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Non-invasive evaluation of physiological stress hormone responses in a captive population of the Greater Bilby (Macrotis lagotis)
Abstract
The Greater Bilby (Macrotis lagotis) in as IUCN listed Vulnerable (C1 ver 3.1) marsupial species in Australia. Proper monitoring of physiological stress hormone responses in the individuals involved in captive breeding programs is important. In this study, we used a fecal cortisol enzyme-immunoassay (EIA) and validated it to measure stress hormone (cortisol) metabolites in adult male and female bilbies (n = 7) in captivity at the Dreamworld Theme park, Queensland. The fecal cortisol EIA was validated via parallelism and recovery of exogenous cortisol added to pooled fecal extracts (greater than 99 % recovery). Female bilbies had higher mean baseline cortisol concentrations than male bilbies however there was no relationship with bilby age. There was high day-to-day variation in fecal cortisol metabolites, larger in females than males. Cortisol levels for most individuals varied widely through time with numerous peaks and troughs following long-term stressors (illnesses, injury and reproductive issues) and known short-term stressors such as use in shows at Dreamworld or public displays in local schools, manual restraint and short-term veterinary procedures (e.g. general anaesthesia). Overall, the higher mean baseline cortisol metabolite levels of long-term stressed individuals appear to be, at least partially, related to their higher response to short-term stressors. This suggests an interaction between responses to short and long-term stressors, which have been explained in relation to habituation and/or facilitation of long-term stressors. In future, non-invasive fecal monitoring of stress hormones could be applied within wild populations to quantify the impact of survey programs on wild populations.
... Recent years, various methods have been developed to quantify the stress levels in animals by measuring the glucocorticoid metabolites present in body secretions and outgrowth like blood, urine, saliva, faeces, hair (Glenk et al., 2014;Narayan et al., 2012;Narayan et al., 2011;Peric et al., 2013). All the methods involved in the quantification of cortisol levels have their advantages and drawbacks. ...
... Another non-invasive method, the measurement of faecal cortisol metabolites (FCM), can be an effective way to quantify stress levels, as it requires no interaction with the animals, striking out the possibility of causing undue stress to animals due to human interactions (Narayan et al., 2012;Palme, 2019). In this study, we quantified faecal glucocorticoid profiles of three Australian marsupial species which belong to the families Potoroidia and Thylacomyidae Stress levels can act as an indicator of the welfare status of the animals, and quantifying stress levels can help researchers gain insights on the effectiveness of the management practices in a noninvasive manner (Narayan et al., 2012). ...
... Another non-invasive method, the measurement of faecal cortisol metabolites (FCM), can be an effective way to quantify stress levels, as it requires no interaction with the animals, striking out the possibility of causing undue stress to animals due to human interactions (Narayan et al., 2012;Palme, 2019). In this study, we quantified faecal glucocorticoid profiles of three Australian marsupial species which belong to the families Potoroidia and Thylacomyidae Stress levels can act as an indicator of the welfare status of the animals, and quantifying stress levels can help researchers gain insights on the effectiveness of the management practices in a noninvasive manner (Narayan et al., 2012). In this study, we aim to determine the variations in FCM levels of marsupials through time and present absolute levels across time, sex, age, groupings and housing arrangement for each species. ...
Australia has the highest small-mammal extinction rate in the world, and there are ample of reasons that have led to the higher extinction rates such as introduction of feral species, wildfires, climate change and uneven population distribution. All these factors instil a dire need to protect the species in captive environment. Stress has a major role to play in maintaining an adequate welfare conditions for wildlife, hence quantification of the stress levels in captive animals help us understand the effectiveness of the methods used to house the animals. In this study, we assessed the stress levels of captive marsupials namely Rufous bettongs (Aepyprymnus rufescens) (n=9), Long-nosed potoroo (Potorous tridactylus) (n=4) and Greater bilby (Macrotis lagotis) (n=5) by measuring the faecal cortisol metabolites using the Enzyme-Immuno assay (EIA) (R4866). We carried out a laboratory validation of the EIA for the Rufous bettongs and long-nosed potoroo by demonstrating parallelism between the pooled faecal samples and the standard curve. We also used the iterative baseline approach to calculate the baseline levels and detect peak concentrations of FCM in all the studied species. No significant differences were found in the FCM and baseline levels in the potoroos. Results highlighted a significant difference (p < 0.05) in the individual FCM and baseline levels of the bettongs. Additionally, significant differences (p < 0.05) were found in the FCM and baseline levels between male and female bettongs. In case of bilbies, the FCM and baseline levels showed no significant differences, however the male and female baseline levels were significantly different (p < 0.05). Our study provides the baseline and peak FCM concentrations in Long-nosed potoroos and Rufous bettongs under captive management. Future studies can use the iterative baseline approach and validated EIA to detect any possible variations in the FCM concentration and baseline values in captive marsupials created due to factors like sex, grouping, and housing conditions.
... Stress can cause an imbalance in the physiological equilibrium of an animal, whereby resources are redirected from normal biological functions in order to cope with the negative stimuli and initiate the 'fight or flight' response (Martin et al. 2011;Dantzer et al. 2014). Short-term, acute stress is a normal aspect of survival in the wild and each animal has its own basal corticosterone level; however, a more extreme acute stress response, or long-term exposure to negative stimuli resulting in chronic stress, can be detrimental to the animal and its recovery (Narayan et al. 2012;Narayan et al. 2018). Chronic stress has the ability to compromise some biological systems, leading to fitness consequences such as immunosuppression and inappetence which can inhibit recovery progress (Dickens et al. 2010;Baxter-Gilbert et al. 2014;Refsnider et al. 2015). ...
... Chronic stress has the ability to compromise some biological systems, leading to fitness consequences such as immunosuppression and inappetence which can inhibit recovery progress (Dickens et al. 2010;Baxter-Gilbert et al. 2014;Refsnider et al. 2015). Continued biological response to stress can also cause impaired reproductive function and hypercortisolism, both of which can have a negative impact on post-release survival (Carlstead & Shepherson 2000;Pryce et al. 2002;Soltis et al. 2003;Narayan et al. 2012). ...
... The major glucocorticoid or stress hormone in turtles (like all herpetofauna) is corticosterone (Case et al. 2005). Non-invasive methods of measuring the stress hormone corticosterone, including the use of faecal corticosterone metabolites (FCM), are becoming more prominent in the assessment of stress (Dehnhard et al. 2001;Weingrill et al. 2004;Franceschini et al. 2008;Narayan et al. 2012;Shepherdson et al. 2013;Watson et al. 2013;Narayan et al. 2018). ...
... • Long term stress is likely to be manifested in stereotypic behaviour (3,4,5) . It is hypothesised that repeating pathways (stereotypic locomotion) stems from the inability to hunt, inability to escape from the enclosure, or frustration born of an insufficient range area (6,7) . ...
... • The use of faecal corticosterone metabolites (FCM) is becoming more prominent in the assessment of physiological stress (5,15) . The combination of non-invasive indicators of stress, including behavioural assays and analysis of FCMs, could aid the early recognition of stress and lower the potential for development of chronic stress. ...
... • Stereotypies are commonly described as invariant, repetitive and abnormal (3,4,5,6,7) . The behaviour documented here has been quantitatively and objectively found to be in line with such definitions using the method of zone sequence analysis. ...
