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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.

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.

ABSTRACT DNA metabarcoding is a contemporary technique in diet composition studies and stands to fill key knowledge gaps left by traditional diet analysis methods. For endangered species such as the African wild dog (Lycaon pictus), the fulfilment of these knowledge gaps presents an opportunity for improved management practices and vulnerability assessments. There are an estimated ~600 African wild dogs remaining in South Africa. These dogs are generally understood to prey upon impala (Aepyceros melampus) and other medium‐sized ungulates. Here, we present the first assessment of DNA metabarcoding as a valuable method for diet composition analysis of this highly social carnivore. DNA from faecal samples collected across seven landscape types in the Kruger National Park (KNP) was extracted and used to determine the presence of seven unique prey taxa, including novel species such as the Cape hare (Lepus capensis). Impala was identified as a prey item in all landscape types, complementing the diet preference prediction made with stable isotope analysis using the same samples and existing understanding of wild dog diet. Given recommended improvements, the application of DNA metabarcoding in wild dog diet analysis shows promising prospects for identifying novel prey species and validating previous records of this endangered canids diet.

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.

To develop successful ex-situ breeding programs for the conservation of threatened wildlife, an understanding of reproductive endocrinology in the species of interest is essential. Fecal hormone metabolite patterns are widely used in this regard, but limited information is available regarding how these reflect blood hormone profiles. This study aimed to longitudinally monitor and compare circulating progestagen (sP) and estrogen (sE) concentrations, as well as fecal progestagen (fPM) and estrogen (fEM) metabolite concentrations with observed reproductive behaviors of African lionesses. For 18 months, blood sampling (n = 309) from five captive lionesses took place 1–7 times per week during positive reinforcement training. In parallel, over a period of 9 months, fecal samples (n = 129) from four of these lionesses were searched for daily and collected when found. Daily behavioral monitoring of all females enabled classification of reproductive stage according to presence/absence of characteristic behavioral events (i.e., “allowing mount”, “copulation”, “flirting run”, “lordosis”, “purring”, and “rolling”). Competitive enzyme immunoassays were used for steroid quantification. In total, 11 an-ovulatory and 16 ovulatory cycles (7 pregnancies and 9 pseudo-pregnancies) were assessed. When compared, the pattern of fecal hormone metabolite concentrations matched reproductive behavior more reliably than circulating steroid concentrations. Both sP and fPM patterns were correlated (n = 51; r = 0.86) and enabled identification of luteal phases, which helped to distinguish between pregnant and non-pregnant females. In contrast, detection of estrus by measurement of sE and fEM was not accurate regardless of the matrix evaluated.

Given the ongoing fragmentation and decline of suitable habitat coupled with recurring disease outbreaks and pack eradications, a major rethink is required to manage the long-term genetic diversity of the endangered African wild dog. Competing financial demands in developing countries mean that conservation strategies need to yield the biggest genetic gains for the smallest economic cost. As such, we propose the development of a hybrid conservation approach to the metapopulation management of African wild dogs, which augments assisted dispersal and natural breeding initiatives with genetic gains afforded by sperm banking and artificial insemination. We discuss how such an approach would address current challenges faced by the species in the context of its unique social biology and how this could integrate with existing conservation management interventions. Moreover, during a period spanning nearly 20 years, we have developed many of the supporting reproductive technologies required to maximise genetic diversity of African wild dogs, in addition to a suite of non-invasive management tools to quantify, monitor and manipulate behaviour, vocalisations, hormones for reproductive function and responses to stressors, as well as immune function to improve the social cohesion, health and reproduction of packs.

Abstract The changing physical properties of the Southern Ocean are known to impact the recruitment and survival of Antarctic krill (Euphausia superba). For oceanic krill predators, the resulting reduced energy intake may lead to population-level effects likely preceded by an alteration in the animals’ body condition. This is especially true for capital breeders that rely on stored energy for successful reproduction. One such Southern Ocean capital breeder, the southern right whale (Eubalaena australis), has been monitored over the past 43 years in their South African wintering ground. Changes in the population have been documented in the past decade, including a decreased reproductive rate and a shift in foraging strategy. To evaluate if a reduced foraging success is an underlying factor, we assessed the temporal variation in morphological body condition through aerial photogrammetry. Results showed a 23% reduction in maternal body condition, potentially contributing to the decreased reproductive rate of the population. To the best of our knowledge, this is the first study to quantify a decadal reduction in the body condition of a capital breeder dependent on Southern Ocean productivity. Understanding the bioenergetic consequences of environmental change is vital to predicting species’ resilience to climate change.

