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Human-wildlife interaction is a broad and complex topic. Due to rapid world population growth, there have been greater human impacts on wildlife through agriculture and land fragmentation. In many countries, significant challenges exist with managing wildlife and its negative impacts on humans and wildlife. This special issue discusses human-wildlife co-existence.

Animal welfare is important for the humane treatment of animals under our care. Zoos and rescue centres manage various charismatic animals, such as big cats, with limited resources. It is therefore essential for caretakers to understand the needs of an individual big cat to ensure its welfare. However, these needs may differ due to a big cat's personality, which may be identified by its coping style in a stressful situation. In addition, stress is one of the major factors affecting animal welfare. There is limited evidence showing strong associations between personality and stress physiology in big cats. This review focuses on the integration of personality and stress physiology of captive big cats, to highlight possible improvements in their husbandry. Our review identifies key factors that may influence big cat responses to stressors. These influencing factors include: i) social interactions; ii) environment; iii) life history and evolutionary traits; iv) genetics; and v) health. The first two factors are relatively well covered in the literature; however, the final three are potentially very promising avenues for future research to better understand how we can improve big cat welfare.

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.

Simple Summary China is the world’s largest producer of food fish, and Chinese consumers have a preference to buy live fish. Live transport of fish is, therefore, a common procedure in aquaculture and is a potential animal welfare hazard. Little has been published on current fish transportation practices in China or the knowledge and attitudes of stakeholders in this industry. Our qualitative study aimed to obtain original information about live transport processes from a cross-section of aquaculture stakeholders in China by conducting individual interviews. Stakeholders were interviewed about their knowledge of live transport and their attitudes towards the welfare of fish. Self-described knowledge of live transport varied between participants with different job types. Most participants had heard of and understood the concept of “animal welfare”, but many understood it to only refer to terrestrial livestock, not fish. This suggests that knowledge of fish welfare in the industry may be less than for other farm animals. The findings of this pilot study contribute to a better understanding of live fish transport from a stakeholder point of view. The findings will also assist in informing, educating, and sensitizing stakeholders to the importance of fish welfare during live transport. Abstract China is the largest food fish producer in the world. Chinese consumers normally purchase fish that are still alive to ensure freshness. Therefore, the live transport of fish is important in China’s aquaculture, although it carries potential risks for animal welfare. This study investigated the attitudes and knowledge of stakeholders within Chinese aquaculture towards the live transport and welfare of fish. Semi-structured interviews were conducted with 12 participants who were involved with the aquaculture industry in China. Most participants self-rated their transport-related knowledge as moderate and had some understanding of animal welfare, although this term was generally considered only relevant to terrestrial animals. Participants’ responses indicated that the live transport of fish occurs frequently in China, generally using sealed tanks, plastic bags, and foam boxes, in purpose-built vehicles. Seasonal changes, such as changes in ambient and water temperature, are considered to be important contributors to successful live transport, as well as sufficient oxygen supplies and stocking density. The use of anesthetics was not commonly reported, particularly in food fish, and fish capture is predominantly by conventional dipnets. The health status of transported fish is determined mostly by morphology (body injury, body or eye color, and fin condition), as well as vigor and swimming ability. Our results indicate that live transport poses a number of welfare risks to fish but that participants in the process associated welfare concerns more with terrestrial animals, not fish.

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.

Wild animals under human care as well as domesticated farm production animals are often exposed to environmental changes (e [...]

Simple Summary Stress evaluation in wildlife is valuable tool for rehabilitation and injury prevention. This pilot study investigated categories of stress in rescued birds. We determined three categories of stressors (preliminary, primary and secondary) using clinical data of rescued birds from Adelaide, South Australia. It was discovered that birds are highly susceptible to impact injuries (e.g., flying into a building window) and vehicle-related injuries as preliminary stressors, which often result in hospitalisation of birds. Immobility and abnormal behaviour represented the most common primary stressor, while the most common secondary stressors included trauma and fracture. Furthermore, the most common outcome in clinics due to exposure of birds to these three stressor categories was euthanasia. Abstract Urbanisation exposes avian wildlife to an array of environmental stressors that result in clinical admission and hospitalisation. The aim of this pilot study was to conduct a retrospective analysis of clinical data and characterise this based on categories of stress experienced by avian wildlife patients. The results from this study indicated that impact injuries (n = 33, 25%) and vehicle-related injuries (n = 33, 25%) were the most common occurring preliminary stressors that resulted in the hospitalisation of avian wildlife. The most common outcome of avian patients that suffered from vehicle-related injuries was euthanasia (n = 15, 45%), as was avian patients that suffered from impact injuries (n = 16, 48%). Immobility (n = 105, 61%) and abnormal behaviour (n = 24, 14%) were the most commonly occurring primary stressors of avian patients. Finally, trauma (n = 51, 32%) and fractures (n = 44, 27%) were the most common occurring secondary stressors in avian patients. The most common outcome of all these stressors was euthanasia. This study provided further evidence towards the notion that human- and urbanisation-related stressors are the main causes of hospitalisation of avian wildlife, but also indicated that birds admitted as a result of human-related stressors are more likely to be euthanised than released. This study also provided a categorisation system for the stressors identified in avian wildlife patients (preliminary, primary and secondary) that may be used to monitor the stress categories of wildlife patients and gain a deeper understanding of the complex notion of stress.