Populations of marine turtle are in global decline and 5 of the 6 species are threatened with extinction. • Rehabilitation facilities seek to rescue, rehabilitate and release recovered individuals, but recovery times can vary and some may require permanent captive care and never return to the wild. • Chronic stress associated with captivity and poor health status may cause some biological systems to become compromised, leading to fitness consequences such as immunosuppression, inappetence, impaired reproductive function and hypercortisolism; all of which can have a negative impact on recovery and post-release survival (1,2). • Long term stress is likely to be manifested in stereotypic behaviour (3,4,5). It is hypothesised that repeating pathways (stereotypic locomotion) stems from the inability to hunt, inability to escape from the enclosure, or frustration born of an insufficient range area (6,7). • Green turtles inhabit large home ranges and undertake migratory movements over large distances = wide variation and sensory stimulation within their environment. Confinement in smaller and unnaturalistic enclosures may therefore be a contributing factor to the development of stereotypical behaviour and locomotion (8,9,10,11). • The use of faecal corticosterone metabolites (FCM) is becoming more prominent in the assessment of physiological stress (5,15). The combination of non-invasive indicators of stress, including behavioural assays and analysis of FCMs, could aid the early recognition of stress and lower the potential for development of chronic stress. Therefore supporting the success of rehabilitation efforts and other conservation interventions. • Trialling the use of zone sequence analysis to identify whether repetitive pathways (stereotypic locomotion) are present within a small group of captive green turtles. • For the first time in a marine turtle species, faecal corticosterone metabolites (FCM) was trialled as a biological marker of stress in a non-invasive alternative to the classical use of blood sampling. • It is hypothesised that the rescued green turtles will exhibit stereotypic locomotion and record detectable levels of baseline FCMs.
... Few studies have investigated the stress physiology of marsupials in relationship to captivity, but stressors identified to date include social isolation (McKenzie and Deane, 2005), social status (Stoddart and Bradley, 1991) and capture and handling . Health-related concerns, such as arthritis, dental disease, upper respiratory tract infections and associated veterinary procedures have also been found to be associated with increased mean FGM concentrations in captive bilbies (Macrotis lagotis; Narayan et al., 2012). ...
... We acknowledge the significant logistical challenges associated with studying the stress response in Australian marsupials, many of which are cryptic, nocturnal and threatened, including resource limitations (for example, time-and labour-intensive trapping and sample collection) and the only recent development of validated assays. Captive populations may provide feasible candidates for assay validation (Hogan et al., 2011;Narayan et al., 2012). Once key stressors can be identified, measures to manage stress can be trialled, and if successful, incorporated into management plans. ...
... Individual differences in the stress response of Australian marsupial species have been explored by Narayan et al. (2012). Individual bilbies reacted differently to short-term stressors depending on the degree to which they had habituated to long-term stressors and their health condition . ...
Many Australian marsupials are threatened species. To effectively manage in situ and ex situ populations, it is important to understand how marsupials respond to threats. Stress physiology (the study of animals’ response to challenging stimuli), a key approach in conservation physiology, can be used to characterise the physiological response of wildlife to threats. We reviewed the literature on the measurement of glucocorticoid (GC), endocrine indicators of stress, to understand the stress response to conservation relevant stressors in Australian marsupials and identified 29 studies. These studies employed a range of methods to measure GC, with faecal glucocorticoid metabolite (FGM) enzyme immunoassay (EIA) being the most common method. The main stressors considered in studies of marsupials were capture and handling. To date, the benefits of stress physiology have yet to be fully harnessed in marsupial conservation. Despite a theoretical base dating back to the 1960s, GCs have only been used to understand how 23 of the 142 extant species of Australian marsupial respond to stressors. These studies include just 7 of the 60 marsupial species of conservation concern (IUCN Near Threatened to Critically Endangered). Furthermore, the fitness consequences of stress for Australian marsupials are rarely examined. Individual and species differences in the physiological stress response also require further investigation because significant species specific variations in GC levels to stressors can shed light on why some individuals or species are more vulnerable to stress factors while others appear more resilient. This review summarises trends, knowledge gaps and future research directions for stress physiology research in Australian marsupial conservation.
... The majority of recent studies have employed broad-spectrum (polyclonal antibody based) enzymeimmunoassay (EIA) or radio-immunoassay (RIA) to measure the metabolites of biologically active (''free cortisol'') in mammalian feces (Cook, 2012). For examples; felids in general (Brown, 2006), Grevy's zebra (Equus grevyi) (Franceschini et al., 2008), spotted hyena (Crocuta crocuta) (Goymann et al., 1999), African elephant (Loxodonta sp.) (Ganswindt et al., 2003), southern white rhinoceros (Ceratotherium simum simum) (Metrione and Harder, 2011), greater bilby (Macrotis lagotis) (Narayan et al., 2012), koala, phascolarctos cinereus) , mountain gorilla (Gorilla beringei beringei) (Nizeyi et al., 2011), cats (Felis catus) and dogs (Canis lupus familiaris) (Schatz and Palme, 2001), cheetah (Acinonyx jubatus) (Terio et al., 1999), clouded leopard (Neofelis nebulosa) (Wielebnowski et al., 2002) and carnivores in general (Young et al., 2004). These studies on diverse mammals highlight that faecal analysis of cortisol metabolites is a valuable method for evaluating status of the HPA-axis under basal conditions as well as useful for quantifying cortisol response to potential acute stimuli (such as injuries and activities), which in turn, can aid in optimizing environmental and management conditions of captive wildlife. ...
... Cross reactivity of the R4866 anti-cortisol antiserum is reported as 100% with cortisol and less than 10% with other steroids tested (Munro and Stabenfeldt, 1985). The same reagents were validated recently for assessing FCM for diverse mammals (Muehlenbein et al., 2012;Narayan et al., 2012;2013). Samples were assayed on Nunc Maxi-Sorp plates (96 wells) and in duplicate. ...
... Detailed information relating to each individual's daily activities must be kept and documented; such as recent research conducted on the captive population of Greater Bilbies (M. lagotis) at the Dreamworld by Narayan et al. (2012). This design would allow for the identification of specific stressful stimuli for tigers, and to evaluate the extent to which these events or activities cause increases in FCM in individuals. ...
... Under 363 chronic stress, a higher level of plasma cortisol is expected due to dysregulation of the HPA axis and 364 prolonged elevation of cortisol(Sapolsky, 1990). This is also supported byNarayan et al. (2012), who 365 found a higher cortisol concentration in koalas suffering from chronic stress and ongoing health 366 issues. Therefore, although insignificant, our result showed that all three biomarkers have the 367 potential to detect chronic stressors. ...
... 386illness associated with oxalate nephrosis and the expected higher cortisol concentration under chronic 387 conditions(Narayan, et al., 2012, Sapolsky, 1990. The use of cortisol:DHEAS and cortisol:DHT to detect chronic stress is not validated in this study due to the insignificant differences. ...