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.

There is increasing evidence of compromised welfare for elephants managed in captivity. Should such facilities eventually close, more elephants will need to be rehabilitated and reintegrated into the wild. The goal of such reintegration would be to restore any physical or psychological aspects of the elephant that may have been compromised in captivity, followed by introduction into a free-roaming system where they can interact with other elephants. However, to achieve this goal, the reintegration methods implemented need to be assessed to ensure that welfare remains the priority. The objective of this study was to test whether parameters generally associated with stress and disturbance in African elephants, respond to changes in potentially stressful environmental conditions, assessed at multiple temporal scales ranging from minutes to months. The main changes in environmental conditions that were investigated included the different phases of reintegration of a group of elephants from captivity into the wild. Stress and disturbance related parameters used for comparisons included physiological responses, namely the extent of temporal gland secretions (eTGS) and faecal glucocorticoid metabolite (fGCM) levels as well as behavioural responses, namely the display of stereotype and stress-related behaviours. Results showed that eTGS significantly increased during the initial release of the elephants compared to when in captivity. Stereotypic behaviours were only recorded during the captive phase and immediately ceased after release. Faecal GCM levels spiked in the first year after release before decreasing back to pre-reintegration levels during the third year. These findings indicate that fGCM levels, the eTGS and disturbance related behaviours all proved effective in explaining the changes in stress and disturbance experienced by elephants during the initial years after being reintegrated from captivity into the wild.

Historically, reproductive health in wildlife species has been evaluated primarily via immunoassay detection of fecal and urinary steroid hormone metabolites. This combination of sample type, biomarker category, and assay has been preferred for decades due to the ease of assessing reproductive health through the evaluation of stable compounds in easily collected biological samples using a cost-effective method. Increasingly, beginning with high performance liquid chromatography (HPLC) and more recently with convergence chromatography and ultra HPLC coupled with mass spectrometry (MS), wildlife studies are incorporating more sensitive and specific high-throughput technologies for the assessment of not only steroid hormone metabolites but proteins as well. Of note, a comprehensive health evaluation requires the measurement of biological readouts that modulate reproduction such as: glucocorticoids, leptin, insulin, thyroid hormones, melatonin, the microbiome, and markers of inflammation. Emerging modulatory biomarkers of reproductive health include acute phase proteins, microRNAs, and reactive oxygen species. Several of these biomarkers require application of newer technologies such as LC-MS/MS and sequencing, which demonstrates the need for the field of wildlife reproductive biology to diversify from its reliance on immunoassays. Importantly, endocrine disrupting chemicals adversely affect many aspects of reproductive function and evaluation of these compounds requires high throughput technology such as LC-MS/MS. The application of sequencing, particularly Next Generation Sequencing of bulk RNA (RNA-Seq) and single cell RNA-Seq, is uncommon in studies of wildlife reproductive health. However, as the cost of these methods decreases and consortiums of wildlife researchers band together to raise funds in support of studies using these technologies, their use will become more routine. Future research should focus on integration of known biomarkers of related systems into comprehensive reproductive assessments and the development of new biomarkers which are sensitive, precise, and employ non-invasive methodologies for the assessment of reproductive health of wildlife species.

Artificial insemination (AI) can aid conservation of African wild dogs (AWD), but methods to determine the appropriate timing of AI in females are not feasible without immobilization. This study determined whether certain behaviors coupled with fecal estrogen (fEM) and progestogen (fPM) metabolite concentrations, could be used as non-invasive parameters to predict the fertile period in female AWDs. Behavior was observed in three alpha females before, during and after the mating period, and feces analyzed for hormone metabolites. During the periovulatory period, females were immobilized 2–3 times to evaluate vulvar condition, blood hormone concentrations and vaginal cytology, and to conduct vaginoscopy. Late estrus (fertile period) could be distinguished from pro-estrus, early estrus, and diestrus using behavior; with a 2- to 5-fold higher rate of male-female affiliative behavior, sexual and non-sexual follow, alpha male initiating behavior, ride-up and copulation (P ≤ 0.05). Sexual behaviors, and male-female resting patterns declined significantly or ceased the day after last mating. Two females showed a 2.5- to 3-fold increase in fPM concentrations during late estrus compared to the pro-estrus period (P ≤ 0.05) and elevated fEM levels that rose in pro-estrus, declined to baseline by late estrus. The one anovulatory female showed no distinct pattern in fPM or fEM concentrations. Vaginal cytology and vaginoscopy could not discriminate between different phases. In summary, behavioral observations coupled with rising fPM and declining fEM can determine the fertile period in African wild dog females, whereas infrequent measurement of blood hormone concentrations, vaginal cytology and vaginoscopy are unreliable.

