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Koala (Phascolarctos cinereus) is an iconic folivorous marsupial native to the sclerophyll forests and woodlands of Australia. Due to the ever-changing habitat, this species is highly vulnerable to anthropogenic factors such as habitat loss and fragmentation, and this is reflected in the increasing number of injured and/or diseased koalas over the years. The majority of adult koalas admitted at wildlife hospitals are deceased, either due to natural causes, or have to be euthanized. Thus, orphaned koala joeys constitute a substantial number of wildlife rescues, and mortality is also prevalent in koala joeys being hand-reared/rehabilitated, with little knowledge about the causes of such high rates of mortality. Wildlife hospitals/rehabilitation centres are inherently stressful, and although the hypothalamic–pituitary–adrenal (HPA) axis plays a vital role in mediating the stress endocrine function (by producing glucocorticoids such as cortisol), there are no studies quantifying glucocorticoids in koala joeys. To contribute to this dearth of research, we sampled a total of seven individuals residing at Port Macquarie Koala Hospital and noted their clinical information. Faecal samples were collected from all seven koala joeys during routine cage cleaning. In total, 123 faecal samples were collected, processed and analysed for cortisol using enzyme-immunoassay (EIA). We used the iterative baseline approach to determine baseline and peak concentrations of FCM in koala joeys. Baseline concentrations ranged between 14.11 ng/g – 51.10 ng/g (healthy – sick), whereas, peak FCM concentrations ranged between 25.65 ng/g – 56.58 ng/g (healthy – sick). There was a significant difference (p < 0.05) between FCM concentrations of healthy and impaired individuals. Healthy individuals displayed relatively consistent FCM concentrations, whereas, diseased individuals displayed a significant increase in FCM concentrations over time. Our study provides the first record of baseline and peak FCM concentrations in rescued koala joeys with their associated clinical condition. Future studies can use the iterative baseline approach to determine FCM concentration in wild koala joeys that can serve as a baseline to compare glucocorticoid levels of rescued joeys.

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.

Direct and indirect anthropogenic factors play a massive role in driving wildlife species towards extinction. Longitudinal retrospective studies identify key ‘factors’ responsible for the decline in numbers of wildlife, however, lack the reasoning behind the events leading to mortality. The overarching aim of this study was to categorize these ‘factors’ into different stressor categories faced by reptiles to understand its impact on an individual, and to compare how each stressor category influences the survival of an individual. The results from this study indicated that almost half of the number of reptiles being hospitalized were due to exposure to preliminary stressors such as lawn mowing incidents and pet attack. Primary and secondary admissions were fairly equal in number, however the mortality rate for secondary admissions was drastically high (~80%). The discussion integrates species’ ecology and stress physiology which can prove to have multi-faceted benefits across the fields of ecology and animal welfare. Ecologists can use the results from this study to comprehend species’ activity patterns to better plan reptilian conservation programs, whereas, for wildlife clinicians and rehabilitators, assignment of stressor categories could be a beneficial tool for bolstering the welfare monitoring program for small native reptiles in clinical settings.

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.

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.

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.

Aims: To assess the use of potassium bromide (KBr) as a therapeutic intervention for perennial ryegrass toxicosis (PRGT) in lambs fed ryegrass seed containing lolitrem B. Methods: Male lambs aged 10–12 months (n = 43) were assigned to receive ryegrass seed containing lolitrem B, at a dose of 0.16 mg/kg/day (Groups 2, 3 and 4), or lucerne chaff and molasses (Groups 1 and 5). Lambs in Groups 2 and 3 were observed for clinical signs and gait changes until defined signs of PGRT were observed, when they were transferred, with lambs in Group 1, to the Testing phase of the trial. Lambs in Group 3 were then treated with a single oral dose of 300 mg/kg bromide. Lambs in Groups 4 and 5 received KBr daily from the start of the trial (540 mg/kg bromide over 3 days then 20 mg/kg daily) and were transferred to the Testing phase after 18 days. Clinical examination and gait assessment, and surface electromyography of the triceps muscle, measuring root-mean-square (RMS) voltages, were carried out on Days 0, 1 and 2 of the Testing phase followed by necropsy, histopathology, measurement of concentrations of bromide in serum and CSF and faecal cortisol metabolites (FCM). Results: In Group 3 lambs, mean composite gait scores decreased between Testing phase Day 0 and Days 1 and 2 (p 800 µg/mL in lambs in Groups 3 and 4 on Day 2. Concentrations of FCM were higher in lambs from Group 2 than in Groups 1 or 5, but were similar in Groups 2, 3 and 4. Histopathological findings in the cerebellum of lambs from Groups 2, 3 and 4 were similar, showing pyknosis of neurons within the granular layer of the cerebellum and Purkinje neuron proximal axonal spheroid formation. Conclusions and clinical relevance: A single oral dose of 300 mg/kg bromide in lambs with neurological signs of PRGT resulted in reduced composite gait scores and reduced RMS voltages, indicating a significant improvement in clinical signs of ataxia, movement disorder and muscle tremor associated with the neurotoxic effects of lolitrem B.