Koalas (Phascolarctos cinereus) are one of the most iconic marsupial species endemic to Australia. However, their population is declining due to threats including habitat loss, disease, dog attacks, and vehicle collisions. These threats also serve as acute or chronic stressors that impact koala welfare and conservation. Cortisol is widely used as a biomarker to study stress in koalas. However, plasma cortisol concentration is less studied due to its limited ability to assess chronic stress and welfare concerns. Dehydroepiandrosterone sulphate (DHEAS) and dihydrotestosterone (DHT) are biomarkers that could potentially detect chronic stress due to their antagonising and inhibitory effects on cortisol. In this study, we used plasma cortisol and the ratio of DHEAS and DHT to cortisol to assess stress in rescued koalas (n = 10) admitted to RSPCA Queensland. Although no significant differences were found between koalas across all biomarkers and the ratios failed to detect chronic stressors, similar trends were found consistently, suggesting the potential use of the biomarkers to assess stress. Across all biomarkers, the highest medians were found in koalas with Chlamydia-related reproductive disease and oxalate nephrosis and the lowest medians were found in koalas with Chlamydia-related conjunctivitis. Higher medians were also found consistently in females (n = 3) and adult koalas. In addition, insignificant negative correlations were found across all biomarkers between age, weight, and body conditioning scores, except for the positive correlation between weight and cortisol and cortisol:DHT. Overall, the consistency of trends and the insignificant differences found across biomarkers in our study suggested that using a single biomarker to assess chronic stress is insufficient, especially for hospital-based studies limited by sample population. Thus, this pilot study provides first step towards developing a koala-specific allostatic load index based on multiple stress biomarkers to understand chronic stress in rescued koalas.
Lay summary
Stress in koalas can be challenging for their welfare and conservation. In this study, we tested plasma glucocorticoids and their ratios as biomarkers of acute and chronic stress. Our finding showed ratios of DHEAS and DHT to cortisol are comparable across stress parameters and animal demographic characteristics. This study serves as a foundational framework for developing a stress index based on multiple biomarkers that could be useful tool for koala welfare.
... Under 363 chronic stress, a higher level of plasma cortisol is expected due to dysregulation of the HPA axis and 364 prolonged elevation of cortisol(Sapolsky, 1990). This is also supported byNarayan et al. (2012), who 365 found a higher cortisol concentration in koalas suffering from chronic stress and ongoing health 366 issues. Therefore, although insignificant, our result showed that all three biomarkers have the 367 potential to detect chronic stressors. ...
... 386illness associated with oxalate nephrosis and the expected higher cortisol concentration under chronic 387 conditions(Narayan, et al., 2012, Sapolsky, 1990. The use of cortisol:DHEAS and cortisol:DHT to detect chronic stress is not validated in this study due to the insignificant differences. ...
Koalas (Phascolarctos cinereus) are one of the most iconic marsupial species endemic to Australia. However, their population is declining due to threats including habitat loss, disease, dog attacks, and vehicle collisions. These threats also serve as acute or chronic stressors that impact koala welfare and conservation. Cortisol is widely used as a biomarker to study stress in koalas. However, plasma cortisol concentration is less studied due to its limited ability to assess chronic stress and welfare concerns. Dehydroepiandrosterone sulphate (DHEAS) and dihydrotestosterone (DHT) are biomarkers that could potentially detect chronic stress due to their antagonising and inhibitory effects on cortisol. In this study, we used plasma cortisol and the ratio of DHEAS and DHT to cortisol to assess stress in rescued koalas (n = 10) admitted to RSPCA Queensland. Although no significant differences were found between koalas across all biomarkers and the ratios failed to detect chronic stressors, similar trends were found consistently, suggesting the potential use of the biomarkers to assess stress. Across all biomarkers, the highest medians were found in koalas with Chlamydia-related reproductive disease and oxalate nephrosis and the lowest medians were found in koalas with Chlamydia-related conjunctivitis. Higher medians were also found consistently in females (n = 3) and adult koalas. In addition, insignificant negative correlations were found across all biomarkers between age, weight, and body conditioning scores, except for the positive correlation between weight and cortisol and cortisol:DHT. Overall, the consistency of trends and the insignificant differences found across biomarkers in our study suggested that using a single biomarker to assess chronic stress is insufficient, especially for hospital-based studies limited by sample population. Thus, this pilot study provides first step towards developing a koala-specific allostatic load index based on multiple stress biomarkers to understand chronic stress in rescued koalas.
... In summary, faecal samples (0.2 g dry weight) were lyophilized (freeze-dried) and extraction carried out using 90% ethanol and heat treatment (80°C for 10 min). Extracts were assayed for FCM by EIA using a polyclonal anti-cortisol antiserum R4866 protocol (Narayan et al., 2012;Hing et al., 2016). The R4866 anti-cortisol antiserum has been reported to cross react 100% with cortisol metabolites and less than 10% with other steroids (K. ...
... Females had higher mean FCM than males, a pattern also reported in other Australian marsupials including the bilby (Macrotis lagotis) (Narayan et al., 2012), koala (Narayan (Dowle, Webster & Deane, 2012). These results may be a reflection of sex differences in glucocorticoid metabolism (Lane, 2006) and could suggest that male and female woylies have different physiological sensitivities to stressors, a pattern that has been observed in other species (Handa et al., 1994). ...
Faecal glucocorticoid metabolites are minimally invasive stress physiology indices that can be used to understand how animals respond to physical and/or psychological challenges (stressors) and inform how to optimize conservation management in view of these stressors. We investigated contextual biological, environmental and parasitological factors influencing variation in baseline faecal cortisol metabolite (FCM) concentration in a critically endangered marsupial, the woylie (syn. brush‐tailed bettong, Bettongia penicillata). Woylies have undergone a rapid and significant population decline, with environmental stressors exacerbating disease suggested to contribute to these ongoing declines. We conducted a longitudinal field study of 15 adult woylies (9 females, 6 males) in a captive, naturalistic facility. FCM concentration in faecal samples (n = 269) collected monthly over 20 months was quantified by enzyme immunoassay in parallel with measures of body condition, sex, season, female reproductive status and the presence of endoparasites and ectoparasites. Linear mixed effect modelling revealed a significant effect of season, sex, body condition index and nematode parasite status on FCM. Overall, mean FCM was lowest in summer and highest in autumn and winter, and females had higher mean FCM than males. There was a significant but weak negative association between body condition and FCM. When woylies were shedding oxyurid nematode eggs they had higher mean FCM compared to when they were not shedding. In future, knowledge of factors that influence FCM fluctuations in woylies may be considered when carrying out potentially stressful conservation interventions that may influence the future survival of this unique and threatened species.
... Extraction of FCMs followed methods previously described by Wielebnowski et al. (2002a) for the clouded leopard (Neofelis nebulosa) and used recently in numerous studies by our research group (Narayan et al., 2012(Narayan et al., , 2013a(Narayan et al., , b, 2014Evans et al., 2013). Briefly, all faecal samples were dehydrated in a lyophilizer, then sieved and pulverized. ...
... Laboratory protocols followed those based on established work on the greater bilby (Macrotis lagotis) described by Narayan et al. (2012), the koala (Phascolactus cinereus) described by Narayan et al. (2013b) and, most recently, on the tiger (Narayan et al., 2013a). This procedure involved the quantification of the FCM concentration of each sample through duplicate assays performed in 96-well Nunc MaxiSorp™ plates. ...
Glucocorticoid quantification using non-invasive methods provides a powerful tool for assessing the health and welfare of wildlife in zoo-based programmes. In this study, we provide baseline data on faecal-based glucocorticoid (cortisol) monitoring of Sumatran tigers (Panthera tigris ssp. sumatrae) managed at the Melbourne Zoo in Victoria, Australia. We sampled five tigers daily for 60 days. Faecal cortisol metabolites (FCMs) in tiger faecal extracts were quantified using enzyme immunoassays that were successfully validated using parallelism and accuracy recovery checks. Two female tigers had significantly higher mean FCM levels than the two males and another female, suggesting that females may have higher FCM levels. A significant elevation was noted in the FCM levels for one female 2 days after she was darted and anaesthetized; however, the FCM levels returned to baseline levels within 3 days after the event. Comparative analysis of FCM levels of tigers sampled at Melbourne Zoo with tigers sampled earlier at two other Australian Zoos (Dreamworld Themepark and Australia Zoo) showed that FCM levels varied between zoos. Differences in the enclosure characteristics, timing of sampling, size and composition of groupings and training procedures could all contribute to this variation. Overall, we recommend the use of non-invasive sampling for the assessment of adrenocortical activity of felids managed in zoos in Australia and internationally in order to improve the welfare of these charismatic big cats.