A nuclear leakage or tactical nuclear weapon use in a limited war could cause immense and long-lasting ecological consequences beyond the direct site of exposure. We call upon all scientists to communicate the importance of the environmental impacts of such an event to all life forms on Earth, including humankind. Changes to ecosystem structure and functioning and species extinctions would alter the biosphere for an unknown time frame. Radiation could trigger cascade effects in marine, atmospheric and terrestrial ecosystems of a magnitude far beyond human capabilities for mitigation or adaptation. Even a “tactical nuclear war” could alter planet Earth’s living boundaries, ending the current Anthropocene era.

The naked mole-rat (Heterocephalus glaber) occurs in colonies with a distinct dominance hierarchy, including one dominant, breeding female (the queen), 1–3 breeding males, and non-reproductive subordinates of both sexes that are reproductively suppressed while in the colony. To non-invasively evaluate reproductive capacity in the species, we first had to examine the suitability of enzyme immunoassays (EIAs) for determining progestogen and androgen metabolite concentrations in the naked mole-rat, using urine and faeces. A saline control and gonadotrophin-releasing hormone (GnRH) were administered to twelve (six males and six females) naked mole-rats which were previously identified as dispersers and housed singly. The results revealed that urine is possibly not an ideal matrix for progestogen and androgen metabolite quantification in naked mole-rats as no signal was detected in the matrix post GnRH administration. A 5α-Progesterone EIA and an Epiandrosterone EIA were identified as suitable for quantifying faecal progesterone metabolites (fPMs) and faecal androgen metabolites (fAMs) in males and females, respectively. The results suggest that there are individual variations in baseline fPM and fAM concentrations, and only two out of six females and no males exhibited an increase in fPM concentrations greater than 100% (−20% SD) post GnRH administration. Conversely, only four out of six females and three out of six males had an increase in fAM concentrations greater than 100% (−20% SD) following GnRH administration. These results imply that some naked mole-rat individuals have a reduced reproductive capacity even when they are separated from the queen.

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.

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.

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.

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.

Pollution is a key threat to biodiversity and ecosystem health within protected areas. Using a non-invasive, multi-matrix approach, sediment, vegetation and faecal material from lion (Panthera leo) and giraffe (Giraffa camelopardalis) were collected and assessed for the simultaneous quantification of 20 trace elements using an optimised method for Inductively Coupled Plasma-–Mass Spectrometry (ICP-MS). Method Linearity was confirmed over an analytical range of 0.1–50 mg/kg for aluminium (Al) and iron (Fe); 0.4–400 µg/kg for vanadium (V), cobalt (Co), molybdenum (Mo), and cadmium (Cd); 0.5–5 µg/kg for mercury (Hg); and 1–1 000 µg/kg for elements arsenic (As), boron (B), barium (Ba), chromium (Cr), copper (Cu), manganese (Mn), nickel (Ni), lead (Pb), antimony (Sb), selenium (Se), tin (Sn), strontium (Sr) and zinc (Zn). Coefficient of determination (R2) was above 0.99 for all elements. Accuracy (% recovery) and precision (% RSD) of replicate measurements for certified reference material controls fell within 20% of expected value at lower concentrations and 15% at higher concentrations for all elements except Al. Results for instrument and method limit of detections (LOD), method limit of quantification (LOQ) and expanded uncertainty were satisfactory. Preliminary data indicate As, Cd, Cr, Cu, Hg, Ni, Pb, Se, and Zn were present in all matrices evaluated. This raises concerns regarding the combined action of multiple elements at concentrations that can adversely affect ecosystem and wildlife integrity. • Sample quantity is reduced due to the power and sensitivity of ICP-MS. • The optimised method is capable of detecting differences in trace element concentrations over large orders of magnitude in animal faeces containing different amounts of organic content. • The method can be applied to the quantification of essential and potentially toxic elements in faeces across a wide range of terrestrial species.