Hormonal assessment tools are important for determining the reproductive success of production animals. This study used non-invasive wool assessment to quantify changes in progesterone and cortisol levels in reproducing female merino sheep. Wool samples were collected from a group of n = 46 maiden merino ewes (22-25 months old), naturally joined under natural light conditions in southern New South Wales (NSW), Australia. Three shearing opportunities were conducted as part of standard on-farm management practices. The wool samples were collected at three different dates during 2017, January (prior to rams being put out with the mob and to provide a baseline level since previous shearing in May 2016), September (during very late stages of gestation-approximately 2 weeks prior to parturition) and December (ewes had given birth and ~2-month-old lambs were at foot). Analysis of cortisol and progesterone was conducted concurrently from the same sample of wool. The hormones in wool samples quantified using commercially available cortisol and progesterone enzyme-immunoassay kits. Wool cortisol concentrations increased significantly (p = 3.04E-14) from pre-joining in January (1.33±0.12 ng/g) to late gestation in September (3.59±0.12 ng/g). Concentration of wool cortisol post-lambing in December (3.27±0.14 ng/g) did not decline significantly (p = 0.124) after gestation however remained significantly higher (p = 3.82E-10) than pre-joining levels. Wool progesterone (PG) concentrations increased significantly (p = 1.83E-33) from pre-joining (0.04±0.005 ng/g) in January to late gestation in September (5.53±0.13 ng/g) with a significant (p = 5.44E-59) decline observed in December (0.05±0.003 ng/g) to post- pregnancy concentrations. No significant difference was shown between pre-joining and post lambing PG concentrations (p = 0.057). Our results showed that non-invasive assessment of hormones in Merino sheep wool reflected significant increase in both cortisol and progesterone guided by pregnancy.

Koalas are rescued from the wild often with incidence of burns from bushfire, injury from animal attacks, vehicle collision, and diseases. Exposure to environmental stressors (trauma and disease) could generate physiological stress and potentially impact the outcomes of clinical management intervention and rehabilitation of rescued wild koalas. It is important to quantify the stress physiology of wild koalas upon registering into clinical care. This study demonstrates the first report of physiological stress assessment in rescued wild koalas (n = 22) to determine the potential influences of habitat-specific demographics, stressor category, and clinical diagnosis. Fecal samples were collected from the koalas at rescue and routinely during hospitalization to provide a longitudinal assessment of the koala's stress response throughout clinical care. Fecal glucocorticoid metabolites (FCM) enzyme-immunoassay was used to index physiological stress non-invasively. Koalas were admitted with exposure to various categories of environmental trauma such as vehicle collision, dog attack, burns from forest fire (this also related to conditions such as copious drinking and flat demeanor), and other injury. The main disease diagnosed was chlamydial infections. In terms of environmental interactions, it was found that habitat-specific demographics, location where the rescued koala was found, especially the rural-urban fringe, influenced FCM levels. Furthermore, there was significant interaction between location, stressor category, and clinical diagnosis for mean FCM levels. However, these factors were not predictive of the clinical outcome (euthanized or released). Overall, the results provide invaluable insights into how wild koalas respond physiologically to environmental trauma and disease and how methods of care, husbandry, and treatment can be used to further reduce the impacts of stress with the ultimate aim of increasing the rehabilitation and future release of rescued koalas to revive the declining mainland populations.

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

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.

Reproductive wastage is a key issue for sheep producers, both regionally and globally. The reproductive output of farm animals can be influenced by physiological and environmental factors. Rapid and reliable quantification of physiological stress can provide a useful tool for designing and testing on-farm management interventions to improve farm animal welfare and productivity. In this study, we quantified physiological stress non-invasively using faecal glucocorticoid metabolites-FGMs analysis and body temperature measurements of 15 superovulated donor merino ewes (Ovis aries) during participation in artificial insemination (AI) program conducted during 2015/2016 Australian summer. We hypothesized that low percentage transferable embryos in donor merino ewes will be associated positively with higher body temperature and/or higher FGMs in these ewes. Temperature humidity index (THI) was calculated and found within high thermal stress range during the two AI trials. Overall, results showed none of the factors (ewe ID, AI trial no., THI or FGMs) were significant for reduced percentage transferrable embryos, except ewe body temperature was highly significant (p = 0.014). Within AI trial comparisons showed significant positive associations between higher FGMs and body temperature with reduced transferrable embryos. These results suggest that Australian merino ewes participating in summer AI trials can experience physiological stress. Prolonged activation of the stress endocrine response and high body temperature (e.g. ensued from heat stress) could impact on ewe reproductive output. Therefore, future research should apply minimally invasive physiological tools to gather baseline information on physiological stress in merino sheep to enable the development of new farm-friendly methods of managing stress.