... Hogan and colleagues (Hogan et al. 2011) validated FCM EIA in the captive wombats (Lasiorhinus latifrons) using ACTH challenge and the study also demonstrated the FCMs responses to regular and forced handling in the wombats in captivity. Faecal glucocorticoid metabolite enzymeimmunoassays were also validated in the greater bilby (Macrotis lagotis) and the techniques were used to evaluate physiological stress in semi-free ranging populations (Narayan et al., 2012;). Narayan et al. (2012) demonstrated the effects of moderate stressors in a captive environment such as illnesses, injury or reproductive issues which resulted in greater physiological stress responses, in comparison to the participation of bilbies in school visits and public displays. ...
... allenge and the study also demonstrated the FCMs responses to regular and forced handling in the wombats in captivity. Faecal glucocorticoid metabolite enzymeimmunoassays were also validated in the greater bilby (Macrotis lagotis) and the techniques were used to evaluate physiological stress in semi-free ranging populations (Narayan et al., 2012;). Narayan et al. (2012) demonstrated the effects of moderate stressors in a captive environment such as illnesses, injury or reproductive issues which resulted in greater physiological stress responses, in comparison to the participation of bilbies in school visits and public displays. applied this FCM EIA to study physiological stress in sub-populations of gr ...
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.
... Extraction of FCMs followed methods previously described by Wielebnowski et al. (2002a) for the clouded leopard (Neofelis nebulosa) and used recently in numerous studies by our research group (Narayan et al., 2012(Narayan et al., , 2013a(Narayan et al., , b, 2014Evans et al., 2013). Briefly, all faecal samples were dehydrated in a lyophilizer, then sieved and pulverized. ...
... Laboratory protocols followed those based on established work on the greater bilby (Macrotis lagotis) described by Narayan et al. (2012), the koala (Phascolactus cinereus) described by Narayan et al. (2013b) and, most recently, on the tiger (Narayan et al., 2013a). This procedure involved the quantification of the FCM concentration of each sample through duplicate assays performed in 96-well Nunc MaxiSorp™ plates. ...
Glucocorticoid quantification using non-invasive methods provides a powerful tool for assessing the health and welfare of wildlife in zoo-based programmes. In this study, we provide baseline data on faecal-based glucocorticoid (cortisol) monitoring of Sumatran tigers (Panthera tigris ssp. sumatrae) managed at the Melbourne Zoo in Victoria, Australia. We sampled five tigers daily for 60 days. Faecal cortisol metabolites (FCMs) in tiger faecal extracts were quantified using enzyme immunoassays that were successfully validated using parallelism and accuracy recovery checks. Two female tigers had significantly higher mean FCM levels than the two males and another female, suggesting that females may have higher FCM levels. A significant
elevation was noted in the FCM levels for one female 2 days after she was darted and anaesthetized; however, the FCM levels returned to baseline levels within 3 days after the event. Comparative analysis of FCM levels of tigers sampled at Melbourne Zoo with tigers sampled earlier at two other Australian Zoos (Dreamworld Themepark and Australia Zoo) showed that FCM levels varied between zoos. Differences in the enclosure characteristics, timing of sampling, size and composition of groupings and training procedures could all contribute to this variation. Overall, we recommend the use of non-invasive sampling for the assessment of adrenocortical activity of felids managed in zoos in Australia and internationally in order to improve the welfare of these charismatic big cats.
... This major contraction in range is primarily attributed to predation from introduced species (e.g., red fox [Vulpes vulpes] and feral cats [Felis catus]), habitat destruction through agriculture and competition with introduced herbivores (Moseby et al. 2009). Whilst information on the bilby's reproductive biology, anatomy and behaviour are available, literature on its stress physiology is only from single captive population (Narayan et al. 2012). There is no information available on FCM levels in semi-wild populations of this marsupial. ...
... We analysed bilby fecal extracts for concentrations of FCM using enzyme immunoassay (EIA) previously described for other mammals (Wielebnowski et al. 2002;Millspaugh and Washburn 2004), and recently used for a captive subpopulation of the greater bilby (Narayan et al. 2012;Evans et al. 2013). Laboratory validation was done using accuracyrecovery checks and parallelism. ...
Rapid and reliable physiological evaluation of stress is necessary for understanding the potential impacts of
environmental changes on managed populations of threatened mammals. In situ populations of Australia’s iconic marsupial, the greater bilby (Macrotis lagotis), are nearing extinction due to the impacts of competition and predation by feral animals and unpredictable climatic events (summer heat waves). In this study, we focussed our aim to identify a non-invasive method to measure adrenal activity in the species and also to
identify potential factors that should be considered when comparing physiological stress in semi-free ranging populations of the species. We validated an enzyme immunoassay (EIA) for detecting fecal cortisol metabolites (FCM) from fresh fecal pellets taken from bilbies within four captive sites and two semi-free ranging populations around Queensland and New South Wales, Australia. Our FCM EIA successfully
detected the ‘raise and fall’ pattern of FCM levels within 3 days of exogenous adrenocorticotropic hormone (ACTH) challenge. Mean FCM levels differed significantly between the captive sites and between sexes. All male bilbies grouped outdoor in captivity expressed the highest mean FCM level in comparison to all captive males that were housed individually or as groups indoors. Also, semi-free ranging bilbies expressed higher mean FCM levels than the captive bilbies. Overall, our study successfully validated a non-invasive tool
for monitoring physiological stress in the greater bilby. In the future, it will be worthwhile to consider factors such as housing conditions, sex and location when comparing the adrenal sensitivity to environmental changes, to help evaluate the success of management interventions (such as predator free enclosures) and support the survival of the species.
... Samples were analysed immediately after freezing so that the length of time spent in the freezer 138 was equal between treatment days. FCMs were then extracted from bilby fecal samples as 139 previously described for other mammals (Millspaugh and Washburn 2004; Wielebnowski, Fletchall 140 et al. 2002) and validated recently for the greater bilby (Narayan, Hero et al. 2012). Briefly, 141 samples were freeze-dried, ground, and 0.2 g of well-mixed fecal powder was boiled in 5 mL 142 aqueous ethanol (90% vol) for 15 minutes. ...
... Intra-(within) and inter-(between) assay coefficients of variation (CV) were 2.3 155 % and 6.4 % for the high-binding internal control and 1.9 % and 14.3 % for the low-binding 156 internal control, respectively (n = 15 plates run in total). Detailed protocols for the FCM EIA has 157 been described in our earlier study (Narayan, Hero et al. 2012). 158 159 ...