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 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.

Intensive pig management involves in a commercial setting the housing and implementation of certain procedures, such as castration and tail docking, which may be stressful for the animal. Good farming practices include the reduction of stress due to management processes, but assessing the level of stress perceived entirely through behavioural observations can be challenging. The monitoring stress-related physiological markers, like glucocorticoids (GC), can be an accurate alternative that would presumably be more objective. In order to avoid an additional stressor by taking blood, a non-invasive approach is advisable. We used an adrenocorticotropic hormone (ACTH) stimulation test and the effect of transport to examine the suitability of different enzyme immunoassays (EIAs) for monitoring adrenocortical function in domestic pigs using saliva and faeces as sample matrices. An assay measuring faecal glucocorticoid metabolites (fGCMs) with a 3ß,11ß-diol group has proven suited to determine adrenocortical activity, showing an overall increase of 180% in fGCM concentrations related to ACTH administration and of 70% related to transport, respectively. A cortisol EIA was used to detect salivary glucocorticoid (sGC) concentrations, revealing a 1100% increase in sGC concentrations after ACTH administration. The stability of fGCM concentrations post-defecation was determined to assess possible changes in measured fGCM concentrations in unpreserved faecal material over time, with fGCM concentrations being relatively stable (maximal 12% change) under natural conditions for approximately two days after defecation. This implicates that untreated faecal material from pigs can be analysed for up to two days after collection without appreciable level of depreciation in fGCM concentrations. Being able to assess the physiological stress response of domestic pigs non-invasively can help to improve the well-being of commercially reared pigs.

The use of carbon dioxide (CO2) exposure as a means of animal euthanasia has received considerable attention in mammals and birds but remains virtually untested in reptiles. We measured the behavioral responses of four squamate reptile species (Homalopsis buccata, Malayopython reticulatus, Python bivitattus, and Varanus salvator) to exposure to 99.5% CO2 for durations of 15, 30, or 90 minutes. We also examined alterations in plasma corticosterone levels of M. reticulatus and V. salvator before and after 15 minutes of CO2 exposure relative to control individuals. The four reptile taxa showed consistent behavioral responses to CO2 exposure characterized by gaping and minor movements. The time taken to lose responsiveness to stimuli and cessation of movements varied between 240-4260 seconds (4-71 minutes), with considerable intra- and inter-specific variation. Duration of CO2 exposure influenced the likelihood of recovery, which also varied among species (e.g., from 0-100% recovery after 30-min exposure). Plasma corticosterone concentrations increased after CO2 exposure in both V. salvator (18%) and M. reticulatus (14%), but only significantly in the former species. Based on our results, CO2 appears to be a mild stressor for reptiles, but the relatively minor responses to CO2 suggest it may not cause considerable distress or pain. However, our results are preliminary, and further testing is required to understand optimal CO2 delivery mechanisms and interspecific responses to CO2 exposure before endorsing this method for reptile euthanasia.

Understanding the reproductive biology of the roan antelope (Hippotragus equinus) (É. Geoffroy Saint-Hilaire, 1803) is crucial to optimise breeding success in captive breeding programmes of this threatened species. In this study, the pattern of faecal androgen metabolite (fAM) production related to reproductive events (calving or birthing, mating, gestation, and lactation), sexual behaviours as well as environmental cues were studied in captive adult male roan antelope. Faecal sample collection and behavioural observations were carried out from August 2017 to July 2018 for three reproductive males participating in a conservation breeding programme at the Lapalala Wilderness Nature Reserve in South Africa. As a prerequisite, the enzyme immunoassay used in this study was biologically validated for the species by demonstrating a significant difference between fAM concentrations in non-breeding adults, breeding adults and juvenile males. Results revealed that in adults males, the overall mean fAM levels were 73% higher during the breeding period compared to the non-breeding periods, and 85% higher when exclusively compared to the lactation/gestation periods, but only 5.3% higher when compared to the birthing period. Simultaneously, fAM concentrations were lower during the wet season compared to the dry season, increasing with a reduction in photoperiod. With the exception of courtship, frequencies of sexual behaviours monitored changed in accordance with individual mean fAM concentrations in male roan antelope, the findings suggest that androgen production varies with the occurrence of mating activity and may be influenced by photoperiod but not with rainfall.