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

Extreme environmental temperature could impact the physiology and ecology of animals. The stress endocrine axis provides necessary physiological stress response to acute (day-day) stressors. Presently, there are no empirical evidences showing that exposure to extreme thermal stressor could cause chronic stress in amphibians. This could also modulate the physiological endocrine sensitivity to acute stressors and have serious implications for stress coping in amphibians, particularly those living in fragmented and disease prone environments. We addressed this important question using the cane toad (Rhinella marina) model from its introduced range in Queensland, Australia. We quantified their physiological endocrine sensitivity to a standard acute (capture and handling) stressor after exposing the cane toads to thermal shock at 35°C for 30min daily for 34 days. Corticosterone (CORT) responses to the capture and handling protocol were measured on three sampling intervals (days 14, 24, and 34) to determine whether the physiological endocrine sensitivity was maintained or modulated over-time. Two control groups (C1 for baseline CORT measurement only and C2 acute handled only) and two temperature treatment groups (T1 received daily thermal shock up to day 14 only and a recovery phase of 20 days and T2 received thermal shock daily for 34 days). Results showed that baseline CORT levels remained high on day 14 (combined effect of capture, captivity and thermal stress) for both T1 and T2. Furthermore, baseline CORT levels decreased for T1 once the thermal shock was removed after day 14 and returned to baseline by day 29. On the contrary, baseline CORT levels kept on increasing for T2 over the 34 days of daily thermal shocks. Furthermore, the magnitudes of the acute CORT responses or physiological endocrine sensitivity were consistently high for both C1 and T1. However, acute CORT responses for T2 toads were dramatically reduced between days 24 and 34. These novel findings suggest that repeated exposure to extreme thermal stressor could cause chronic stress and consequently suppress the physiological endocrine sensitivity to acute stressors (e.g. pathogenic diseases) in amphibians.

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.

Understanding immune function is critical to conserving wildlife in view of infectious disease threats, particularly in threatened species vulnerable to stress, immunocompromise and infection. However, few studies examine stress, immune function and infection in wildlife. We used a flow cytometry protocol developed for human infants to assess phagocytosis, a key component of innate immunity, in a critically endangered marsupial, the woylie (Bettongia penicillata). The effects of stress physiology and Trypanosoma infection on phagocytosis were investigated. Blood and faecal samples were collected from woylies in a captive facility over three months. Trypanosoma status was determined using PCR. Faecal cortisol metabolites (FCM) were quantified by enzyme-immunoassay. Mean phagocytosis measured was >90%. An interaction between sex and FCM influenced the percentage of phagocytosing leukocytes, possibly reflecting the influence of sex hormones and glucocorticoids. An interaction between Trypanosoma status and FCM influenced phagocytosis index, suggesting that stress physiology and infection status influence innate immunity.

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.

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.

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.

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).

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.

Repeatability of physiological response variables, such as the stress hormone corticosterone, across numerous sampling occasions is an important assumption for their use as predictors of behaviour, reproduction and fitness in animals. Very few studies have actually tested this assumption in free-living animals under uncontrolled natural conditions. Non-invasive urine sampling and standard capture handling protocol have enabled the rapid quantification of baseline corticosterone and short-term corticosterone stress responses in anuran amphibians. In this study, established non-invasive methods were used to monitor physiological stress and urinary testosterone levels in male individuals of the terrestrial breeding Fijian ground frog (Platymantis vitiana). Adult male frogs (n = 20) were sampled at nighttime on three repeated occasions at intervals of 14 days during their annual breeding season on Viwa Island, Fiji. All frogs expressed urinary corticosterone metabolite responses to the capture and handling stressor, with some frogs showing consistently higher urinary corticosterone responses than others. Ranks of corticosterone values at 0, 4 and 8 h, and the corrected rank were highly significant (r = 0.75-0.99) between the three repeated sampling occasions. Statistical repeatabilities were high for baseline corticosterone (r = 0.973) and for corticosterone values at 2 h (r = 0.862), 4 h (r = 0.861), 6 h (r = 0.820) and 8 h (r = 0.926), and also for the total (inclusive of baseline corticosterone values) and the corrected integrated responses (index of the acute response) [r = 0.867 and r = 0.870]. Urinary testosterone levels also showed high statistical repeatability (r = 0.78). Furthermore, variation in baseline and short-term corticosterone stress responses was greater between individuals than within individuals. Baseline urinary corticosterone was significantly negatively correlated with the corrected integrated corticosterone response (r = -0.3, p0.001) but non-significantly with body-condition (r = -0.04) and baseline urinary testosterone (r = -0.07). In contrast, the corrected integrated corticosterone response was positively correlated (non-significantly) with baseline urinary testosterone (r = +0.04) and body-condition (r = +0.08). Urinary testosterone levels and body-condition were significantly negatively correlated (r = -0.23, p0.001). The results suggest that male frogs with higher levels of testosterone could have depleted energy reserve during the breeding period. The acute corticosterone responses help in replenishing energy that is needed for breeding and survival. The results also provide some support to the 'cort-fitness' hypothesis as highlighted by the negative correlation between baseline corticosterone and body-condition. It is most likely that the acute corticosterone response is adaptive and linked positively with reproductive fitness and survival in male anurans.

