Comparing the agreement of a commercial cortisol kit with a biologically validated assay in evaluating faecal cortisol metabolite levels in koala joeys.

Orphaned koala joeys constitute a substantial number of wildlife rescues. Mortality is highly prevalent in rehabilitating joeys, with little knowledge about the causes of mortality. The hypothalamic-pituitary-adrenal axis plays a vital role in mediating stress by producing glucocorticoids (e.g. cortisol), however, no studies have quantified glucocorticoids in koala joeys. Traditional cortisol enzyme immuno-assay (e.g. R4866) are limited in supply and are process intensive, whereas, modern enzyme immuno-assay (EIA) kits (e.g. Arbor Assay cortisol kit) are available world-wide and provide rapid results. Biological validation is unsuitable to be performed in recuperating joeys due to ethical considerations, hence, we compared the results from biologically validated R4866 assay with the commercially available Arbor Assay cortisol kit. Thirty-four faecal samples were collected, processed and analysed for faecal cortisol metabolites (FCM) using both, R4866 assay and Arbor Assay kit. The joeys presented a suite of clinical conditions which provided the natural variation in stress response for comparing the assay sensitivity and range. The results indicated that there were no significant differences between the FCM values measured by both the assays. Furthermore, the Bland-Altman plot indicated a very strong agreement between the FCM concentrations measured by the two assays. This study is only a step towards recommending the routine use of commercial kit in clinical settings with basic resources, for rapid quantification of stress in koala patients. It is crucial for future studies to perform laboratory validation procedures to confirm the efficacy of the commercial kit before practical use for FCM monitoring in koalas.

Heat stress in horses: a literature review.

Healthy adult horses can balance accumulation and dissipation of body heat to maintain their body temperature between 37.5 and 38.5 °C, when they are in their thermoneutral zone (5 to 25 °C). However, under some circumstances, such as following strenuous exercise under hot, or hot and humid conditions, the accumulation of body heat exceeds dissipation and horses can suffer from heat stress. Prolonged or severe heat stress can lead to anhidrosis, heat stroke, or brain damage in the horse. To ameliorate the negative effects of high heat load in the body, early detection of heat stress and immediate human intervention is required to reduce the horse's elevated body temperature in a timely manner. Body temperature measurement and deviations from the normal range are used to detect heat stress. Rectal temperature is the most commonly used method to monitor body temperature in horses, but other body temperature monitoring technologies, percutaneous thermal sensing microchips or infrared thermometry, are currently being studied for routine monitoring of the body temperature of horses as a more practical alternative. When heat stress is detected, horses can be cooled down by cool water application, air movement over the horse (e.g., fans), or a combination of these. The early detection of heat stress and the use of the most effective cooling methods is important to improve the welfare of heat stressed horses.

Are physiological and behavioural responses to stressors displayed concordantly by wild urban rodents?

Understanding wild animal responses to stressors underpins effective wildlife management. In order for responses to stressors to be correctly interpreted, it is critical that measurements are taken on wild animals using minimally invasive techniques. Studies investigating wild animal responses to stressors often measure either a single physiological or behavioural variable, but whether such responses are comparable and concordant remains uncertain. We investigated this question in a pilot study that measured responses of wild-caught urban brown and black rats (Rattus norvegicus, Rattus rattus) to fur-based olfactory cues from a predator, the domestic cat (Felis catus); a novel herbivore, the koala (Phascolarctos cinereus); and a familiar herbivore and competitor, the common brushtail possum (Trichosurus vulpecula). Physiological responses, measured by assaying faecal glucocorticoid metabolites, were compared to behavioural responses observed via video recordings. We found that physiological and behavioural responses to stressors were expressed concordantly. There was no sizeable physiological response observed, and the behavioural response when considered across the night was negligible. However, the behavioural response to the predator and competitor cues changed across the observation period, with activity increasing with increasing hours of exposure. Our results indicate that responses of wild rodents to cues are nuanced, with stress responses modulated by behaviour changes that vary over time according to the severity of the perceived threat as animals gather further information. If the physiological response alone had been assessed, this moderated response may not have been evident, and in terms of wildlife management, vital information would have been lost.

Dose-response relationship of tryptophan with large neutral amino acids, and its impact on physiological responses in the chick model.

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 (T3) and thyroxine (T4). 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 < .0001, r2 = 0.819) and linear increases in 5-HT (P < .0001, r2 = 0.945), T3 (P = .0003, r2 = 0.403) and T4 (P < .0001, r2 = 0.937) levels. In conclusion, the results from the current study demonstrated that, although incremental levels of Trp/LNAA did not affect bird growth performance or fearfulness, it increased 5-HT, T3 and T4, and decreased CORT levels in a linear dose-dependent manner. Manipulation of Trp feeding levels could be applied to manage stressful conditions in birds.