Anthropogenic land transformation is a consequence of human population growth and the associated agricultural, residential, and industrial needs. This study aimed to investigate the effects of anthropogenic activity and human-mediated land transformation on capture/recapture frequencies, species richness, and diversity of native small mammal community assemblages in the Magaliesberg Biosphere, North West province, South Africa. Five anthropogenically transformed land-use types were investigated: an animal rehabilitation and ecotourism center, an agricultural farmstead, a residential farmstead, a mine-adjacent agricultural farmstead, and a protected nature conservancy. We used live traps to sample small mammals during the dry and wet seasons over three consecutive years and compared population numbers and species composition across study sites and seasons. Capture/recapture frequencies differed significantly between sites and seasons, with the highest capture frequencies recorded at the agricultural and residential farmsteads. Species richness and diversity were highest at the residential and mine-adjacent farmsteads, both of which experienced intermediate levels of anthropogenic disturbance throughout the sampling period. The study shows that while natural and protected landscapes with low levels of disturbance are preferred, transformed landscapes can also be managed effectively to benefit native small mammal populations by regulating the frequency and intensity of human-mediated activities.

Abstract Background African horse sickness (AHS) is of importance to health and international trade in horses worldwide. During export from and transit through AHS endemic countries or zones, physical and chemical measures to protect horses from the vectors of AHS virus (AHSV) are recommended by the World Organization for Animal Health. Protection of containerized air transport systems for horses (jet stalls) with alphacypermethrin insecticide-treated high density polyethylene mesh is effective in reducing the Culicoides midge vector attack rate. In order to determine the effect of this mesh on jet stall ventilation and horse welfare under temperate climatic conditions, jet stall microclimate, clinical variables and faecal glucocorticoid metabolite (FGM) levels of 12 horses were monitored during overnight housing in either a treated or untreated stall in two blocks of a 2 × 3 randomized crossover design. Results Temperature difference between the treated stall and outside was significantly higher than the difference between the untreated stall and outside at 1/15 time points only (P = 0.045, r = 0.70). Relative humidity (RH) difference between the treated stall and outside did not differ from the untreated stall and outside. Temperature and RH in the treated stall were highly and significantly correlated with outside temperature (r = 0.96, P

As the effects of global climate change become more apparent, animal species will become increasingly affected by extreme climate and its effect on the environment. There is a pressing need to understand animal physiological and behavioural responses to climatic stressors. We used the reactive scope model as a framework to investigate the influence of drought conditions on vervet monkey (Chlorocebus pygerythrus) behaviour, physiological stress and survival across 2.5 years in South Africa. Data were collected on climatic, environmental and behavioural variables and physiological stress via faecal glucocorticoid metabolites (fGCMs). There was a meaningful interaction between water availability and resource abundance: when food availability was high but standing water was unavailable, fGCM concentrations were higher compared to when food was abundant and water was available. Vervet monkeys adapted their behaviour during a drought period by spending a greater proportion of time resting at the expense of feeding, moving and social behaviour. As food availability decreased, vervet mortality increased. Peak mortality occurred when food availability was at its lowest and there was no standing water. A survival analysis revealed that higher fGCM concentrations were associated with an increased probability of mortality. Our results suggest that with continued climate change, the increasing prevalence of drought will negatively affect vervet abundance and distribution in our population. Our study contributes to knowledge of the limits and scope of behavioural and physiological plasticity among vervet monkeys in the face of rapid environmental change.

The endangered African wild dog (AWD; Lycaon pictus) is a highly social canid living in packs with a separate male and female hierarchy. Immobilisation, handling and translocations are acute stressors for AWDs, however such interventions are often needed for species management. In addition, new pack formation or temporary pack separation can lead to an increase in intra-pack aggression. The goal of this double-blinded placebo-controlled study conducted in captive zoo populations was to evaluate whether dog appeasing pheromone (DAP) reduces behavioural stress and faecal glucocorticoid metabolite levels (fGCM) normally associated with pack separation, immobilisation and reintroduction (SIR), and to assess whether this reduces aggressive behaviours and faecal androgen metabolite levels (fAM). Four packs (n = 11 males) were treated with DAP and 4 packs (n = 12 males) were treated with a placebo solution, applied at the end of anaesthesia. Behavioural interactions as well as fGCM and fAM were determined from 3 days before until 4-6 days after SIR. No effect of DAP on fGCM was observed, however, fAM increased after SIR in placebo but not DAP treated animals. Moreover, on the day of reintroduction, DAP treated packs tended to have lower rates of contact-dominance and active-submission behaviour, but higher rates of non-contact dominance behaviour. As these effects could decrease the risk of agonistic interactions, DAP may be a useful tool to help manage new pack formations and temporary pack separation.

