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Glucocorticoids (GCs) are primarily involved in mobilising energy reserves to various physiological processes including reproduction. During situations of stress, GCs also help in coping with stress by diverting energy away from processes such as growth and reproduction. Hence, studying annual and seasonal changes in GCs of wild populations can help in understanding their role in stress management and reproduction. The quantification of GCs in wild animals involves capturing, handling and restraining, which could be stressful. Moreover, different species may exhibit differential sensitivity to different stressors. Hence, determining species-specific sensitivities and responses to different stressors may help in developing effective conservation measures. In this context, we studied the annual and seasonal variations in corticosterone metabolites of the Bombay night frog, Nyctibatrachus humayuni. In addition, the effects of handling, marking and short-term captivity (24 h) on corticosterone metabolite levels of N. humayuni were determined. Our results show that urinary corticosterone metabolites (UCM) varied significantly annually and between the sexes; in males, the levels were highest during the breeding season, whilst in females, the levels were highest just before the breeding season. Interestingly, UCM levels of both the sexes were not affected by tagging with visual implant elastomer (VIE), and by short-term captivity, suggesting that these manipulations were not stressful in terms of corticosterone responses.

Koalas (Phascolarctos cinereus) are one of Australia’s most charismatic native small marsupial species. Unfortunately, populations of koalas are rapidly declining throughout Australia as they continue to face increasing pressure from a changing ecosystem. All wildlife species to some degree will use their hypothalamic–pituitary–adrenal (HPA) axis in response to stress. Depending on the duration of activation, the stress response can lead to either acute or chronic side effects and is modulated through the neuroendocrine stress system with the release of catecholamines and glucocorticoids (e.g., cortisol). It is well known that rehabilitation sanctuaries are inherently stressful for all animals, in particular for rescued wild koalas, as it is an unfamiliar environment where the animals cannot predict or control what will happen to them. In this pilot study, we set out to quantify faecal and fur cortisol metabolites in wild rescued koalas undergoing wildlife rehabilitation. Absolute levels of acute and chronic stress were indexed non-invasively, with faecal samples taken to evaluate acute stress, and fur samples taken to evaluate chronic stress. Sampling occurred sporadically over four months (the start of September 2018 to the end of December 2018), and was performed on three rescued koalas (Maree, Tai, and Solstice) being held at the rehabilitation centre. Results of this study show that between the three koalas, the highest recorded faecal cortisol result was 241 ng/g, and the lowest recorded faecal cortisol result was 4 ng/g, whereas the highest recorded fur cortisol result was 1.75 ng/g, and the lowest recorded fur cortisol result was 0.10 ng/g. Statistically, there was a significant difference between all three koalas and their faecal cortisol responses, as well as their fur cortisol responses. Statistically for Maree and Solstice, there was a significant difference in their faecal cortisol response between days when a stressor was recorded, and days when a stressor was not recorded. However, statistically for Tai, this was not the case, as there was no significant difference in his faecal cortisol response between days when a stressor was recorded, and days when a stressor was not recorded. In summary, the hypothesis that faecal glucocorticoids and fur glucocorticoids between koalas will differ based on individual responses to stressors was true as a whole, but individually, this hypothesis was true for Maree and Solstice, but untrue for Tai. The use of biological samples such as faeces and fur to obtain readings of glucocorticoids is a method of measuring absolute levels of physiological stress that is still evolving for koalas, and there is no current glucocorticoid baseline with which to compare the results of this study; although, measuring faecal and fur glucocorticoids is the first step in understanding how koalas undergoing wildlife rehabilitation respond to stressors.

In anurans, vocalisation is used to communicate within and between the sexes during reproduction. Knowledge of vocal repertoire and its diversity is essential in understanding its significance in sexual selection. In this context, we studied the vocal behaviour of Nyctibatrachus humayuni and quantified the urinary metabolites of androgens and corticosterone to understand the associations among hormones, body condition index and vocalisation. Our results show that males of N. humayuni produce an amplitude-modulated call consisting of a train of pulses. Interestingly, a high degree of inter-individual variation was observed in the call parameters. The dominant frequency and pulse interval were negatively correlated with male body size, whereas pulse duration and pulse duty cycle were positively correlated with it. Dominant frequency was negatively correlated with androgens and corticosterone, whereas pulse interval was negatively correlated with corticosterone. Further, pulse duty cycle, pulse duration and pulses per call were positively associated with corticosterone. Surprisingly, none of the acoustic parameters was associated with male body temperature and body condition. A significant variation was evident in the vocal effort, dominant frequency and amplitude of advertisement calls produced under different social situations. Calls produced during territory establishment had lower dominant frequency but higher vocal effort, whereas those produced during courtship had higher amplitude and vocal effort than the calls produced in the absence of a competitor/female. Our results suggest that greater inter-male variability in the advertisement call of N. humayuni could be important in female choice as it may help females to assess the quality of a male. Further, androgens and corticosterone could be important in modulating crucial parameters of the advertisement call.

Herpetological research involving amphibians is increasingly using mark and recapture methods, employing various techniques such as toe-clipping and visible implant alphanumeric tags. Visual Implant Elastomer (VIE) is a new method available for herpetological surveys, involving a coloured dye inserted into the epidermal skin surface of frogs. Previously, there has been only one study which demonstrated that the VIE method does not generate a significant physiological stress response (using a faecal glucocorticoid method) in a captive amphibian species. Physiological stress can also be quantified non-invasively using urinary corticosterone metabolite (UCM) enzyme-immunoassay in amphibians. In this study, we tested the physiological stress response of a common amphibian species, wild caught Asian toads (Duttaphrynus melanostictus), by comparing UCM responses to capture handling, sham control or VIE marking method. Adult males (n = 38) were captured and sampled for baseline UCM (t = 0 h) then marked either using the VIE or sham (saline control), or only handled during capture. Subsequently, urine samples were collected at t = 2, 12 and 24 h for toads within each group. UCM levels were quantified using an enzyme immunoassay (EIA) to determine differences among treatment groups and over time following capture. Toads generated acute stress responses to all three groups, showing a change in UCMs between baseline samples, 12 h, and 24 h samples. The mean UCM levels were not significantly different between the VIE method and the control groups (capture handling or sham operated). These results indicate that VIE method of tagging is no more stressful than routine handling of amphibians, hence in this context, the method does not have any additional welfare implications. Future research should explore the limitations of VIE tagging for long-term mark recapture studies, however, our current findings support its application as a minimally-invasive method for marking amphibians.