Natural weathering conditions can influence faecal cortisol metabolite (FCM) measurements in wildlife if fresh faeces cannot be collected immediately following defaecation. In this study, we evaluated this issue in a threatened Australian marsupial, the greater bilby (Macrotis lagotis). Fresh (<12 h since defaecation) faecal samples (n = 19 pellets per bilby) were collected one morning from seven adult bilbies kept in captivity. One control faecal sample (Day 1) from each bilby was immediately frozen. The remaining faecal pellets were randomly positioned outdoors. Subsequently, we froze one faecal pellet every 24 h for 19 days. FCM levels in bilby faeces were quantified using an enzyme-immunoassay. Mean FCM levels showed variation (daily mean coefficients of variation [CV %]) of 56.83–171.65% over 19 days. Overall, FCM levels were affected by exposure time; however, multiple comparisons showed that no significant change in FCM occurred after environmental exposure (no significant difference in meanFCMbetween control (Day 1) with any of the exposure days (Days 2–19). Individuals and sex also affected FCM levels. We found no correlation between mean daily CVs with daily minimum–maximum temperatures or rainfall. Our results indicate that FCM in bilby faeces is fairly stable to long-term environmental exposure (19 days). In future, freshly excreted bilby faeces (where the sample maintains a distinct odour for 9–13 days) should be used to study FCM levels in wild bilbies.
... Land clearing, landscape fragmentation and impacts from associated built infrastructure, such as roads, pose major threats to the koala population. Since European settlement in 1788, Australia has lost 92% of Eucalyptus and Acacia (Narayan et al., 2012). Outside prime habitat loss, high physiological stress levels in wild koala populations are due to anthropogenic activities and can exacerbate disease and even death (Narayan, 2019). ...
Post-closure care of landfills is intended to reduce adverse impacts on the surrounding environment, and an important component of this process is capping. Landfills represent large areas of land that cannot be built on, and commercial use of landfill after closure has a lot of challenges. It is common for landfill sites to be green spaces initially and subsequently transformed into recreational parks or sports fields in highly populated regions. The emergence of phytocapping as a demonstrated and regulatory accepted alternative capping technology has led to a focus on ecological rehabilitation as a viable after-use for landfills due to their inherent characteristics that are similar to a natural system. Ecological rehabilitation is seldom considered a possible after-use for landfills, even though most landfills end up vegetated in the long term. Identification of ecological value present in a location is highly valuable to drive the decision-making process and, in turn, improve connectivity, ecological health, and biodiversity of a region. This study employs Koalas as an umbrella species to assess the ecological value and connectivity potential of a landfill. By using the Koala as a representative species, this research aims to evaluate the broader ecological implications and connectivity opportunities associated with a landfill site. This study uses buffer analysis and overlay analysis for preliminary identification of the potential for landfills in Koala habitat creation and connectivity. The majority of landfills in the South East Queensland (SEQ) region exhibit significant potential for ecological value through rehabilitation efforts. The findings demonstrate that landfill rehabilitation can be used to provide habitat for threatened species and improve the connectivity of bio-corridors. To maintain environmental sustainability, ecological conservation, and ecological connectedness, regulators and stakeholders are advised to place a stronger emphasis on landfill after-use. The results show why ecological rehabilitation should be given higher emphasis in the waste management industry and point to the missed opportunity for habitat creation, connectivity, expansion and increase of green space.
... In addition to acclimation to a new environment, injury and illness can induce a stress response (via an increase in cortisol). Narayan and colleagues [58] found increases in fecal cortisol metabolites in the greater bilby (Macrotis lagotis) after injury and illness. In threebanded armadillos (Tolypeutes matacus), veterinary procedures evoked stress responses which were demonstrated by an increase in fecal cortisol metabolites [59]. ...
The ocean sunfish (mola; Mola mola) is the heaviest bony fish in the world. This slow-moving fish often is injured by fishing boats that use drift gillnets attributing to its listing as Vulnerable by the IUCN. The Monterey Bay Aquarium (Monterey, CA, USA) has a program that brings in smaller molas from the ocean and acclimates them for an exhibit. When they grow too large for the million-gallon Open Seas exhibit, they are returned to Monterey Bay through a “reverse” acclimatization. Our overall goal was to use skin swabs to evaluate mola stress physiology to better understand the effects of this program. Our objectives were to validate this non-invasive method by taking opportunistic swabs throughout acclimatization and during stressful events. We swabbed each individual (n = 12) in three different body locations. Swabs were analyzed using a cortisol enzyme immunoassay. We averaged the three swabs and examined the absolute change of cortisol from the first taken upon handling to during treatments and the different acclimation stages. We considered elevated cortisol concentrations to be ≥1.5-fold higher than the first sample. Overall, mean (±SEM) cortisol varied among individuals (564.2 ± 191.5 pg/mL swab (range, 18.3–7012.0 pg/mL swab). The majority (four of six) of molas swabbed within the first week or month had elevated skin cortisol compared to their first sample. All seven molas that were being treated for an injury or illness had elevated skin cortisol (range, 1.7- to 127.6-fold higher) compared to their post-acclimation sample. This is the first step in validating the use of non-invasive skin swabs for glucocorticoid analysis in the mola. Further biochemical analysis is needed to determine the specific steroids that are being measured.
... The relationship between captivity and the physiological response to stressors, as evidenced by changing GC concentrations, has been studied in several species [13,[50][51][52], yet it remains under-investigated for wildlife that enter rehabilitation. As animals that enter captivity for rehabilitation are already compromised, the physiological impacts of captivity may vary compared to healthy captive animals, hence, captivity may further compound their already compromised fitness. ...
Volunteer wildlife rehabilitators rescue and rehabilitate thousands of native animals every year in Australia. However, there is little known about how exposure to novel stimuli during rehabilitation could affect the physiology of wildlife. We investigated this question in a species that commonly enters rehabilitation, the common brushtail possum (Trichosurus vulpecula). We evaluated five enzyme immunoassays (EIA) to determine the most suitable for measuring faecal glucocorticoid metabolites (FGM) as a proxy for evaluating the response of brushtail possums to potential stressors during rehabilitation. An adrenocorticotrophin hormone (ACTH) challenge was conducted on wild-caught possums to determine the best-performing EIA based on the successful detection of FGM peaks in at least two of three possums. While a number of assays met these criteria, the 11-oxoaetiocholanolone (abbreviation: 72a) EIA was selected as it had the largest amplitude of change in response to the ACTH challenge. This assay was then used to measure FGM concentrations in 20 possums during rehabilitation. There was high variation in baseline FGM concentrations and response to captivity between possums. Significant changes in FGM levels were detected in most possums during captivity, but were not reliably associated with potentially stressful events that were identified by rehabilitators. The probability of an FGM peak occurring within five days of a potentially stressful event was about 50%, regardless of the type of event. Our study has demonstrated that injured and orphaned possums show changes in FGMs during captivity and rehabilitation and has identified events that can induce a physiological response in some individuals. We recommend that research now focus on the relationship between these responses during rehabilitation and pre- and post-release survival.
... Moreover, to determine if these behavioural changes are reflective of underlying welfare changes in these species, it would be of value to explore a greater suite of welfare indices. The assessment of stress hormone concentration has been used in previous studies on other species [91][92][93][94][95][96] however, as far as the authors are aware, the non-invasive viable assessment of cortisol concentration is yet to be fully validated for these species [46]. This would be of significant value for future research to support better welfare outcomes for species housed in these settings. ...