Giraffes are an important tourist attraction, and human presence to wildlife is increasing. This has an impact on an animal’s behavior and its endocrine correlates. Studies on other species show alterations in movement patterns, vigilance, and stress-related hormone levels in the presence of humans. Limited information is available on how anthropogenic activities alter giraffe’s behavior, social structure, and related endocrine parameters. The purpose of this study was to obtain insight into anthropogenic influences on giraffe’s behavior and adrenal activity. We used GPS devices mounted onto giraffes to compare the distance walked in the presence or absence of human observers. We also conducted behavioral observations to assess their vigilance and collected fecal samples to analyze their fecal glucocorticoid metabolite (fGCM) concentrations. Giraffes walked significantly further distances in the presence of humans, but the cumulative time that observers were present decreased the hourly distance walked with an observer present, suggesting that the giraffes were becoming habituated. The number of observers present significantly increased the percentage of time spent on observing an observer as well as the number of unhabituated individuals present in the herd. The percentage of time spent observing a human observer did not decrease with the increase of habituation. Last, fGCM concentrations increased with human presence but decreased when individuals became habituated to human presence. More research is needed to understand the effect of anthropogenic influences in different scenarios (e.g., tourism, vehicles, hunting, etc.).

Fynbos is a unique endemic vegetation type belonging to the Cape Floral Kingdom in the Western Cape Province of South Africa, representing the smallest of the six floral kingdoms in the world. Nowadays, only a few game reserves in this region support populations of African elephants (Loxodonta africana), and thus, little information exists regarding the suitability of the nutritionally poor Fynbos vegetation for these megaherbivores. Using already established non-invasive methods, the monitoring of individual body conditions and fecal glucocorticoid metabolite (fGCM) concentrations, as a measure of physiological stress, was performed to examine a herd of 13 elephants in a Western Cape Province Private Game Reserve, during two monitoring periods (April and June 2018), following a severe drought. The results indicate that overall median body condition scores (April and June: 3.0, range 2.0–3.0) and fGCM concentrations (April: 0.46 µg/g dry weight (DW), range 0.35–0.66 µg/g DW; June: 0.61 µg/g DW, range 0.22–1.06 µg/g DW) were comparable to those of other elephant populations previously studied utilizing the same techniques. These findings indicate that the individuals obtain sufficient nutrients from the surrounding Fynbos vegetation during the months monitored. However, a frequent assessment of body conditions and stress-associated fGCM concentrations in these animals would assist conservation management authorities and animal welfare practitioners in determining ways to manage this species in environments with comparably poorer nutritional vegetation.

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.

Due to the strong individual cost of being predated, potential prey species alter their behavior and physiology in response to predation risk. Such alterations may cause major indirect consequences on prey populations that are additive to the direct demographic effects caused by prey being killed. However, although earlier studies showed strong general effects of the presence of apex predators, recent data suggest that indirect effects may be highly context dependent and not consistently present. We combined behavioral data with data on endocrine stress and stable isotopes to assess landscape level effects of lion (Panthera leo) presence on two prey species in South Africa, impala (Aepyceros melampus) and blue wildebeest (Connochaetes taurinus). We also evaluated if there was any seasonal variation in such effects. In addition, we provide results from a physiological validation for an enzyme-linked immunoassay (EIA) that can be used for non-invasive monitoring of glucocorticoid stress metabolite concentrations in impala from fecal pellets. We did not find any significant differences in vigilance behavior, fecal glucocorticoid metabolite concentrations, δ13C values or isotope niche breadth between animals living with and without lions for either species. However, wildebeest living in a reserve with lions spent more time foraging compared to wildebeest in a lion-free environment, but only during the wet season. Values of fecal δ15N suggest a shift in habitat use, with impala and wildebeest living with lions potentially feeding in less productive areas compared to animals living without lions. For both species, characteristics of the social groups appeared to be more important than individual characteristics for both foraging and vigilance behavior. Our results highlight that antipredator responses may be highly dynamic and scale-dependent. We urge for further studies that quantify at what temporal and spatial scales predation risk is causing indirect effects on prey populations.