Asiatic black bears (Ursus thibetanus) face chronic stress in bile farms. In this study, we investigated whether bile-farmed bears show significantly high levels of stress at rescue and whether stress levels reduce over time in a bear sanctuary where the bears are supported with environmental enrichment and veterinary care to improve animal welfare. We measured stress hormone levels using faecal cortisol metabolites (FCM) in 16 Asiatic black bears freshly rescued from bile farms in Vietnam. Fresh faeces were collected from each bear on the rescue truck and on a weekly basis for a 22-week study period at a bear sanctuary in Vietnam. Results showed that for all 16 rescued bears (with one exception) individual FCM levels from truck samples were above mean baseline FCMs of bears previously rehabilitated to a bear sanctuary. This suggested the majority of the rescued bears were still capable of showing a stress endocrine response during the rescue operation despite being exposed to conditions causing chronic stress in bears on bile farms. Results showed that mean FCM levels of the rescued bears differed significantly between time-periods (higher at the rescue [on truck samples] compared to week 22 samples) and mean FCM levels showed an overall decline over the first 22 weeks after they arrived at the bear sanctuary. The bears also demonstrated acute FCM stress responses to management interventions at the sanctuary, such as veterinary health checks and transportation. In conclusion, rescued bears tend to modulate their stress endocrine response after rehoming at the bear sanctuary. This is an important result, indicating that the rescue effort and rehabilitation of bile-farm bears is effective. Whether this also coincides with behavioural adjustments in rehabilitating bears (eg lessening of stereotypic behaviour) warrants further investigation.

The koala (Phascolactos cinereus) is currently listed by both the IUCN and the Australian Governments’ Threatened Species Scientific Committee as vulnerable to extinction with a decreasing population trend. This listing can be attributed to climate change and its impact on ecosystems, and anthropomorphic environmental change due to extensive land clearing and habitat fragmentation. These have both been proven to induce stress, which influences the onset of disease. This novel study performed a retrospective analysis whereby records for 12,543 wild, rescued koalas in New South Wales (NSW), Australia were studied in order to determine trends in koala sightings, clinical admissions and injury diagnoses over a period of 29 years (1989-2018). Results indicated that between all three study locations (Port Stephens, Port Macquarie and Lismore), the most common reason koalas were admitted into care was because of disease, the most common disease diagnosed was signs of chlamydia, and the most common outcome for koalas admitted into care was released. At Port Stephens, mature and female koalas were diagnosed with a disease more than any other age or sex, while juvenile and male koalas were released (back into the wild) more than any other age or sex. Additionally, there were fewer koalas with a disease and fewer koalas released in Port Stephens as each year progressed. At Port Macquarie, mature and male koalas were diagnosed with a disease more than any other age or sex, while juvenile and female koalas were released more than any other age or sex. Additionally, there were more koalas with a disease and fewer koalas released in Port Macquarie as each year progressed. At Lismore, adult and female koalas were diagnosed with a disease more than any other age or sex, while joey and male koalas were released more than any other age or sex. Additionally, there were more koalas with a disease and fewer koalas released in Lismore as each year progressed. Determining trends in clinical admissions and diagnosis over such a substantial period of time is an important factor in preventing the continuing decline of koalas throughout Australia, and in particular NSW. It is important to note that there are cultural differences between koala rescue groups in the three study locations (Port Stephens, Port Macquarie and Lismore). These differences may be reflected in the outcomes of koala patients as each group are driven by their own management team. It is essential that any further decline of koala populations is prevented, however this can only be achieved through informed recommendations through research studies such as these. These recommendations should lead to government legislation which can provide stronger protection to koala habitat.

Phenotypic plasticity, broadly defined as the capacity of one genotype to produce more than one phenotype, is a key mechanism for how animals adapt to environmental (including thermal) variation. Vertebrate glucocorticoid hormones exert broad-scale regulation of physiological, behavioral, and morphological traits that influence fitness under many life-history or environmental contexts. Yet the capacity for vertebrates to demonstrate different types of thermal plasticity, including rapid compensation or longer acclimation in glucocorticoid hormone function, when subject to different environmental temperature regimes remains poorly addressed. Here, we explore whether patterns of urinary corticosterone metabolites respond (i.e., evidence of acclimation) to repeated short-term and sustained long-term temperature exposures in an amphibian, the cane toad (Rhinella marina). In response to three repeated short (30-min) high-temperature (37°C) exposures (at 10-d intervals), toads produced urinary corticosterone metabolite responses of sequentially greater magnitude, relative to controls. However, toads subjected to 4 wk of acclimation to either cool (18°C)- or warm (30°C)-temperature environments did not differ significantly in their urinary corticosterone metabolite responses during exposure to a thermal ramp (18°-36°C). Together, these results indicate that adult toads had different, including limited, capacities for their glucocorticoid responses to demonstrate plasticity to different regimes of environmental temperature variation. We advocate further research as necessary to identify plasticity, or lack thereof, in glucocorticoid physiology, to better understand how vertebrates can regulate organismal responses to environmental variation.

Sarcoptic mange is caused by the mite Sarcoptes scabiei and has recently been recognized as an emerging infectious disease of wildlife worldwide. The mite is one of the main causes of population decline in southern hairy-nosed (Lasiorhinus latifrons) and bare-nosed wombats (Vombatus ursinus). This review focuses on Sarcoptes scabiei infestations in wombats and provides insights into why the disease may be so prevalent in wombats. Current treatment practices and trials conducted in the field to reduce the incidence of sarcoptic mange in wombats are described and critically reviewed. Current and potential future avenues of research are discussed.