This study investigated the behaviour of two Elasmobranch species; Southern fiddler ray (Trygonorrhina dumerilii, n = 1) and Port Jackson shark (Heterodontus portusjacksoni, n = 4) and two teleost species; moonlighter (Tilodon sexfasciatus, n = 1) and banded morwong (Cheilodactylus spectabilis, n = 1) living within a single enclosure. For this study, two treatments were compared, the original enclosure design, and then after the enclosure had been renovated to more closely represent the species natural habitats, with a raised front viewing glass to prevent visitor interaction. Behaviours such as resting, swimming and abnormal behaviours such as surface and perimeter swimming (elasmobranchs only) were recorded as well as location within the enclosure, for 10 days pre and 10 days post renovation. The Port Jackson sharks significantly reduced the performance of abnormal behaviours after renovation, and significantly increased the time spent near the exhibit front. The Southern fiddler ray increased resting post renovation, while the teleost species also spent more time near the exhibit front. Although a small sample size was used, the results suggest that a more naturalistic environment with multiple micro-habitats and effective visitor barriers allows for a greater proportion of the day spent exhibiting natural behaviours, greater space use and reduced stereotypes.
... The use of faecal sampling is due to the fact that it is almost a completely non-invasive procedure that is able to be performed by untrained personnel [20]. Faeces are able to be collected fairly easily from animals within the field after careful observation of recent defecation [21]. The only concern however, is that unlike other biological samples such as fur, right after collection, faeces need to be stored at −20 • C [22]. ...
Koalas (Phascolarctos cinereus) are one of Australia's most charismatic native small marsupial species. Unfortunately, populations of koalas are rapidly declining throughout Australia and they continue to face increasing pressure from a changing ecosystem. Negative stimulants in the environment can elicit stress responses through activation of the hypothalamic-pituitary-adrenal (HPA) axis. Depending on the duration of the negative stimulant, the stress response can lead to either acute or chronic side effects, and is shown through the activation of the neuroendocrine stress system and the release of glucocorticoids (e.g., cortisol). Wild koalas entering clinical care face novel stressors that can be out of a wildlife carer's control. In this pilot study, we monitored physiological stress in three wild koalas at a wildlife rehabilitation centre in New South Wales, Australia. Acute and chronic stress was 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, from the start of September 2018 to the end of December 2018. Results attempt to understand the stress response of koalas to negative stimulants in the environment by comparing faecal glucocorticoids on days where a known stressor was recorded with days where no known stressor was recorded. Furthermore, variations in faecal and fur glucocorticoids were compared between the three koalas in this study. To our knowledge, this is the first evidence of stress tracking of wild rescued koalas in a sanctuary. We suggest that further monitoring of baseline, acute and chronic stress will be needed to better understand how koalas respond to negative stimulants associated with clinical care.
... The popularity of faecal sampling is due to the fact that it is almost a completely noninvasive procedure that is able to be performed by untrained personnel (Sheriff et al., 2011). Faeces are able to be collected fairly easily from animals within the field after careful observation of recent defecation (Narayan et al., 2012). The only concern however, is that unlike other biological samples such as fur, right after collection faeces need to be stored at -20 C (Narayan & Vanderneut, 2019). ...
Koalas (Phascolarctos cinereus) are one of Australia's most charismatic native small marsupial species. Unfortunately, populations of koalas are rapidly declining throughout Australia and they continue to face increasing pressure from a changing ecosystem. Negative stimulants in the environment can elicit stress responses through activation of the hypothalamic-pituitary-adrenal (HPA) axis. Depending on the duration of the negative stimulant, the stress response can lead to either acute or chronic side effects, and is shown through the activation of the neuroendocrine stress system and the release of glucocorticoids (e.g., cortisol). Wild koalas entering clinical care face novel stressors that can be out of a wildlife carer's control. In this pilot study, we monitored physiological stress in three wild koalas at a wildlife rehabilitation centre in New South Wales, Australia. Acute and chronic stress was 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, from the start of September 2018 to the end of December 2018. Results attempt to understand the stress response of koalas to negative stimulants in the environment by comparing faecal glucocorticoids on days where a known stressor was recorded with days where no known stressor was recorded. Furthermore, variations in faecal and fur glucocorticoids were compared between the three koalas in this study. To our knowledge, this is the first evidence of stress tracking of wild rescued koalas in a sanctuary. We suggest that further monitoring of baseline, acute and chronic stress will be needed to better understand how koalas respond to negative stimulants associated with clinical care.
... Many conservation programs have utilized FGM as a means of monitoring the health of captive and semicaptive populations of threatened wildlife species (Madliger et al. 2016). These include programs using FGM to guide the conservation management of threatened marsupial species, including the koala (Phascolarctos cinereus; Davies et al. 2013, Narayan et al. 2013), Gilbert's potoroo (Potorous gilbertii;Stead-Richardson et al. 2010), the numbat (Myrmecobius fasciatus; Hogan et al. 2012), and the greater bilby (Macrotis lagotis; Narayan et al. 2012). While the majority of these studies have been on captive populations or small wild populations, some recent in situ studies have focused on FGM in mammals in response to environmental stressors (George et al. 2014, Rakotoniaina et al. 2016. ...
Urbanization significantly impacts the health and viability of wildlife populations yet it is not well understood how urban landscapes differ from non‐urban landscapes with regard to their effects on wildlife. This study investigated the physiological response of eastern grey kangaroos (Macropus giganteus) to land use at a landscape scale. Using fecal glucocorticoid metabolites (FGM) we compared stress levels of kangaroo populations in urban and non‐urban environments. We modeled FGM concentrations from 24 kangaroo populations against land use (urban or non‐urban) and other anthropogenic and environmental factors, using a linear modeling approach. We found that land use was a significant predictor of FGM concentrations in eastern grey kangaroos with significant differences in concentrations between urban and non‐urban populations. However, the direction of the relationship differed between northern and southern regions of Australia. In the northern study sites, kangaroos in urban areas had significantly higher FGM levels than their non‐urban counterparts. In contrast, in southern sites, where kangaroos occur in high densities in many urban areas, urban kangaroos had lower FGM concentrations than non‐urban kangaroos. Rainfall and temperature were also significant predictors of FGM and the direction of the relationship was consistent across both regions. These results are consistent with the contrasting abundance and persistence of kangaroo populations within the urban matrix between the two study regions. In the northern region many populations have declined over the last two decades and are fragmented, also occurring at lower densities than in southern sites. Our study indicates that it is the characteristics of urban environments, rather than the urban environment per se, which determines the extent of impacts of urbanization on kangaroos. This research provides insights into how the design of urban landscapes can influence large mammal populations.
... Taking these factors into account, it is difficult to ascertain whether the changes in corticosterone levels observed in the present study were due to a stress response from confinement in a trap (ranging from 2-16 h), or a response to stressors experienced in the period leading up to the pre-and post-trapping events. Further research into lag time in woylies would assist with interpretation and ensuring that measures accurately reflect the physiological events of interest (Narayan et al. 2012). ...