Ozobranchus spp. are leeches that feed solely on turtle blood. They are common ectoparasites found on a range of marine turtle species, with some species of the leech being implicated as vectors of fibropapilloma-associated turtle herpesvirus (FPTHV). Green (Chelonia mydas) and hawksbill (Eretmochelys imbricata) turtles are the two commonly occurring species in the inner granitic islands of the Seychelles. Routine monitoring of nesting turtles on Cousine Island, Seychelles, allowed for opportunistic sightings of leeches on two hawksbill females. In both cases infestation was low, with three leeches collected off one female turtle and five off the other. No obvious signs of papillomas secondary to infection of FPTHV were seen. All of the turtle leeches collected were determined to be Ozobranchus margoi as they had five pairs of lateral digiform branchiae. The specimens were deposited in the Seychelles Natural History Museum on Mahé. To the best of our knowledge this is the first record of Ozobranchus margoi recorded in the inner granitic Seychelles on hawksbill turtles.

Background: Faecal hormone metabolite measurement is a widely used tool for monitoring reproductive function and response to stressors in wildlife. Despite many advantages of this technique, the delay between defaecation, sample collection and processing may influence steroid concentrations, as faecal bacterial enzymes can alter steroid composition post-defaecation. Objectives: This study investigated changes in faecal glucocorticoid (fGCM), androgen (fAM) and progestagen (fPM) metabolite concentrations in faeces of a male and female African elephant (Loxodonta africana) post-defaecation and the influence of different faeces-drying regimes. Method: Subsamples of fresh faeces were frozen after being dried in direct sun or shade for 6, 20, 24, 48 and 72 h and 7 and 34 days. A subset of samples for each sex was immediately frozen as controls. Faecal hormone metabolite concentrations were determined using enzyme immunoassays established for fGCM, fAM and fPM monitoring in male and female African elephants. Results: Hormone metabolite concentrations of all three steroid classes were stable at first, but changed distinctively after 20 h post-defaecation, with fGCM concentrations decreasing over time and fPM and fAM concentrations steadily increasing. In freeze-dried faeces fGCM concentrations were significantly higher than respective concentrations in sun-dried material, which were in turn significantly higher than fGCM concentrations in shade-dried material. In contrast, fAM concentrations were significantly higher in sun- and shade-dried faeces compared to freeze-dried faeces. Higher fPM concentrations were also found in air-dried samples compared to lyophilised faeces, but the effect was only significant for sun-dried material. Conclusion: The revealed time restriction for collecting faecal material for hormone monitoring from elephants in the wild should be taken into account to assure reliable and comparable results. However, if logistics allow a timely collection, non-invasive hormone measurement remains a powerful and reliable approach to provide information about an elephant’s endocrine status.

In mammals, glucocorticoid (i.e. GC) levels have been associated with specific life-history stages and transitions, reproductive strategies, and a plethora of behaviors. Assessment of adrenocortical activity via measurement of glucocorticoid metabolites in feces (FGCM) has greatly facilitated data collection from wild animals, due to its non-invasive nature, and thus has become an established tool in behavioral ecology and conservation biology. The aim of our study was to validate a fecal glucocorticoid assay for assessing adrenocortical activity in meerkats (Suricata suricatta), by comparing the suitability of three GC enzyme immunoassays (corticosterone, 11β-hydroxyetiocholanolone and 11oxo-etiocholanolone) in detecting FGCM increases in adult males and females following a pharmacological challenge with adrenocorticotropic hormone (ACTH) and biological stimuli. In addition, we investigated the time course characterizing FGCM excretion, the effect of age, sex and time of day on FGCM levels and assessed the potential effects of soil contamination (sand) on FGCM patterns. Our results show that the group specific 11β-hydroxyetiocholanolone assay was most sensitive to FGCM alterations, detecting significant and most distinctive elevations in FGCM levels around 25 h after ACTH administration. We found no age and sex differences in basal FGCM or on peak response levels to ACTH, but a marked diurnal pattern, with FGCM levels being substantially higher in the morning than later during the day. Soil contamination did not significantly affect FGCM patterns. Our results emphasize the importance of conducting assay validations to characterize species-specific endocrine excretion patterns, a crucial step to all animal endocrinology studies using a non-invasive approach.