Physiological responses of wildlife species to zoo visitors should be studied to better understand how wildlife perceive human encounters. We conducted an experimental test of the effect of changes in zoo visitor encounter experiences on the glucocorticoid (GC) response of koalas (Phascolarctos cinereus) in a Sydney zoo. Koalas were housed in a multiple-bay enclosure (two to three koalas per bay) for photography sessions with zoo visitors (no touching of koalas permitted by visitors). Following a one-week no-photography baseline period, photography sessions were rotated between three enclosure bays for four weeks (Intensive photography), then between five enclosure bays for an additional four weeks (Standard photography). A sixth enclosure bay was never included in the photography sessions (control bay); koalas in this bay showed no significant change in fecal cortisol metabolites (FCMs) during the course of the study. In the five experimental bays differences were seen between male and female koalas. Males had higher mean FCMs than females, and individual FCM traces showed that two male koalas that were related and of similar age responded strongly to the experimental manipulation. These two males showed a peak in FCMs at the beginning of the Intensive photography period, then a decline when photography sessions returned to the Standard protocol. No systematic pattern in response to photography sessions was observed in females. Our results demonstrate successful application of a non-invasive endocrinology tool for assessing the stress biology and welfare of captive zoo wildlife.

Faecal glucocorticoid metabolites are minimally invasive stress physiology indices that can be used to understand how animals respond to physical and/or psychological challenges (stressors) and inform how to optimize conservation management in view of these stressors. We investigated contextual biological, environmental and parasitological factors influencing variation in baseline faecal cortisol metabolite (FCM) concentration in a critically endangered marsupial, the woylie (syn. brush-tailed bettong, Bettongia penicillata). Woylies have undergone a rapid and significant population decline, with environmental stressors exacerbating disease suggested to contribute to these ongoing declines. We conducted a longitudinal field study of 15 adult woylies (9 females, 6 males) in a captive, naturalistic facility. FCM concentration in faecal samples (n = 269) collected monthly over 20 months was quantified by enzyme immunoassay in parallel with measures of body condition, sex, season, female reproductive status and the presence of endoparasites and ectoparasites. Linear mixed effect modelling revealed a significant effect of season, sex, body condition index and nematode parasite status on FCM. Overall, mean FCM was lowest in summer and highest in autumn and winter, and females had higher mean FCM than males. There was a significant but weak negative association between body condition and FCM. When woylies were shedding oxyurid nematode eggs they had higher mean FCM compared to when they were not shedding. In future, knowledge of factors that influence FCM fluctuations in woylies may be considered when carrying out potentially stressful conservation interventions that may influence the future survival of this unique and threatened species.

Simple Summary Rapidly expanding global human population has led to increased supply chain demands on animal-based farming systems and the desire for environmentally friendly products. This has also resulted because of socio-political pressure and increased public concerns over the impacts of conventional agriculture on the environment. In order to be sustainable, animal production systems must also advance animal welfare, avoiding physically and psychologically stressful situations for the animals and apply innovative methods of reducing contribution of farming practices to global climate change while also functioning at optimum productivity. Consequently, to achieve a practical and effective improvement towards environmental sustainability, animal-based agriculture should consider animal welfare assessment, objective measures of physiological stress, climate change evaluation and animal productivity in a multi-dimensional and holistic approach. Abstract In this retrospective study, we conducted a desktop-based analysis of published literature using the ScienceDirect™ search engine to determine the proportion of livestock research within the last 7 years (2015–2021) that have applied animal welfare assessment combining objective measures of physiological stress and evaluation of climate change factors in order to provide an account of livestock productivity. From the search results, 563 published articles were reviewed. We found that the majority of the literature had discussed animal production outcomes (n = 491) and animal welfare (n = 453) either individually or in conjunction with another topic. The most popular occurrence was the combination of animal welfare assessment, objective measures of stress physiology and production outcomes discussed collectively (n = 218). We found that only 125 articles had discussed the impact of climate change (22.20%) on livestock production and/or vice versa. Furthermore, only 9.4% (n = 53) of articles had discussed all four factors and published research was skewed towards the dairy sector. Overall, this retrospective paper highlights that although research into animal welfare assessment, objective measures of stress and climate change has been applied across livestock production systems (monogastrics and ruminants), there remains a shortfall of investigation on how these key factors interact to influence livestock production. Furthermore, emerging technologies that can boost the quantitative evaluation of animal welfare are needed for both intensive and extensive production systems.

An individual merino sheep’s output of wool production is influenced by synergistic interactions of sheep genetics, climate, farm management, and nutrition available to the whole flock. The price paid to the producer for this wool commodity is determined via numerous tested parameters and /or subjective appraisal of the raw greasy wool. This research investigated the level of variation in wool cortisol (a physiological stress biomarker) and wool micron (MIC) in Merino ewes (Ovis aries), pre-partum and post-lambing (lactation/lambs at foot), using maiden ewe (n = 38) managed in an outdoor paddock in a commercial farm. The key findings of this study are; (1) wool quality indicators showed a significant variation between pre- and post- parturition including significant reduction in MIC and (2) there was a negative correlation between wool cortisol levels and wool micron pre-parturition (rs = − 0.179,p p

Glucocorticoid hormone profiles are increasingly used as physiological markers to infer the strength of species interactions that can influence fitness and ensuing population dynamics of animals. Here we investigated two aims. First, we measured the effect of a 90-min capture stress protocol on the plasma corticosterone responses of a large native Australian lizard, the lace monitor (Varanus varius). Second, we compared the basal and postcapture stress corticosterone responses of lace monitors in habitats where they were exposed to high or low densities of the European red fox (Vulpes vulpes), an introduced competitor. Lace monitors responded to the capture stress protocol by significantly increasing plasma levels of corticosterone above basal at 45- and 90-min-postcapture blood-sampling intervals. In habitats with high fox densities, lace monitors produced a significantly greater basal and capture-stress-induced corticosterone response compared to individuals in low-fox density habitat. A significant interaction among fox density, time postcapture, and body condition was also found to influence plasma corticosterone values. These results suggest competition with red fox, perhaps via nutritional stress and increased hypersensitivity of the adrenocortical axis in lizards. At present, without further research, we do not understand whether such responses mediate lizard fitness or whether they have adaptive or maladaptive consequences for lizard populations in response to red fox competition. Nevertheless, our results help broaden understanding of the physiological implications arising from species interactions and specifically how introduced competitors could mediate diverse impacts on native biodiversity.

