concentrations as the variables best able to explain variability in faecal glucocorticoid metabolite concentrations. However, the models explained only a fraction (26% and 12%, respectively) of the observed variability and graphical analysis suggests a biologically relevant difference in faecal glucocorticoid metabolite concentrations between captive and free-ranging animals during nonreproductive periods. Thus, captivity may have affected glucocorticoid output in our focal animals, potentially weakening the expected relationship between reproductive activity and faecal glucocorticoid metabolite variability. Due to the ease of faecal and observational sample collection, a large number of studies monitoring adrenocortical activity in wildlife are conducted using only captive settings, with inferences unquestioned when applied to free-ranging scenarios. Our study cautions against this practice, as particular housing or management conditions may influence the pattern of adrenocortical activity.