Non-invasive techniques can be applied for monitoring the physiology and behaviour of wildlife in Zoos to improve management and welfare. Thermal imaging technology has been used as a non-invasive technique to measure the body temperature of various domesticated and wildlife species. In this study, we evaluated the application of thermal imaging to measure the body temperature of koalas (Phascolarctos cinereus) in a Zoo environment. The aim of the study was to determine the body feature most suitable for recording a koala&rsquo, s body temperature (using coefficient of variation scores). We used a FLIR530TM IR thermal imaging camera to take images of each individual koala across three days in autumn 2018 at the Wildlife Sydney Zoo, Australia. Our results demonstrated that koalas had more than one reliable body feature for recording body temperature using the thermal imaging tool&mdash, the most reliable features were eyes and abdomen. This study provides first reported application of thermal imaging on an Australian native species in a Zoo and demonstrates its potential applicability as a humane/non-invasive technique for assessing the body temperature as an index of stress.

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.

The hypothalamic–pituitary–adrenal (HPA) axis and the hypothalamic–pituitary–gonadal (HPG) axis systems are inversely related in humans and animals. Although livestock animals, such as sheep (Ovis aries), tend to be well adapted to their environment, it is known that the livestock production processes subject animals to a multitude of physical and psychological stressful stimuli that have the potential to elevate the HPA axis activity. Chronic stress is one of the major challenges in sheep production, as it is difficult to detect and can result in prolonged dysfunction of the HPA axis, causing downstream negative physiological effects such as immunosuppression, increased susceptibility to disease and reproductive dysfunction. The elevation of HPA axis activity during chronic stress has been suggested as the primary neuroendocrine mechanism underlying the aetiology of reproductive dysfunction in sheep. Research in sheep has demonstrated that glucocorticoids act on the HPG axis at the level of the hypothalamus and hypophyseal portal system to decrease gonadotrophin secretion and at the level of the pituitary gland to reduce responsiveness and sensitivity of gonadotroph cells and their receptors to GnRH. Sheep farming enterprises rely on the breeding efficacy of ewes to optimise lambing percentage and reproductive success in order to ensure a profitable business. This review discusses the influences of the HPA axis on the HPG axis and defines any significant reproductive function consequences caused by stress in ewes and places them into perspective for sheep management and productivity.

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.

Australia has a rich terrestrial and marine biodiversity and high species endemism. However, the oceanic continent is facing the biodiversity extinction crisis. The primary factors are anthropogenic induced environmental changes, including wildlife habitat destruction through urbanisation and predation by feral animals (e.g. red foxes and feral cats), increased severity of diseases (e.g. chytridiomycosis and chlamydia), and increased occurrence of summer heat waves and bush fires. Stress physiology is a dynamic field of science based on the studies of endocrine system functioning in animals. The primary stress regulator is the hypothalamo-pituitary adrenal (interrenal) axis and glucocorticoids (corticosterone and/or cortisol) provide stress index across vertebrate groups. This review paper focuses on physiological stress assessments in Australian wildlife using examples of amphibians, reptiles, birds and marsupials. I provide a thorough discussion of pioneering studies that have shaped the field of stress physiology in Australian wildlife species. The main findings point towards key aspects of stress endocrinology research, such as quantification of biologically active levels of glucocorticoids, development of species-specific GC assays and applications of stress physiology approaches in field ecology and wildlife conservation programs. Furthermore, I also discuss the importance of chronic stress assessment in wildlife populations. Finally, I provide a conceptual framework presenting key research questions in areas of wildlife stress physiology research. In conclusion, wildlife management programs can immensely benefit from stress physiology assessments to gauge the impact of human interventions on wildlife such as species translocation and feral species eradication.