Rapid and reliable physiological evaluation of stress is necessary for understanding the potential impacts of environmental changes on managed populations of threatened mammals. In situ populations of Australia’s iconic marsupial, the greater bilby (Macrotis lagotis), are nearing extinction due to the impacts of competition and predation by feral animals and unpredictable climatic events (summer heat waves). In this study, we focussed our aim to identify a non-invasive method to measure adrenal activity in the species and also to identify potential factors that should be considered when comparing physiological stress in semi-free ranging populations of the species. We validated an enzyme immunoassay (EIA) for detecting fecal cortisol metabolites (FCM) from fresh fecal pellets taken from bilbies within four captive sites and two semi-free ranging populations around Queensland and New South Wales, Australia. Our FCM EIA successfully detected the ‘raise and fall’ pattern of FCM levels within 3 days of exogenous adrenocorticotropic hormone (ACTH) challenge. Mean FCM levels differed significantly between the captive sites and between sexes. All male bilbies grouped outdoor in captivity expressed the highest mean FCM level in comparison to all captive males that were housed individually or as groups indoors. Also, semi-free ranging bilbies expressed higher mean FCM levels than the captive bilbies. Overall, our study successfully validated a non-invasive tool for monitoring physiological stress in the greater bilby. In the future, it will be worthwhile to consider factors such as housing conditions, sex and location when comparing the adrenal sensitivity to environmental changes, to help evaluate the success of management interventions (such as predator free enclosures) and support the survival of the species.

Koalas (Phascolarctos cinereus) are the only extant representatives of Australia's unique marsupial family Phascolarctidae and were listed as nationally Vulnerable in 2012. Causes of mortality are diverse, although the disease chlamydiosis, dog attacks, collisions with cars, and loss of habitat represent the principal reasons for the continued species decline. Koala breeding facilities in Queensland and New South Wales, Australia have been established for conservation and tourism. Non-invasive monitoring of physiological stress is important for determining the sub-lethal effects of environmental stressors on the well-being, reproduction and survival of Koalas in Zoos and also in the wild. In this study, we developed a faecal cortisol metabolite (FCM) enzyme-immunoassay (EIA) for monitoring physiological stress in Koalas from two established Zoos in Australia and also within a free-living sub-population from Queensland. Biological validation of the FCM EIA was done using an adrenocorticotropic hormone (ACTH) challenge. We discovered excretory lag-times of FCM of 24 h in females (n=2) and 48 h in male (n=2) Koalas in response to the ACTH challenge. FCM levels showed an episodic and delayed peak response lasting up to 9 days post ACTH challenge. This finding should be taken into consideration when designing future experiments to study the impacts of short-term (acute) and chronic stressors on the Koalas. Laboratory validations were done using parallelism and recovery checks (extraction efficiency) of the cortisol standard against pooled Koala faecal extracts. Greater than 99% recovery of the cortisol standard was obtained as well as a parallel displacement curve against Koala faecal extracts. FCM levels of the captive Koalas (n=10 males and 13 females) significantly differed by sex, reproductive condition (lactating versus non-lactating Koalas) and the handling groups. Handled male Koalas had 200% higher FCM levels than their non-handled counterparts, while females were not affected by handling as long they were not undergoing lactation. There was no significant difference in FCM levels between the captive and wild Koalas (n=9 males and 7 females). Overall, these results provide foundation knowledge on non-invasive FCM analysis in this iconic Australian marsupial. Non-invasive stress endocrinology opens up opportunities for evaluating the sub-lethal physiological effects of management activities (including caging, translocation) on the nutritional status, reproductive behaviors and disease status of captive and managed in situ Koala populations.

Translocation can be stressful for wildlife. Stress may be important in fauna translocation because it has been suggested that it can exacerbate the impact of infectious disease on translocated wildlife. However, few studies explore this hypothesis by measuring stress physiology and infection indices in parallel during wildlife translocations. We analysed faecal cortisol metabolite (FCM) concentration and endoparasite parameters (nematodes, coccidians and haemoparasites) in a critically endangered marsupial, the woylie (Bettongia penicillata), 1–3 months prior to translocation, at translocation, and 6 months later. FCM for both translocated and resident woylies was significantly higher after translocation compared to before or at translocation. In addition, body condition decreased with increasing FCM after translocation. These patterns in host condition and physiology may be indicative of translocation stress or stress associated with factors independent of the translocation. Parasite factors also influenced FCM in translocated woylies. When haemoparasites were detected, there was a significant negative relationship between strongyle egg count and FCM. This may reflect the influence of glucocorticoids on the immune response to micro- and macro-parasites. Our results indicate that host physiology and infection patterns can change significantly during translocation, but further investigation is required to determine how these patterns influence translocation success.