Context Temperament can affect an individual’s fitness and survival if it also influences behaviours associated with predator avoidance, interactions with conspecifics, refuge selection and/or foraging. Furthermore, temperament can determine an individual’s response to novel stimuli and environmental challenges, such as those experienced through translocation. Increasing our understanding of the effect of temperament on post-translocation fitness is thus necessary for improving translocation outcomes. Aims The aim was to test whether differences in an individual’s behaviour or physiology could help predict body mass changes post-translocation in the woylie (brush-tailed bettong, Bettongia penicillata ogilbyi). In the absence of predation (due to release into a predator-free exclosure), body mass was used as a proxy for an individual’s success in securing resources in the new habitat, and therefore fitness. Methods Forty woylies were translocated from two predator-free exclosures to a larger exclosure, all in Western Australia. Behavioural and physiological measures were recorded during trapping, processing, holding, and release, and again at re-capture ~100 days post-release. Key results Translocated woylies generally increased in body mass post-translocation. This suggests that, in the absence of predation, the selected candidates were able to cope with the stress of translocation and possessed the behavioural plasticity to successfully find resources and adapt to a novel environment. The strongest predictors of body mass gain were sex, heart rate lability and escape behaviour when released (a convoluted escape path). Conclusions There was no significant difference in body mass between males and females pre-translocation but females showed greater mass gain post-translocation than did males, which could reflect greater investment in reproduction (all females had pouch young). Heart rate lability and escape behaviour are likely to reflect reactivity or fearfulness, a significant temperament trait in the context of translocation success. Implications Behavioural measures that can be easily incorporated into the translocation process – without increasing stress or affecting welfare of individuals – may hold promise for predicting the fate of translocated animals.
... Cross reactivity of the R4866 anticortisol antiserum is reported as 100% with cortisol and less than 10% with other steroids tested 42 . The sample assay reagents (supplied by Coralie Munro at the University of California, Davis, USA) have been used for monitoring FCMs in koalas 35 and other mammals 21,43 . Parallelism for koala faecal extracts on the FGM EIA was reported in earlier study 35 . ...
Australian small mammals such as koalas must cope with immense pressure from anthropogenic induced stressors or trauma such as bushfires, vehicle collision impacts and habitat disturbance and land clearance. In addition, they must cope with diseases such as chlamydia. To date, there is no published literature on physiological stress levels in wild koala populations compared with identified environmental stressors. This study investigated physiological stress levels within sub-populations of wild koalas encountering environmental trauma and disease from New South Wales (NSW), Queensland (QLD) and South Australia (SA).
Physiological stress was determined using a faecal glucocorticoid (or cortisol) metabolites (FGMs) enzyme-immunoassay (EIA) from 291 fresh faecal samples collected from wild koalas at the point of rescue. A healthy breeding sub-population from a forest reserve in QLD acted as a control group.
Clearance of prime Eucalyptus habitat had the largest impact on FGMs, followed by bushfire related factors (e.g. flat demeanour, dehydration and burns injury). Koalas with other sources of physical injury (dog-attacks and vehicle collisions) and disease (chlamydia) also had higher FGMs compared to healthy wild koalas. Healthy wild koalas expressed the lowest median levels of FGMs.
Overall, the results highlight that anthropogenic-induced stressors tend to increase physiological stress in wild koalas. Thus, the ultimate stressors such as habitat clearance and bush fire events could increase the incidences of proximate stressors such as dog attacks and vehicle collisions, and increase risks of foliage shortage, diseases and mortality. Therefore, there is need for ecological monitoring, conservation management actions and policy changes to curb the koala population crisis, especially within on-going and future land and road development programs.
... Validation of the fecal cortisol metabolites (FCM) extraction method is described in (15) and follows the previously described extraction protocols of (28)(29)(30). FCM concentrations were determined using a polyclonal anti-cortisol antiserum (R4866) diluted to 1:15,000, horseradish peroxidase (HRP) conjugated cortisol 1: 80,000 and cortisol standards (1.56-400 pg well −1 ). Sample extracts were then assayed in duplicate on Nunc Maxisorp TM plates (96 wells). ...
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. Faecal 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. Faecal 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 demeanour), 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 (euthanised 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.
... Non-invasive collection methods have proven to be an effective tool for monitoring reproduction in a variety of wildlife species (Monfort 2003;Schwarzenberger 2007;Schwarzenberger and Brown 2013;Kersey and Dehnhard 2014), including marsupial species (Hamilton et al. 2000;Matson et al. 2008;Ditcham et al. 2009;Takahashi et al. 2009;Hogan et al. 2010aHogan et al. , 2010bHogan et al. , 2010cHogan et al. , 2011Hogan et al. , 2012Pollock et al. 2010;Lambert et al. 2011;Descovich et al. 2012aDescovich et al. , 2012bKeeley et al. 2012;Narayan et al. 2012Narayan et al. , 2013Du et al. 2017;Swinbourne et al. 2017b). In the SHNW, the non-invasive collection and analysis of faecal samples has provided helpful (Paris et al. 2002;Hogan et al. 2010aHogan et al. , 2010bHogan et al. , 2010c albeit limited information regarding female reproduction based on faecal progesterone metabolites that appear to be stable and readily measured. ...
Southern hairy-nosed wombats (SHNWs: Lasiorhinus latifrons) do not breed well in captivity. To better understand their reproduction, daily urine samples were collected from nine captive females and analysed for volume (mL), specific gravity and a qualitative index of the number of epithelial cells, then stored at -20°C until samples could be analysed for progesterone metabolites (P4M). The mean oestrous cycle length was 35.1±2.4 days; however, individual cycle length ranged from 23 to 47 days. The mean luteal phase length was 20.8±1.3 days (range: 12 to 33 days). Urinary P4M was divided into four oestrous cycle stages: (1) early follicular phase, (2) late follicular phase, (3) early luteal phase, (4) late luteal phase, and analysed against urinary characteristics. During the late follicular phase, urine volume decreased (P=0.002) while urine specific gravity (P=0.001) and concentration of epithelial cells (P=0.004) both increased. The level of variability in oestrous cycle length suggests that some captive females may exhibit abnormal cycles; however, the changes in the urinary characteristics associated with the different stages of the oestrous cycle appear to offer a possible non-invasive means of monitoring the reproductive status of captive SHNWs.
... Owing to the small sample size (2 males and 2 females), it is possible that the sex difference in cortisol secretion might be masked in their study. Previously, Narayan et al. (2012) reported sex variation in FCM levels in the semi-free ranging Eastern Greater Bilby (Macrotis lagotis), with female FCM concentrations being constantly higher than males for over 21 days. Moreover, there was some evidence that the sex-related differences existed in both plasma and FCM levels in captive koalas (Davies et al., 2013;Narayan et al., 2013). ...
This study reports the validation and use of enzyme immunoassays (EIA) to measure changes in plasma and urinary luteinizing hormone, testosterone metabolites (UTM) and cortisol metabolites (UCM) in captive southern hairy-nosed wombats (Lasiorhinus latifrons). GnRH agonist and ACTH agonist challenges were conducted to validate urinary testosterone (male wombat only) and cortisol (male and female wombats) EIAs. Following intra-muscular injection of 8 - 12 μg buserelin (n = 4 males), there was a significant increase in both plasma (P < 0.001) and urinary testosterone concentrations (P < 0.001) 60 min and 21 h after administration, respectively. Plasma LH levels were elevated (p < 0.05) at 20 min but there was no significant increase found in urinary LH concentrations after injection. Intra-muscular injection of Synacthen® Depot (250 μg) (n = 3 males, 3 females) resulted in a significant increase (p < 0.05) in plasma cortisol secretion 15 min and in urinary cortisol concentrations 3 h post injection, respectively. Sex-related differences in cortisol secretion were also reported in this study. These findings indicate that (1) urinary LH might not be an appropriate index for describing the reproductive status in captive male L. latifrons, and (2) the UTM and UCM assays appear to be suitable for the assessment of the testicular steroidogenic capacity and the adrenocortical activity in captive southern hairy-nosed wombats, respectively.