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.

There is increasing interest in using nonblood measures of glucocorticoids to assess the physiological response to chronic stress conditions. In sheep, cortisol has been measured in various matrices including saliva, feces, and wool, but comprehensive studies of the relationship between plasma concentrations of cortisol and concentrations in these nonblood matrices are lacking. Therefore, we tested the hypothesis that administration of cortisol to sheep would result in elevated concentrations of cortisol in blood, saliva, feces, and wool. Merino ewes were administered with saline or 2 mg/kg BW/d hydrocortisone acetate (HCA) by intramuscular (i.m.) injection for 28 d. This treatment was imposed to mimic circulating cortisol concentrations experienced during periods of chronic stress. Cortisol and cortisone were directly measured in plasma, saliva, and wool before, during, and after treatment with saline or HCA. A 14-d pre-treatment and a 14-d post-treatment period were used to measure time taken for glucocorticoid concentrations in each of the matrices to return to baseline levels. Cortisol was also measured in feces before, during, and after treatment. Wool growth was also measured. Before treatment, there was no difference in the concentration of cortisol or cortisone in plasma, saliva, feces, or wool in animals treated with saline or HCA. In contrast, treatment with HCA increased (P < 0.05) concentrations of both cortisol and cortisone in plasma, saliva, and wool and of cortisol in feces. In plasma, cortisol concentrations were higher than cortisone (P < 0.05), whereas saliva cortisol and cortisone concentrations did not differ significantly. In wool, the concentration of cortisone was about 19-fold higher than that of cortisol during treatment and post-treatment periods. Treatment with HCA inhibited wool growth. These results demonstrate that an increase in glucocorticoids in the blood of sheep is reflected in increases in saliva (after 7 d of treatment), feces (21 d), and wool (14 d). Therefore, measures of glucocorticoids in these matrices may provide a measure of activation of the adrenal glands over time in sheep, thereby providing a retrospective indicator of chronic stress. With respect to wool, it appears that cortisol is predominantly metabolized to cortisone in the skin or wool follicle and is stored as cortisone. Therefore wool cortisone may also provide an important measure in quantifiying chronic stress in sheep.

The use of noninvasive techniques to evaluate stress responses in animals has become an increasingly popular method of animal welfare assessment in both production animals and wildlife. In particular, using fecal samples to measure fecal cortisol metabolites (FCMs) as a quantitative measure has proven ideal as samples can be collected remote to the animal after defecation without the need for invasive procedures. Colorimetric enzyme immunoassays (EIAs) have been shown to have a high level of selectivity and sensitivity for FCM concentration analysis, equivalent to the traditionally used radioimmunoassay. Regardless of the assay system used, species- and sample-specific validation must be undertaken to ensure the reliability of results, particularly where sampling is undertaken in a novel species or where environmental conditions might impact FCM stability in the fecal sample. To determine the limit of environmental exposure acceptable for analysis of FCM concentrations in ovine scat samples collected from a paddock under conditions of stable heat and humidity, this study quantified FCMs in ovine feces shortly after defecation (2–7 h) and after timed environmental exposure (1–9 d). Samples were determined to show stable FCM concentrations for up to 5 d by this analysis. Understanding the impacts of environmental exposure, and therefore the viability of remote fecal collection methods for quantitative analysis of FCM by EIA, is important to assess the utility of noninvasive measures of endocrine status in animals where the exact timing of defecation may not be known.

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.