Evaluation of physiological stress in Australian wildlife: Embracing pioneering and current knowledge as a guide to future research directions.

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.

Fecal glucocorticoid metabolite response of captive koalas (Phascolarctos cinereus) to visitor encounters.

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.

One size does not fit all: Monitoring faecal glucocorticoid metabolites in marsupials.

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 0.125-4.5days 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.

Does physiological response to disease incur cost to reproductive ecology in a sexually dichromatic amphibian species?

It is well known that the disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) has contributed to amphibian declines worldwide. The impact of Bd varies, with some species being more susceptible to infection than others. Recent evidence has shown that Bd can have sub-lethal effects, whereby increases in stress hormones have been associated with infection. Could this increased stress response, which is a physiological adaptation that provides an increased resilience against Bd infection, potentially be a trade-off with important life-history traits such as reproduction? We studied this question in adult male frogs of a non-declining species (Litoria wilcoxii). Frogs were sampled for (1) seasonal hormone (testosterone and corticosterone), color and disease profiles, (2) the relationship between disease infection status and hormone levels or dorsal color, (3) subclinical effects of Bd by investigating disease load and hormone level, and (4) reproductive and stress hormone relationships independent of disease. Testosterone levels and color score varied seasonally (throughout the spring/summer months) while corticosterone levels remained stable. Frogs with high Bd prevalence had significantly higher corticosterone levels and lower testosterone levels compared to uninfected frogs, and no differences in color were observed. There was a significant positive correlation between disease load and corticosterone levels, and a significant negative relationship between disease load and testosterone. Our field data provides novel evidence that increased physiological stress response associated with Bd infection in wild frogs, could suppress reproduction by down-regulating gonadal hormones in amphibians, however the impacts on reproductive output is yet to be established.

Sexual dimorphism in baseline urinary corticosterone metabolites and their association with body-condition indices in a peri-urban population of the common Asian toad (Duttaphrynus melanostictus).

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.

Repeated thermal stressor causes chronic elevation of baseline corticosterone and suppresses the physiological endocrine sensitivity to acute stressor in the cane toad (Rhinella marina).

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.

Non-invasive wool hormone assessment of Australian merino rams (Ovis aries): a pilot investigation of cortisol and testosterone.

Introduction: Non-invasive hormone assessment is growing in interest as producers and livestock researchers seek new methods to assess animal welfare. Non-invasive wool assessment offers long-term, historic reflections of hormone concentration at the scale of weeks and months - and are not limited by sampling stress - thus making wool an appropriate tissue for long-term hormone analysis. This pilot study quantified cortisol and testosterone concentrations of ram fleece and determined if there is a significant difference between segments of the sample staple, and whether there is a correlation between hormones. Cortisol is a glucocorticoid produced within the adrenal glands and secreted in anticipation of or in response to a stressor. Testosterone is an androgen mainly synthesised within the testes of males and responsible for several critical functions including regulation of muscle growth, libido and spermatogenesis. Methods: In our study, 70 topknot wool samples were collected from rams on a commercial stud property in Dirranbandi, Queensland, Australia. Of these animals, 12 samples were selected at random to undergo cortisol and testosterone quantification. In the laboratory, a single, intact staple was isolated from the total sample, divided into 10 mm segments and prepared for their respective (cortisol or testosterone) immunoassays. Results: No significant difference (p > 0.05) was found between wool segments for either cortisol or testosterone, however, statistical differences (p < 0.05) were found between individuals for both hormones. A strong positive correlation (R2 = 0.9173, p < 0.05) was found between wool cortisol and testosterone concentrations. Discussion: In summary, this study reveals the major future possibilities for non-invasive wool hormone assessment in merino rams.

Loss of a grooming enrichment impacts physical, behavioural, and physiological measures of welfare in grazing beef cattle.

Pasture-based beef cattle are raised in a range of production environments. Some paddocks may contain trees and other objects that allow for grooming, hence being naturally enriching, whilst others may be barren without these opportunities. Additionally, it is not uncommon for cattle to move between these enriched and barren environments as part of routine management. While the benefits of enrichment are well studied, how this 'enrichment loss' impacts cattle welfare as access to stimuli is removed is unknown. This trial assessed the impacts of the loss of an enriching object (grooming brush) on grazing beef cattle welfare and production characteristics. When grooming brush access was blocked, cattle became dirtier, showed reduced average daily gain, and had elevated faecal cortisol metabolites, although this varied according to the degree of initial individual brush use. Additionally, allogrooming and grooming on other objects were reduced when access to the brush was returned, potentially indicating a rebound effect. These results demonstrate that the loss of adequate grooming objects can impair the overall welfare of grazing cattle; however, further work is needed to determine exactly which natural or artificial objects provide adequate grooming opportunities.