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.

Amphibians respond to environmental stressors by secreting corticosterone, a stress hormone which promotes physiological and behavioral responses. Capture handling can be used to stimulate physiological stress response in amphibians. The use of single blood sampling and presentation of mean data often limits the quantification of within and between individual variation in baseline and short-term corticosterone stress responses in amphibians. It is important for studies of amphibian physiological ecology to determine whether baseline and short-term corticosterone stress responses are consistent or not. We quantified repeatability (r), a statistical measure of consistency, in baseline and short-term corticosterone stress responses to a standard capture and handling stress protocol in free-living adult male cane toads (Rhinella marina). Corticosterone metabolite concentrations were measured entirely non-invasively in male toad urine samples via an enzyme-immunoassay. During the first sampling occasion, urine samples were collected manually from individual male toads (n = 20) immediately upon field capture. Toads were handled for 5 min then transferred to plastic bags (constituting a mild stressor), and urine samples were collected hourly over 8 h in the field. The toads were resampled for baseline (0 h) urine corticosterone with hourly urine sampling over 8 h (for quantification of the stress induced corticosterone) at 14 day intervals on three consecutive occasions. Within and between sample variations in urinary corticosterone metabolite concentrations were also quantified. All toads expressed a corticosterone stress response over 8 h to our standard capture and handling stress protocol. Variations both within and between toads was higher for corrected integrated corticosterone concentrations than corticosterone concentrations at baseline, 3 or 6 h. Baseline urinary corticosterone metabolite concentration of the male toads was highly repeatable (r = 0.877) together with high statistical repeatabilities for 3 h (r = 0.695), 6 h (r = 0.428) and 8 h (r = 0.775) corticosterone metabolite concentrations, and for the total and corrected integrated corticosterone responses (r = 0.807; r = 0.743 respectively). This study highlights that baseline and short-term corticosterone stress responses are repeatable in free-living amphibians. Future studies should utilize this non-invasive tool to explore repeatability among seasons and across years, and determine its functional significance in relation to behavioral ecology and reproduction in amphibians generally.

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.

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.

This review focuses on non-invasive endocrinology, which is a key component of amphibian conservation physiology. It enables rapid assessment of reproductive and stress hormones in free-living and captive populations. It also provides a direct physiological measure of population sensitivity to extreme environments and their sub-lethal impacts on reproduction, health and survival., Non-invasive endocrinology utilizes non-invasive biological samples (such as faeces, urine, hair, aquatic media, and saliva) for the quantification of hormones in wildlife. Urinary-based enzyme immunoassay (EIA) and radio-immunoassay have enabled the rapid quantification of reproductive and stress hormones in amphibians (Anura: Amphibia). With minimal disturbance, these methods can be used to assess the ovarian and testicular endocrine functions as well as physiological stress in captive and free-living populations. Non-invasive endocrine monitoring has therefore greatly advanced our knowledge of the functioning of the stress endocrine system (the hypothalamo–pituitary–interrenal axis) and the reproductive endocrine system (the hypothalamo–pituitary–gonadal axis) in the amphibian physiological stress response, reproductive ecology, health and welfare, and survival. Biological (physiological) validation is necessary for obtaining the excretory lag time of hormone metabolites. Urinary-based EIA for the major reproductive hormones, estradiol and progesterone in females and testosterone in males, can be used to track the reproductive hormone profiles in relationship to reproductive behaviour and environmental data in free-living anurans. Urinary-based corticosterone metabolite EIA can be used to assess the sublethal impacts of biological stressors (such as invasive species and pathogenic diseases) as well as anthropogenic induced environmental stressors (e.g. extreme temperatures) on free-living populations. Non-invasive endocrine methods can also assist in the diagnosis of success or failure of captive breeding programmes by measuring the longitudinal patterns of changes in reproductive hormones and corticosterone within captive anurans and comparing the endocrine profiles with health records and reproductive behaviour. This review paper focuses on the reproductive and the stress endocrinology of anurans and demonstrates the uses of non-invasive endocrinology for advancing amphibian conservation physiology. It also provides key technical considerations for future research that will increase the accuracy and reliability of the data and the value of non-invasive endocrinology within the conceptual framework of conservation physiology.

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.