Synergism between extrinsic and intrinsic factors is crucial for the seasonality of reproduction. Environmental factors such as photoperiod and temperature activate the hypothalamus-pituitary-gonadal axis leading to the secretion of steroid hormones that are crucial for reproduction. Sex steroids are not only essential for the maturation of gonads, but also for development of secondary sexual characters in males and reproductive behaviour of both the sexes. In the present study, we quantified the urinary testosterone (UTM) and corticosterone (UCM) metabolites in males and urinary estradiol metabolites (UEM) and UCM in females of Nyctibatrachus humayuni for two consecutive years to determine annual and seasonal variation in the levels of sex steroids, corticosterone and body condition index (BCI). The results show that sex steroids were highest during the breeding season and lowest during the non-breeding season in both the sexes. An increase in UTM and UEM was observed in males and females respectively during the breeding season. Testicular histology showed the presence of all stages of spermatogenesis throughout the year indicating that spermatogenesis is potentially continuous. Ovarian histology showed the presence of vitellogenic follicles only during the breeding season indicating that oogenesis is strictly seasonal. In males, UCM levels were highest during the breeding season, while in females their levels were highest just prior to the breeding season. In males, BCI was highest during the pre-breeding season, declined during the breeding season to increase again during the post-breeding season. In females, BCI was comparable throughout the year. In males, UTM levels were positively correlated with UCM levels but negatively correlated with BCI. Interestingly, UEM, UCM and BCI were not correlated in females. These results indicate that N. humayuni exhibits an associated pattern of reproduction. Quantification of urinary progesterone metabolites (UPM) during the breeding season showed UPM levels were higher in post-spawning females, suggesting the significance of progesterone in ovulation. Further, non-invasive enzyme immunoassay has been successfully standardized in N. humayuni for the quantification of urinary metabolites of steroid hormones.

Tryptophan (Trp) has been associated with the regulation of several behavioral and physiological processes, through stimulation of serotonergic activity. Tryptophan utilization at the metabolic level is influenced by the competitive carrier system it shares with large neutral amino acids (LNAA). This study was carried out using meat-type chicken as a model, to investigate the dose response effects of Trp/LNAA on fear response (tonic immobility; TI) and hormonal responses, including corticosterone (CORT), serotonin (5-HT), triiodothyronine (T) and thyroxine (T). A total of 12 cages (48 birds) were assigned to each of the six experimental groups at 29–42 days of age. Experimental diets were formulated to have incremental levels of Trp/LNAA (0.025, 0.030, 0.035, 0.040, 0.045, and 0.050). The results revealed that, Trp/NAA had no significant effect on growth performance and TI of the birds. However, elevation of Trp/LNAA was concurred with a linear reduction in CORT (P

The survival of animal species and individuals is largely determined by their ability to express physiological stress responses to predictable and unpredictable environmental challenges. Currently, there is no empirical evidence presenting the stress endocrine responses of female frogs during breeding between different reproductive groups. In this study, non-invasive urine sampling and standard capture and handling protocol were used to quantify baseline and short-term corticosterone stress responses in vitellogenic and non-vitellogenic female Fijian ground frog (Platymantis vitiana) during the annual breeding period. Urinary estrogen and progesterone metabolites were also quantified in the same frog urine samples. Repeated sampling of the female frogs (n=20) on three occasions enabled repeatability (r) of reproductive and stress hormones to be quantified. All female frogs generated urinary corticosterone responses to the standard capture and handling stressor. Both baseline and short-term corticosterone responses were significantly higher in magnitude in the vitellogenic females in comparison to the non-vitellogenic female frogs. Vitellogenic females also showed significantly higher levels of urinary estrogen and progesterone metabolites in comparison to the non-vitellogenic females. Baseline urinary corticosterone, short-term corticosterone responses, urinary estrogen, and progesterone metabolites were highly repeatable for both female groups. The results highlight the importance of reproductive and stress hormones during the breeding period in female ground frogs. Future studies should determine the role of potential biological stressors (such as interactions with invasive species) that could be mediating the observed differences in stress endocrine responses of the vitellogenic and non-vitellogenic female frogs.

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.

Non-invasive measurement of urinary corticosterone and testosterone metabolites in amphibians provides opportunities for endocrine studies of responses to physiological and psychological stressors. Typically, corticosterone metabolite concentrations increase in frog urine within 1-2h of a mild capture and handling stress protocol. However, no study has investigated the effect of duration of manual restraint on the changes in corticosterone and reproductive hormones in amphibians. We quantified urinary corticosterone and testosterone metabolite responses for 8h following various durations of manual restraint (control, 5, 15 or 30min) in adult male cane toads (Rhinella marina) under controlled laboratory conditions. All toads had a corticosterone stress response over 8h to our standard capture and handling stressor. The mean corticosterone stress response was significantly higher after 15 or 30min restraint in comparison to the control (no restraint) or to 5min restraint. Manual restraint for 5, 15 or 30min caused a significant reduction in urinary testosterone concentrations over 8h. We also provide a novel method of quantifying plasticity in corticosterone stress responses in amphibians with respect to restraint duration using the concept of a "reaction norm". The reaction norm, which was calculated as slope of the regression line of integrated corticosterone response against restraint duration, was 9.69 (pg corticosterone/μg creatinineh)/min for male toads. In summary, corticosterone and testosterone responses to restraint are affected by restraint duration in male toads. Glucocorticoid reaction norms can be applied to study the change in physiological stress hormonal response with respect to restraint duration in other amphibian species.