India's charismatic wildlife species are facing immense pressure from anthropogenic-induced environmental perturbations. Zoos play a major role in the conservation of threatened species, but their adaptation in captivity is posing a major challenge globally. Stress from inadequate adaptation could lead to suppression of cognitive functioning and increased display of stereotypic behaviour. It is thus necessary to measure biological traits like behaviour, stress physiology, and contextual factors driving the animals maintained at zoos. In this study, we assessed stereotypic behaviour and stress physiology employing standard behaviour scoring, non-invasive stress monitoring, and their contextual drivers in a sub-population of two large felid species managed in six Indian zoos. The prevalence and intensity of stereotypic behaviours and levels of faecal corticosterone metabolites (FCM) were ascertained among 41 Royal Bengal tigers Panthera tigris tigris and 21 Indian leopards Panthera pardus fusca between April 2014 and March 2015. Behavioural observations showed that tigers spent more time stereotyping (12%) than leopards (7%) during daylight hours. Stress levels assessed using FCM revealed that tigers (23.6 ± 1.62 ng/g) had marginally lower level of corticosterone metabolites than leopards (27.2 ±1.36 ng/g). Stereotypic behaviour increased significantly with FCM level when the effect of heath status was controlled in tigers, and the effects tree cover, stone, den and keeper attitude controlled in leopards. Comparison of stereotypes of tigers with various biological and environmental factors using binary logistic regression revealed that stereotypic prevalence decreased with increased enclosure size, and enclosure enrichments like presence of pools and stones, when managed socially with conspecifics, and with positive keeper attitude, these factors accounting for 43% of variations in stereotypic prevalence among tigers. Stereotype among leopards was significantly absent when associated with increased tree cover and presence of pool, and den in the enclosure, age and among zoo-born than wild-born ones. These factors explain 81% of variations in stereotypic prevalence in them. A comparison of FCM levels with context-dependent factors revealed that stress levels among tigers decreased significantly with enclosure size and with individuals from nil to low, and severity of health issues. These factors explain 64% of variations in FCM levels. In leopards, the presence of stones in the enclosure and keepers with positive attitude resulted in significant decrease in FCM levels, these factors together accounting for 94% of variations. Multiple regressions on selected variables based on Factor Analysis of Mixed Data showed that in tigers the intensity of stereotype decreased significantly with enclosure size, sociality and positive keeper attitude and FCM level with health problems. Similarly, analyses in leopards revealed that intensity of stereotype decreased significantly with tree cover, age and FCM level with positive keeper attitude. Overall, our study suggests that to reduce stereotypes and stress level, tigers in captivity should be managed in larger enclosures enriched with pool, and stones, and in appropriate social conditions with adequate veterinary care. Leopards should be managed in enclosures with dense tree cover, pool, stones and den. Positive keeper attitude plays a crucial role in the welfare of both the species in captivity. Our study is promising and is comparable with their natural behaviour in the wild; for example, tigers require larger natural habitats, while leopards can manage even with smaller isolated patches but with dense vegetation cover.

Captive breeding programs are increasingly being used as a management option for threatened mammals. The greater bilby Macrotis lagotis, for example, is a vulnerable species which is maintained in captivity at several facilities in Australia. Non-invasive evaluation of stress hormones (cortisol in mammals) via excretory metabolites can be used to monitor physiological stress responses of captive individuals. In this study, we validated an enzyme-immunoassay (EIA) to measure cortisol metabolites in fresh faecal samples of adult male and female bilbies (n = 7) held in captivity at the Dreamworld Theme Park, Queensland, Australia. The faecal cortisol EIA was validated via parallelism and the recovery of exogenous cortisol added to pooled faecal extracts (>99% recovery). Female bilbies had higher average faecal cortisol metabolite concentrations and higher day-to-day variation than male bilbies; however, there was no relationship with bilby age. Cortisol metabolites for most individuals varied widely through time, with numerous peaks and troughs in response to long-term stressors (illnesses, injury and reproductive issues) and short-term stressors, such as use in shows at Dreamworld or public displays in local schools, manual restraint and short-term veterinary procedures (e.g. general anaesthesia). Overall, the higher mean cortisol metabolite concentrations of individuals suffering long-term stress was related to a greater response to short-term stressors. This suggests an interaction between responses to short-term and long-term stressors which is perhaps due to habituation and/or facilitation of long-term stressors. Non-invasive faecal monitoring of stress hormones could provide further information on the implications of captive breeding programs and the release of animals reared in captivity.

Studies of baseline (unstressed) and short-term corticosterone stress responses in free-living amphibianscan provide crucial information on the physiological responses of different populations to environmentalchange. In this study, we compared baseline and urinary corticosterone metabolite responses of free-living adult males and females of two closely related Fijian frogs of thePlatymantisgenus (Family:Ceratobatrachidae). Fijian ground frogs (Platymantis vitiana) live on the ground while Fijian tree frogs(Platymantis vitiensis) are arboreal. We captured free-living frogs and applied our moderate stress proto-col (5 min handling during urine sampling at hourly intervals), with urinary corticosterone metaboliteconcentrations measured by enzyme-immunoassay. Mean urinary corticosterone metabolite concentra-tions in male and female Fijian ground frogs increased from 0 to 2 h and continued to increase to peakconcentrations 5 6 h after capture. Mean baseline corticosterone concentration was signi“cantly differ-ent between sexes (higher in males than females) only for Fijian ground frogs. There was no signi“cantdifference between sexes in the integrated corticosterone responses for both species. Mean baseline andurinary corticosterone metabolite responses of Fijian tree frogs were lower than those of Fijian groundfrogs. Corticosterone levels increased for 4 5 h in both species and began to decrease again 7 h after ini-tial capture. Corticosterone responses were consistently higher for Fijian ground frogs than Fijian treefrogs. Individuals in both species showed markedly variable corticosterone responses over the 8 h dura-tion of the stressor, with some individuals showing low stress responses and others showing high stressresponses. The magnitude of the corrected integrated response of the ground frogs was almost twice thatof the tree frogs. These differences in baseline and short-term corticosterone stress responses betweenthese two species could be a consequence of ecological differences including micro-habitat, predatorinteractions and/or competitive interactions with the introduced cane toad (Rhinella marina). Compari-sons of corticosterone responses between populations and species provide a valuable tool for measuringthe physiological responses of the amphibians to environmental change. 2012 Elsevier Inc. All rights reserved.

