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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.

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.

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

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).

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

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

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