In vertebrates, the increase in plasma androgens and corticosteroids is essential for the expression of reproductive behaviour. In male anurans, the interaction between hypothalamus-pituitary-gonadal and hypothalamus-pituitary-interrenal axes plays a pivotal role in calling behaviour and energy mobilisation through the secretion of testosterone and corticosterone respectively. To explain the association among body condition, testosterone, corticosterone and calling behaviour the energetic-hormone-vocalisation (EHV) model has been proposed. The model predicts that with continued participation in chorus activity within and across nights, levels of circulating androgens, corticosterone and vocal effort tend to increase and should be positively correlated in calling males. Consequently, decreasing energy reserves should be inversely correlated with corticosterone level in calling males. Depleted energy reserves lead to the peaking of circulating corticosterone, which suppresses androgen production and calling behaviour. In the present study, we used Nyctibatrachus humayuni with unique reproductive behaviour to test the model by quantifying calling behaviour and urinary metabolites of testosterone and corticosterone. We also computed the body condition index (BCI) to assess the association among energetics, levels of testosterone, corticosterone and calling behaviour. The results show that calling males had higher levels of urinary testosterone metabolites (UTM) than non-calling males indicating the importance of testosterone in controlling the calling behaviour. Surprisingly, urinary corticosterone metabolite (UCM) levels were comparable between calling and non-calling males. Further, calling males had higher body condition estimates than non-calling males. The vocal effort was neither associated with UTM, UCM nor BCI. However, a positive association was observed between UTM and UCM levels in calling males indicating the requirement of higher energy for advertisement. Analysis of UTM and UCM levels throughout the breeding season revealed that breeding basal of UTM was significantly higher than that of UCM. Interestingly, UCM levels were maintained at a lower threshold during the breeding season. These observations are in line with some of the predictions of EHV model.

Extreme temperature can cause metabolic, immune and behavioural changes in amphibians. Short-term stress hormonal response via increased secretion of corticosterone enables amphibians to make necessary physiological and behavioural adjustments for coping with stressors. The effect of temperature on short-term corticosterone responses has not been studied in amphibians. In this study, this relationship was evaluated in adult male cane toads (Rhinella marina). We acclimated male toads (n=24 toads per group) at low, medium and high temperature (15, 25 or 35°C) under controlled laboratory conditions for a 14 day period. After thermal acclimation, short-term corticosterone responses were evaluated in the toads subjected to a standard capture and handling stress protocol over a 24h period. Corticosterone metabolites in toad urine were measured via enzyme-immunoassay. During acclimation, mean baseline urinary corticosterone level increased after transfer of the toads from wild into captivity and returned to baseline on day 14 of acclimation for each of the three temperatures. At the end of the 14 days of thermal acclimation period, baseline corticosterone level were highest for toad group at 35°C and lowest at 15°C. All toads generated urinary corticosterone responses to the standard capture and handling stressor for each temperature. Both individual and mean short-term corticosterone responses of the toads were highest at 35°C and lowest at 15°C. Furthermore, Q(10) values (the factor by which the reaction rate increases when the temperature is raised by 10°) were calculated for mean corrected integrated corticosterone responses as follows; (15-35°C) Q(10)=1.51, (15-25°C) Q(10)=1.60; (25-35°C) Q(10)=1.43. Both total and corrected integrated corticosterone responses were highest for toads at 35°C followed by 25°C and lowest for the 15°C toad group. Overall, the results have demonstrated the thermodynamic response of corticosterone secretion to short-term capture and handling stress in an amphibian species.

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.