Annual cycles of reproductive steroid metabolites were measured in urine collected from free-living and captive tropical endangered Fijian ground frogs (Platymantis vitiana) a terrestrial breeding. Free-living frogs were sampled on Viwa Island, Fiji and captive frogs were maintained in an outdoor enclosure in Suva, Fiji. Urinary estrone, progesterone and testosterone metabolite concentrations increased in male and female frogs after hCG challenges, with clear peaks in steroid concentrations 2 or 3 days after the challenges. There were annual cycles of testosterone metabolites in wild and captive males, and of estrone and progesterone metabolites in wild and captive females. Peaks of steroid concentrations in the wet season corresponded with periods of mating and egg laying in females in December and January. Steroid concentrations declined in January and February when maximum egg sizes in females were also declining. Body weights of wild male and vitellogenic female frogs showed annual cycles. Body weights of non-vitellogenic female frogs varied significantly between months, although there was no clear pattern of annual changes. Body weights of the 3 captive male frogs and 4 captive female frogs were similar to those of the wild frogs. Estrone metabolites were 80% successful in identifying non-vitellogenic females from males. The results suggest that the Fijian ground frog is a seasonal breeder with an annual gonadal cycle, and this species is likely to be photoperiodic. Urinary steroid measurements can provide useful information on reproductive cycles in endangered amphibians.

Field endocrinology research through the quantification of glucocorticoids or stress hormones in free-living wildlife is crucial for assessing their physiological responses towards pervasive environmental changes. Urinary corticosterone metabolite (UCM) enzyme-immunoassay (EIA) has been validated for numerous amphibian species as a non-invasive measure of physiological stress. Body-condition indices (BCIs) have also been widely used in amphibians as an indirect measure of animal health. Field endocrinology research on amphibian species in Asia is limited. In this study, we validated a UCM EIA in a peri-urban sub-population of the common Asian toad (Duttaphrynus melanostictus) in Pune, Maharashtra, India. We determined the baseline levels of UCMs in male (n=39) and female (n=19) toads. Secondly, we used a standard capture handling protocol to quantify changes in UCMs during short-term captivity. We also determined BCIs in the male and female toads using Fulton's index (K) and residual condition index (RCI). The results showed that mean baseline levels of UCMs were significantly higher in male toads than in females. There was no significant change in mean levels of UCMs of males and females between capture and captivity (0-12h). This highlights plausible habituation of the species to the peri-urban environment. Associations between UCMs with BCIs (K and R) were positive in male toads but negative in females. In conclusion, our UCMs EIA can be applied with BCIs to assess health of the Asian toads. We also suggest that direct fitness parameters such as sperm and oocyte quality, reproductive ecology and immunocompetence measurements should be applied in combination with these conservation physiology tools to quantify the fitness consequences of pervasive environmental changes on native amphibians.

We have successfully employed non-invasive fecal based glucocorticoid monitoring to evaluate fecal cortisol metabolites in male and females Sumatran tigers from Australian Zoos. Our study provides baseline FCM data which will be crucial for evaluting the health and welfare of big cats in zoos around the world., Glucocorticoid quantification using non-invasive methods provides a powerful tool for assessing the health and welfare of wildlife in zoo-based programmes. In this study, we provide baseline data on faecal-based glucocorticoid (cortisol) monitoring of Sumatran tigers (Panthera tigris ssp. sumatrae) managed at the Melbourne Zoo in Victoria, Australia. We sampled five tigers daily for 60 days. Faecal cortisol metabolites (FCMs) in tiger faecal extracts were quantified using enzyme immunoassays that were successfully validated using parallelism and accuracy recovery checks. Two female tigers had significantly higher mean FCM levels than the two males and another female, suggesting that females may have higher FCM levels. A significant elevation was noted in the FCM levels for one female 2 days after she was darted and anaesthetized; however, the FCM levels returned to baseline levels within 3 days after the event. Comparative analysis of FCM levels of tigers sampled at Melbourne Zoo with tigers sampled earlier at two other Australian Zoos (Dreamworld Themepark and Australia Zoo) showed that FCM levels varied between zoos. Differences in the enclosure characteristics, timing of sampling, size and composition of groupings and training procedures could all contribute to this variation. Overall, we recommend the use of non-invasive sampling for the assessment of adrenocortical activity of felids managed in zoos in Australia and internationally in order to improve the welfare of these charismatic big cats.

Climatic warming is a global problem and acute thermal stressor in particular could be considered as a major stressor for wildlife. Cane toads (Rhinella marina) have expanded their range into warmer regions of Australia and they provide a suitable model species to study the sub-lethal impacts of thermal stressor on the endocrine physiology of amphibians. Presently, there is no information to show that exposure to an acute thermal stressor could initiate a physiological stress (glucocorticoid) response and secondly, the possible effects on reproductive hormones and performance. Answering these questions is important for understanding the impacts of extreme temperature on amphibians. In this study, we experimented on cane toads from Queensland, Australia by acclimating them to mildly warm temperature (25°C) and then exposing to acute temperature treatments of 30°, 35° or 40°C (hypothetical acute thermal stressors). We measured acute changes in the stress hormone corticosterone and the reproductive hormone testosterone using standard capture and handling protocol and quantified the metabolites of both hormones non-invasively using urinary enzyme-immunoassays. Furthermore, we measured performance trait (i.e. righting response score) in the control acclimated and the three treatment groups. Corticosterone stress responses increased in all toads during exposure to an acute thermal stressor. Furthermore, exposure to a thermal stressor also decreased testosterone levels in all toads. The duration of the righting response (seconds) was longer for toads that were exposed to 40°C than to 30°, 35° or 25°C. The increased corticosterone stress response with increased intensity of the acute thermal stressor suggests that the toads perceived this treatment as a stressor. Furthermore, the results also highlight a potential trade-off with performance and reproductive hormones. Ultimately, exposure acute thermal stressors due to climatic variability could impact amphibians at multiple eco-physiological levels through impacts on endocrine physiology, performance and potentially fitness traits (e.g. reproductive output).