Changes in serum and urinary corticosterone and testosterone during short-term capture and handling in the cane toad (Rhinella marina).

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 (0h) with a second urine sample taken at 0.5h and hourly between 1 and 8h. 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 0.5h, reaching the highest level between 6 and 8h. The mean urinary CORT concentration increased with a lag-time of 1h and continued to increase up to 8h. The mean level of serum T decreased between 0 and 7h and increased between 7 and 8h. Mean urinary T concentration decreased with a lag-time of 0.5h. Urinary T levels did not change between 4 and 8h. Mean serum T levels reached 50% of the original 0h value at 1h while mean serum CORT levels reached 200% of the original 0h value within 0.5h. Mean urinary T levels reached 50% of the original 0h value within 3h while mean urinary CORT levels reached 200% of the original 0h value within 3h. 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.

Non-invasive monitoring of glucocorticoid physiology within highland and lowland populations of native Australian Great Barred Frog (Mixophyes fasciolatus).

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 1-10days post injection. Urine samples and body condition measurements were collected from lowland (60m) and highland (660m and 790m) 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.

Non-invasive evaluation of physiological stress in an iconic Australian marsupial: the Koala (Phascolarctos cinereus).

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.

Faecal cortisol metabolites in Bengal (Panthera tigris tigris) and Sumatran tigers (Panthera tigris sumatrae).

The tiger (Panthera tigris) faces a great risk of extinction as its wild numbers have plummeted due to poaching and habitat destruction so ex-situ conservation programs are becoming ever more necessary. Reliable non-invasive biomarkers of the stress hormone (cortisol) are necessary for assessing the health and welfare of tigers in captivity. To our knowledge, non-invasive stress endocrinology methods have not been tested as widely in tigers. The first aim of this study was to describe and validate a faecal cortisol metabolite enzyme-immmunoassay (FCM EIA) for two tiger sub-species, the Bengal tiger (Panthera tigris tigris) and the Sumatran tiger (Panthera tigris sumatrae). Individual tigers (n=22) were studied in two large Zoos in Queensland, Australia (Dreamworld Theme Park and Australia Zoo). Fresh faecal samples (<12 h old) were collected each morning from both Zoos over a study period of 21 days. Biological validation was conducted separately by collecting feces 5 days before and 5 days after blood was taken from four male and five female tigers. Results showed that mean FCM levels increased by 138% and 285% in the male and female tigers within 1 day after bloods were taken, returning to baseline in 5 days. Laboratory validations of the FCM EIA were done using an extraction efficiency test and parallelism. Results showed >89% recovery of the cortisol standard that was added to tiger faecal extract. We also obtained parallel displacement of the serially diluted cortisol standard against serially diluted tiger faecal extract. Our second aim was to determine whether the FCM levels were significantly different between tiger sub-species and sex. Results showed no significant difference in mean FCM levels between the Bengal and Sumatran tiger sub-species. Mean levels of FCMs were significantly higher in females than in male tigers. Those male and female tigers with reported health issues during the study period expressed higher FCM levels than the reportedly healthy tigers. Interestingly, those tigers that took part in some activity (such as walks, photos, presentations and guest feeds) expressed moderately higher FCM levels at Dreamworld and lower FCM levels at Australia Zoo in comparison to those tigers that did not take part in such activities. These results indicate potential habituation in some tigers for routine activity through specialized training and pre-conditioning. In conclusion, the FCM EIA described in this study provides a reliable non-invasive method for evaluating the stress status of tigers in Zoos.

Are baseline and short-term corticosterone stress responses in free-living amphibians repeatable?

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 5min then transferred to plastic bags (constituting a mild stressor), and urine samples were collected hourly over 8h in the field. The toads were resampled for baseline (0h) urine corticosterone with hourly urine sampling over 8h (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 8h 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 6h. Baseline urinary corticosterone metabolite concentration of the male toads was highly repeatable (r=0.877) together with high statistical repeatabilities for 3h (r=0.695), 6h (r=0.428) and 8h (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.

Repeatability of baseline corticosterone and short-term corticosterone stress responses, and their correlation with testosterone and body condition in a terrestrial breeding anuran (Platymantis vitiana).

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, p < 0.001) 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, p < 0.001). 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.