Toe-clipping, the removal of one or more toes, is a common method used to individually mark free-living animals. Whilst this method is widely used in studies of amphibians, the appropriateness of the method, and its potential detrimental effects have been the subject of debate. Here, we provide for the first time, evidence that toe-clipping is a stressor in a wild amphibian. We measured urinary corticosterone responses of male cane toads ( Rhinella marina ) to capture and handling only, and to toe-clipping under field conditions. Urinary testosterone concentrations and white blood cell proportions were also measured. Urinary corticosterone metabolite concentrations increased 6 h after capture and handling only and remained high for 24 h; corticosterone returned to baseline levels after 48 h and remained low at 72 h post capture and handling. Corticosterone concentrations in toads subjected to toe-clipping increased at 6 h to significantly higher concentrations than after capture and handling only, then decreased more slowly than after capture and handling, and were still elevated (approximately double basal level) 72 h after toe-clipping. Testosterone did not change significantly after capture and handling only, whereas after toe-clipping testosterone decreased at 6 h and remained low at 72 h. There were weak short-term effects of toe-clipping compared with capture and handling only on white blood cell proportions. We have clearly shown that toe-clipping is a distinctly stronger stressor than capture and handling alone. This indicates that there is an ethical cost of toe-clipping, and this should be considered when planning studies of amphibians.

Urinary corticosterone metabolite responses to capture have recently been shown for the first time in amphibians, and in the present study urinary corticosterone metabolite responses to capture and to confinement in captivity were measured in adult cane toads ( Rhinella marina ) in Queensland, Australia. An adrenocorticotropic hormone (ACTH) challenge was used to provide a biological validation for urinary corticosterone metabolite concentrations measured by radioimmunoassay (RIA). Urinary corticosterone metabolite increased 1–2 days after ACTH but not saline injection and then returned to initial values, indicating that the RIA could detect changes in corticosterone secretion in toads. Urinary corticosterone metabolite responses to short-term capture and restraint in plastic bags were first apparent 2 h after capture of wild toads. Toads held communally in captivity for 5 days had elevated urinary corticosterone metabolite concentrations. Mean corticosterone concentrations declined significantly after a further 7 days in individual housing chambers. There was no sex difference in urinary corticosterone metabolite responses of toads to ACTH challenge, short-term capture or captivity. The relative amount of variation in the mean corticosterone responses was quantified by calculating coefficients of variation (CV) for each mean corticosterone response. Mean corticosterone at 0 min was more variable for captive toads than wild toads. Furthermore, initial corticosterone concentrations (0 min) were more variable than concentrations during the ACTH challenge, short-term capture and captivity. There was little change in the amount of variation of mean corticosterone levels between male and female toads with increasing time in captivity (12–29 days). This study has shown individual corticosterone responses of amphibians for the first-time, and has provided a novel method for quantifying the relative amount of variation in amphibian corticosterone responses.

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.

Marsupial research, conservation, and management can benefit greatly from knowledge about glucocorticoid (GC) secretion patterns because GCs influence numerous aspects of physiology and play a crucial role in regulating an animal's response to stressors. Faecal glucocorticoid metabolites (FGM) offer a non-invasive tool for tracking changes in GCs over time. To date, there are relatively few validated assays for marsupials compared with other taxa, and those that have been published generally test only one assay. However, different assays can yield very different signals of adrenal activity. The goal of this study was to compare the performance of five different enzyme immunoassays (EIAs) for monitoring adrenocortical activity via FGM in 13 marsupial species. We monitored FGM response to two types of events: biological stressors (e.g., transport, novel environment) and pharmacological stimulation (ACTH injection). For each individual animal and assay, FGM peaks were identified using the iterative baseline approach. Performance of the EIAs for each species was evaluated by determining (1) the percent of individuals with a detectable peak 0.125–4.5 days post-event, and (2) the biological sensitivity of the assay as measured by strength of the post-event response relative to baseline variability (Z-score). Assays were defined as successful if they detected a peak in at least 50% of the individuals and the mean species response had a Z ⩾ 2. By this criterion, at least one assay was successful in 10 of the 13 species, but the best-performing assay varied among species, even those species that were closely related. Furthermore, the ability to confidently assess assay performance was influenced by the experimental protocols used. We discuss the implications of our findings for biological validation studies.