Australian native fauna are thought to be well adapted to fire-prone landscapes, but bushfires may still pose considerable challenges or stressors to wildlife. We investigated the impact of bushfire on the stress physiology of the woylie (brush-tailed bettong, Bettongia penicillata) a critically endangered Australian marsupial, and assessed whether fitness indices (body condition and parasite load) influenced stress physiology before and after the fire. We hypothesised that there would be a significant change in stress physiology indicators (in the form of faecal cortisol metabolites, FCM) following the fire, compared with the months previous. We trapped woylies (n = 19) at Whiteman Park Reserve in Perth, Western Australia, two days after a major bushfire and measured FCM concentration by enzyme immunoassay. Population-level comparisons of FCM were made between these samples and those collected in previous months (n = 58). While mean FCM varied by month of sample collection, it was not higher after the fire. We suggest that woylies may be able to maintain homeostasis through change (allostasis), at least in the period immediately after the fire. This is supported by our finding that FCM did not relate significantly to body condition or parasite load. Our results potentially highlight the physiological and behavioural adaptations of woylies to fire, which could be further explored in future studies.

Rapid colour changes in vertebrates have fascinated biologists for centuries, herein we demonstrate dynamic colour change in an anuran amphibian, the stony creek frog (Litoria wilcoxii), which turns from brown to bright (lemon) yellow during amplexus. We show this by comparing the colour of baseline (unpaired males) and amplecting (paired) males. We also investigate the possible role of stress and stress hormones on this colour change. Frogs were subjected to four different levels of stressors (handling, toe-clipping, saline injection and adrenocorticotropic hormone [ACTH] injection) and the colour change was measured using digital photography. A comparison of baseline colour and stress hormone (corticosterone) levels was also conducted to give further insight to this topic. From the images, the Red Blue Green (RGB) colour values were calculated, and a principal components analysis (PCA) was used to create a single colour metric (the major axis) as an index of colour in the visible spectrum. A moderate stressor (toe-clipping) led to a significant change in colour (within 10 min) similar to that of amplecting males. Surprisingly, neither a mild stressor (handling and saline injection) nor the maximum stressor (handling and ACTH injection) led to a lightening response. This study confirms that the dynamic male colour change in this species in response to medium stressors adds new knowledge to the understanding of the functional mechanisms of dynamic colour change in amphibians.

This study used non-invasive endocrinology to examine baseline corticosterone at different altitudes in a free-living Australian amphibian: the Great Barred Frog ( Mixophyes fasciolatus ). An enzyme immunoassay (EIA) was performed on urine samples and validated biologically using an adrenocorticotropic hormone (ACTH) challenge. Frogs were injected with ACTH on day 0 and recaptures occurred 1–10 days post injection. Urine samples and body condition measurements were collected from lowland (60 m) and highland (660 m and 790 m) sub-populations of M. fasciolatus in South East Queensland (SEQ), close to their post-breeding period during autumn 2011. We simultaneously sampled these sub-populations for Batrachochytrium dendrobatidis ( Bd ), a pathogenic fungus responsible for mass mortalities of amphibians worldwide. The ACTH challenge successfully validated the urinary corticosterone EIA in M. fasciolatus , with a peak urinary corticosterone response to ACTH injection on day 2 and a return to baseline levels by day 6. Polymerase chain reaction (PCR) analysis of 50 individuals returned only 1 positive result for Bd . Simple linear regression showed a strong positive relationship between baseline urinary corticosterone concentrations and altitude and no relationship with body condition. We hypothesize that higher baseline corticosterone concentrations within highland sub-populations of male M. fasciolatus could be associated with increased environmental challenge at high altitudes and geographic range limits. Whether this pattern is an indication of chronic stress in highland populations or life-time fitness and survival, warrants future investigation.

Sexual signalling using dynamic skin colouration is a key feature in some vertebrates; however, it is rarely studied in amphibians. Consequently, little is known about the hormonal basis of this interesting biological phenomenon for many species. Male stony creek frogs (Litoria wilcoxii) are known to change dorsal colouration from brown to lemon yellow within minutes. This striking change is faster then what has been seen most amphibians, and could therefore be under neuronal regulation, a factor that is rarely observed in amphibians. In this study, we observed colour changes in wild frogs during amplexus to determine the natural timing of colour change. We also investigated the hypothesis that colour change is mediated by either reproductive or neuro- hormones. This was achieved by injecting frogs with epinephrine, testosterone, saline solution (control 1) or sesame oil (control 2). A non-invasive approach was also used wherein hormones and controls were administered topically. Male frogs turned a vivid yellow within 5 minutes of initiation of amplexus and remained so for 3–5 hours before rapidly fading back to brown. Epinephrine-treated frogs showed a significant colour change from brown to yellow within 5 minutes, however, testosterone-treated frogs did not change colour. Our results provide evidence of the role neuronal regulation plays in colour change systems.

Batrachochytrium dendrobatidis (Bd), a cutaneous amphibian fungus that causes the lethal disease chytridiomycosis, has been implicated as a cause of many amphibian declines. Bd can tolerate low temperatures with an optimum thermal range from 17-24°C. It has been shown that Bd infection may result in species extinction, avoiding the transmission threshold presented by density dependent transmission theory. Prevalence of Bd during autumn and winter has been shown to be as low as 0% in some species. It is currently unclear how Bd persists in field conditions and what processes result in carry-over between seasons. It has been hypothesised that overwintering tadpoles may host Bd between breeding seasons. The Great Barred Frog (Mixophyes fasciolatus) is a common, stable and widespread species in Queensland, Australia, and is known to carry Bd. Investigation into Bd infection of different life stages of M. fasciolatus during seasonally low prevalence may potentially reveal persistence and carry-over methods between seasons. Metamorphs, juveniles, and adults were swabbed for Bd infection over three months (between March and May, 2011) at 5 sites of varying altitude (66 m-790 m). A total of 93 swabs were analysed using Polymerase Chain Reaction (PCR) real-time analysis. PCR analysis showed 6 positive (1 excluded), 4 equivocal and 83 negative results for infection with Bd. Equivocal results were assumed to be negative using the precautionary principle. The 5 positive results consisted of 4 emerging (Gosner stage 43-45) metamorphs and 1 adult M. fasciolatus. Fisher's exact test on prevalence showed that the prevalence was significantly different between life stages. All positive results were sampled at high altitudes (790 m); however prevalence was not significantly different between altitudes. Infection of emerging metamorphs suggests that individuals were infected as tadpoles. We hypothesise that M. fasciolatus tadpoles carry Bd through seasons. Thus, Mixophyes fasciolatus may act as disease reservoirs at multiple life stages.