[This corrects the article DOI: 10.1371/journal.pone.0128706.].

During the past several years, non-invasive monitoring of steroid metabolites in faeces of elephants has become an increasingly popular technique to generate more information about the causal relationship between hormones and behaviour in both living elephant species. This is important knowledge which can be used to optimise local conservation and wildlife management by finding new strategies for better elephant population management and control. In this context, however, information about an actual involvement of the hypothalamic-pituitary-adrenal axis during assumable stressful events is still limited, especially for wildlife populations. One difficulty in discovering such information is often the lack of reliable data for hormone baseline levels. Therefore, the aim of this study was to determine baseline concentrations of faecal glucocorticoid metabolites that could be expected within age classes and between seasons in African elephants (Loxodonta africana) in the Kruger National Park (KNP ). A total of 374 faecal samples were collected from randomly located family herds in the southern KNP between May 2002 and August 2005. The samples were analysed for immunoreactive concentrations of faecal glucocorticoid metabolites using a validated enzyme immunoassay for 3α,11oxo-cortisol metabolites(3α,11oxo-CM). All samples were grouped according to the estimated age class of the subject using a field method based on bolus diameter, and regarding the ecological season collected. No significant differences in faecal 3α,11oxo-CM concentrations were found across age classes (H3 = 7.54; p = 0.057), but the mean 3α,11oxo-CM concentration of samples collected in the dry season (n = 196) was significantly higher than in the wet season (n = 178) (u = 15206.50; p = 0.032), which indicates a possible physiological stress situation due to a decline in food quantity and quality. The information generated in this study represents a reliable data set for baseline concentrations of faecal glucocorticoid metabolites for elephants within the KNP and can be used to measure the stressrelated effects of translocations, management actions and the impact of chosen land use activities.

Tiger (Panthera tigris), an endangered species, is under severe threat from poaching, habitat loss, prey depletion and habitat disturbance. Such factors have been reported causing local extermination of tiger populations including in one of the most important reserves in India, namely Sariska Tiger Reserve (STR) in northwestern India. Consequently, tigers were reintroduced in STR between 2008 and 2010, but inadequate breeding success was observed over the years, thus invoking an investigation to ascertain physiological correlates. In the present study, we report glucocorticoid stress responses of the reintroduced tigers in relation to anthropogenic disturbance in the STR from 2011 to 2013. We found anthropogenic disturbance such as encounter rates of livestock and humans, distance to roads and efforts to kill domestic livestock associated with an elevation in fecal glucocorticoid metabolite (fGCM) concentrations in the monitored tigers. In this regard, female tigers seem more sensitive to such disturbance than males. It was possible to discern that tiger's fGCM levels were significantly positively related to the time spent in disturbed areas. Resulting management recommendations include relocation of villages from core areas and restriction of all anthropogenic activities in the entire STR.

Enzyme immunoassays (EIA) that measure faecal testosterone metabolites (fTM) are useful tools to monitor gonadal activity. The aim of this study was to validate an "in-house" epiandrosterone EIA to monitor fTM in spotted hyenas. FTM were characterised in a male and a female hyena that each received an injection of 3H-testosterone. High-performance liquid chromatography (HPLC) analyses revealed a cluster of highly polar enzyme-hydrolysable hormone metabolite conjugates. We performed hydrolysis using β-glucuronidase to deconjugate metabolites and improve sensitivity of the assay. Because β-glucuronidase from Helix pomatia has been reported to bias testosterone measurements in some species, we compared the enzymatic activity of the commonly used β-glucuronidase extracted from H. pomatia with the same enzyme from Escherichia coli. Our results showed that β-glucuronidases from both sources produced similar results from spotted hyena faeces. We therefore hydrolysed samples with H. pomatia enzymes. HPLC analyses also demonstrated that following hydrolysis the epiandrosterone EIA measured significant amounts of immunoreactive metabolites corresponding to radiolabelled metabolites in both sexes. Additionally, HPLC and GC-MS analyses confirmed the presence of epiandrosterone in faeces of spotted hyenas. The biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM levels in response to a testosterone injection within 16 h, (2) no biological responsiveness to an adrenocorticotropic hormone (ACTH) injection and (3) significant differences in fTM levels between juvenile males and adult immigrant males in a free-ranging wild population. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor gonadal activity in spotted hyenas.