Non-invasive endocrine monitoring with minimally invasive biological samples, such as urine, is being used widely for conservation biology research on amphibians. Currently, it is unknown how closely urinary measurements correspond with the traditional serum hormone measurements. We compared urinary and serum concentrations of corticosterone (CORT) and testosterone (T) in adult male cane toads (Rhinella marina) using a standard capture and handling (short-term stressor) protocol. Free-living male cane toads were captured and sampled for baseline urine (0. h) with a second urine sample taken at 0.5. h and hourly between 1 and 8. h. A single blood sample was collected from each toad after the final urine sampling and capture handling. The mean serum CORT concentration increased between 0 and 0.5. h, reaching the highest level between 6 and 8. h. The mean urinary CORT concentration increased with a lag-time of 1. h and continued to increase up to 8. h. The mean level of serum T decreased between 0 and 7. h and increased between 7 and 8. h. Mean urinary T concentration decreased with a lag-time of 0.5. h. Urinary T levels did not change between 4 and 8. h. Mean serum T levels reached 50% of the original 0. h value at 1. h while mean serum CORT levels reached 200% of the original 0. h value within 0.5. h. Mean urinary T levels reached 50% of the original 0. h value within 3. h while mean urinary CORT levels reached 200% of the original 0. h value within 3. h. The inter-individual variation in baseline serum and urinary CORT and T levels were highly comparable, suggesting that baseline urine sample provides a reliable indicator of the physiological status of the animal. Overall, the results have demonstrated that urine sampling and standard capture handling protocol provide reliable measures of baseline corticosterone and testosterone, as well as short-term stress hormone responses in amphibians.

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.

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.

Urinary corticosterone metabolite enzyme-immunoassay (EIA) can be used for the non-invasive assessment of baseline levels and corticosterone responses in amphibians. In this study, urinary corticosterone responses of wild male cane toads (Rhinella marina) to confinement and repeated handling were measured to quantify individual variation in corticosterone responses for the first time in an amphibian species. Urine samples were collected at 0 h in the wild, hourly from 2 to 8 h after transfer into captivity, and again at 12 and 24 h in captivity. Toads were then held in captivity and subjected to the same sampling protocol on three occasions at 14 days intervals to quantify variation in corticosterone metabolite responses within and between toads. Baseline and individual corticosterone metabolite responses in male cane toads were generally consistent, with high statistical repeatabilities for 0 h (r = 0.630), 6 h (r = 0.793), 12 h (r = 0.652) and 24 h (r = 0.721) corticosterone metabolite concentrations, and for the total and corrected integrated corticosterone responses (r = 0.567, p = 0.033; r = 0.728, p = 0.014 respectively). Urinary corticosterone responses appear to be a stable, repeatable trait within individuals. Corticosterone responses in amphibians can be more readily measured when urine rather than plasma samples are collected, and the protocol established in the current study can now be applied to the study of variation in corticosterone responses in other amphibians.

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.

Since European settlement more than 10 % of Australia’s native fauna have become extinct and the current picture reflects 46 % are at various vulnerability stages. Australia’s iconic marsupial species, koala (Phascolarctos cinereus) is listed as vulnerable under national environmental law. Human population growth, road expansion and extensive land clearance have fragmented their eucalyptus habitat and reduced the ability of koalas to move across the tree canopy; making the species most vulnerable on the ground. Disease-principally chlamydia, road death, dog-attack and loss of habitat are key environmental pressures and the reasons why koalas are admitted for veterinary care. It is important to understand the dynamics of the physiological impacts that the koala faces from anthropogenic induced environmental challenges, especially on its essential biological functions (e.g. reproduction and immune system function). This review explores published literature and clinical data to identify key environmental stressors that are operating in mainland koala habitats, and while the focus is mostly on the koala, much of the information is analogous to other wildlife; the review may provide the impetus for future investigations involving other vulnerable native wildlife species (e.g. frogs). Oxalate nephrosis associated renal failure appears to be the most prevalent disease in koala populations from South Australia. Other key environmental stressors included heat stress, car impacts and dog attacks. It is possible that maternal stress, nutritional deprivation, dehydration and possible accumulation of oxalate in eucalyptus leaf increase mostly during drought periods impacting on fetal development. We hypothesize that chronic stress, particularly in urban and fringe zones, is creating very large barriers for conservation and recovery programs. Chronic stress in koalas is a result of the synergistic interplay between proximate environmental stressor/s (e.g. heat stress and fringe effects) acting on the already compromised kidney function, immune- and reproductive suppression. Furthermore, the effects of environmental pollutants in the aggravation of diseases such as kidney failure, reproductive suppression and suppression of the unique marsupial immune system should be researched. Environmental policies should be strengthened to increase human awareness of the threats facing the koala, increased funding support towards scientific research and the protection and creation of reserve habitats in urban areas and fringe zones. Global climate change, nutritional deprivation (loss of food sources), inappropriate fire management, invasive species and the loss of genetic diversity represent the complexities of environmental challenges impacting the koala biology.