Amphibians, like other animals, generate corticosterone or cortisol glucocorticoid responses to stimuli perceived to be threatening. It is generally assumed that the corticosterone response of animals to capture and handling reflects the corticosterone response to stimuli such as the sight of a predator that are thought to be natural stressors. Fijian ground frogs ( Platymantisvitiana ) are preyed upon by the introduced cane toads ( Rhinellamarina ), and we used ground frogs to test the hypothesis that the sight of a predator will induce a corticosterone stress response in an amphibian. Urinary corticosterone metabolite concentrations increased in male ground frogs exposed to the sight of a toad for 1, 3 or 6 h, whereas corticosterone did not change in frogs exposed to another male ground frog, a ball, or when no stimulus was present in the test compartment. The frogs exposed to a toad initially moved towards the stimulus then moved away, whereas frogs exposed to another frog moved towards the test frog and remained closer to the frog than at the start of the test. Tonic immobility (TI) was measured as an index of fearfulness immediately after the test exposure of the frogs to a stimulus. The duration of TI was longer in frogs exposed to a toad than to another frog or to a ball. The results provide novel evidence that the sight of a predator can induce a corticosterone response and lead to increased fearfulness in amphibians. In addition, they show that endemic frogs can recognise an introduced predator as a threat.

Natural weathering conditions can influence faecal cortisol metabolite (FCM) measurements in wildlife if fresh faeces cannot be collected immediately following defaecation. In this study, we evaluated this issue in a threatened Australian marsupial, the greater bilby (Macrotis lagotis). Fresh (

Physiological stress assessment is important for in-situ conservation and captive management of threatened wildlife. Leukocyte (white blood cell) evaluation, especially the neutrophil : lymphocyte (N : L) ratio, provides a logical representation of experimentally elevated corticosterone (stress hormone) in amphibians. Urinary corticosterone enzyme-immunoassay (EIA) is a rapid non-invasive tool for assessing stress responses in amphibians. To our knowledge, no one has explored the relationship between N : L ratio and urinary corticosterone in wild amphibians in a non-experimental way. This study provides a comparative assessment of relative leukocyte numbers, N : L ratios and urinary corticosterone responses of the endangered Fijian ground frog (Platymantis vitiana) during transportation and captivity. Adult frogs (n = 40) were collected from Viwa, Fiji Island for captive breeding. Frogs showed significant changes in leukocyte proportions during transportation and captivity. N : L ratios were higher 6 h after transportation and over 5 and 15 days in captivity. Urinary corticosterone responses of the frogs were also higher 6 h after transportation and after 5 and 15 days in captivity. All leukocyte proportions, N : L ratios and urinary corticosterone concentrations of the frogs returned near baseline levels after the frogs were kept in an environmentally enriched outdoor enclosure for over 25 days. These results highlight the value of leukocyte evaluation and urinary corticosterone EIAs as physiological tools for evaluating stress in amphibians.

Stressors generally decrease testosterone secretion and inhibit reproduction in animals. Urinary testosterone and corticosterone metabolite concentrations were measured in adult male cane toads (Rhinella marina) at the time of capture from the wild and during 24 h of confinement with repeated handling. Mean urinary testosterone concentrations increased 2 h after capture, were significantly elevated above initial concentrations at 5 h, and then declined. Mean testosterone concentrations remained elevated 24 h after capture. Mean urinary corticosterone concentrations increased after capture, were significantly elevated above initial concentrations at 2 h, and remained elevated thereafter. This is the first report in amphibians of an increase in testosterone excretion after capture from the wild, with previous studies showing either no change or decline in testosterone. This finding may be associated with the mating strategy and maintenance of reproductive effort in the cane toad, a species that shows explosive breeding and agonistic male–male interactions during breeding. The finding that testosterone excretion increases rather than decreases after capture in male cane toads shows that it should not be generally assumed that reproductive hormone secretion will decrease after capture in amphibians.

This study was based on the development of a non-invasive glucocorticoid enzyme-immunoassay for the assessment of stress in wild and captive endangered Fijian ground frogs (Platymantis vitiana). Enzyme-immunoassays were developed and validated for the first time to non-invasively measure both cortisol and corticosterone metabolites in frog urine. Frog urine showed parallel displacement with corticosterone but not cortisol standards, therefore corticosterone enzyme immunoassays were used to examine stress in wild and captive frogs. Urinary corticosterone metabolite concentrations increased in frog urine (n = 4) at 6 h, 1 day and 2 days after injection with adrenocorticotropic hormone (0.44 μg g–1 bodyweight), indicating that the corticosterone enzyme-immunoassay could detect changes in circulating corticosterone in frogs. Urinary concentrations of corticosterone were measured in wild frogs (n = 18) after capture in the field. The first measurement beyond the initial sample was at 2–3 h. Mean urinary corticosterone concentrations rose after the initial sample and were significantly elevated in samples collected 3–4 h after capture. This is the first demonstration of a urinary corticosterone response to capture in amphibians. Urinary corticosterone metabolite concentrations for all months combined were lower in captive males than in wild males, and differed between vitellogenic, non-vitellogenic and captive females. Concentrations did not differ between captive and wild females. In conclusion, urinary corticosterone enzyme immunoassays can be used in frogs for assessing stress responses to capture and natural stress profiles of both captive and wild populations.