... In summary, faecal samples (0.2 g dry weight) were lyophilised (freeze-dried), and extraction was carried out using 90% ethanol and heat treatment (80 C for 10 min). Extracts were assayed for FCM by EIA using a polyclonal anticortisol antiserum R4866 protocol (Narayan et al. 2012;Hing et al. 2016). Results were expressed as FCM concentration (pg g -1 ) on a dry-weight basis. ...
Australian native fauna are thought to be well-adapted to fire-prone landscapes, but bushfires may still pose considerable challenges or stressors to wildlife. We investigated the impact of bushfire on the stress physiology of the woylie (brush-tailed bettong, Bettongia penicillata) a critically endangered Australian marsupial, and assessed whether fitness indices (body condition and parasite load) influenced stress physiology before and after the fire. We hypothesised that there would be a significant change in stress physiology indicators (in the form of faecal cortisol metabolites; FCM) following the fire, compared to the months previous. We trapped woylies (n=19) at Whiteman Park Reserve in Perth, Western Australia two days after a major bushfire and measured FCM concentration by enzyme immunoassay. Population level comparisons of FCM were made between these samples and those collected in previous months (n=58). While mean FCM varied by month of sample collection, it was not higher after the fire. We suggest that woylies may be able to maintain homeostasis through change (allostasis), at least in the period immediately after the fire. This is supported by our finding that FCM did not relate significantly to body condition or parasite load. Our results potentially highlight the physiological and behavioural adaptations of woylies to fire which could be further explored in future studies.
... In other contexts, captive mammals may not respond physiologically to anthropogenic stressors; this appears to be the case with numbats, Myrmecobius fasciatus (Hogan et al., 2012). Alternatively, regular exposure to humans may lead to potential habituation, as is the case with the greater bilby (Macrotis lagotis): when used regularly for educational displays (school visits and shows) there was no significant increase in FGMs (Narayan et al., 2011). ...
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.
... Extracts were assayed for FCM by enzyme immunoassay (EIA) using a polyclonal anti-cortisol antibody R4866 protocol [39]. We demonstrated parallelism (between dilutions of pooled faecal extracts and the cortisol standard curve), recovery of exogenous cortisol added to extracts (y = 1.1026× − 0.8897, R 2 = 0.9929), sensitivity of the assay (2.04 ± 0.39 pg/well, n = 15) and degree of intra-assay variation (CV 4% for high binding internal control and CV 6% for low binding internal control) and inter-assay variation (CV 2% for high binding internal control, CV 12% for low binding internal control). ...
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.
... An understanding of the relationship between physiological stress and disease in wildlife will help us improve the planning, design, execution and evaluation of wildlife management interventions. It is crucial that we characterise how wildlife respond to research and conservation interventions such as capture and handling (de Villiers et al. 1995;Narayan et al. 2012) and translocation (Kahn et al. 2007) to assess risk, minimise harm and increase the efficacy of these activities. For example, it has been suggested that the stress of translocation is associated with increased risk of infectious disease in translocated wildlife (Teixeira et al. 2007;Dickens et al. 2010;Sainsbury and Vaughan-Higgins 2012), including recrudescence of latent and normally innocuous pathogens as well as increased vulnerability to diseases at the release site to which the translocated animals may not have been previously exposed (Mihok et al. 1992). ...
Wildlife populations are under increasing pressure from a variety of threatening processes, ranging from climate change to habitat loss that can incite a physiological stress response. The stress response influences immune function with potential consequences for patterns of infection and transmission of disease among and between wildlife, domesticated animals and humans. This is concerning because stress may exacerbate the impact of disease on species vulnerable to extinction with consequences for biodiversity conservation globally. Furthermore, stress may shape the role of wildlife in the spread of Emerging Infectious Diseases (EID) such as Hendra virus (HeV) and Ebola. However, we still have a limited understanding of the influence of physiological stress on infectious disease in wildlife. We highlight key reasons why increased attention to the relationship between stress and wildlife disease could benefit conservation, animal and public health and discuss approaches for future investigation. In particular, we recommend increased attention be given to the influence of anthropogenic stressors including climate change, habitat loss and management interventions on disease dynamics in wildlife populations.
... Faecal glucocorticoid metabolite enzyme-immunoassays were also validated in the greater bilby (Macrotis lagotis) and the techniques were used to evaluate physiological stress in semi-free ranging populations . Narayan et al. (2012) demonstrated the effects of moderate stressors in a captive environment such as illnesses, injury or reproductive issues which resulted in greater physiological stress responses, in comparison to the participation of bilbies in school visits and public displays. applied this FCM EIA to study physiological stress in sub-populations of greater bilbies in natural reserves and captive populations. ...
... There are many factors that can affect the concentration of FCMs in a faecal deposit that need to be taken into consideration when planning experiments, and in analysing results. Age (Blas, Baos, Bortolotti, Marchant, & Hiraldo, 2006;Dufty Jr & Belthoff, 1997;Wilcoxen, Boughton, Bridge, Rensel, & Schoech, 2011), sex (Narayan, Hero, Evans, Nicolson, & Muccib, 2012;Narayan et al., 2013) reproductive state (Kenagy & Place, 2000;Whittier, Corrie, & Limpus, 1997), body condition (Heath & Dufty Jr, 1998;Smith, Wingfield, & Veit, 1994), diet (Dantzer, McAdam, Palme, Boutin, & Boonstra, 2011), and not only seasonal variations but even daily rhythms (Breuner, Wingfield, & Romero, 1999;Millspaugh et al., 2001;Romero & Remage-Healey, 2000) all have evidence that they can significantly alter the physiological stress response. Although farm animals are often kept in largely monotonous groups, if any comparisons are to be made between groups or studies it is important to note down all these factors. ...
Sheep are a valuable production animal for Australia’s meat and wool industry. Animal health and management should be prioritised for production animals to reduce stress and health problems. To quantify the stress imposed on animals, cortisol is often measured. The most common means of doing so is to take blood samples, a practise which in itself imposes stress and can give misleading results. More recently, faecal cortisol metabolites (FCMs) have been shown to be an effective non-invasive, feedback-free, measure of stress in animals, both in the wild and captivity. This honours thesis aims to validate field-based sampling methods to assess the underlying stress levels in sheep grazing naturalised pastures, and make comparisons with animals exposed to two types of toxic ‘pasture’. The relationship between this exposure and their FCM levels will be considered. To undertake this research, an R4866 polyclonal cortisol antibody horseradish peroxidase enzyme immunoassay system was validated for use in sheep. Field and laboratory precision of the assay were assessed by measuring FCMs in sub samples taken from different areas of sheep faecal deposits. Data showed accuracy and reliability to give highly consistent results from sheep faeces where extraction precision was found to be 14.22 ± 1.24%. Mean FCM concentrations of sectioned sub-samples showed little intra-sample variation of FCMs within a faecal deposit (mean CV = 11.74 ± 2.48%). Further validation was done during exposure of fresh faecal samples to natural climatic conditions for 9 d. Mean FCM concentrations showed little variation for up to 3 d. Examination of FCMs in sheep exposed to two types of toxic ‘pastures’: the first in experimental trials, animals were ingesting legume pastures which caused primary photosensitisation and the second where animals were exposed to a toxin that causes perennial ryegrass toxicosis. These studies identified that affected animals in both trials had significantly higher FCM levels than their controls (P < 0.05). In conclusion, these validated non-invasive tools can be applied to assess physiological stress in sheep on farms to minimise risk of stress related production loss assist in addressing animal welfare concerns.
... Extracts were assayed for FCM by enzyme immunoassay (EIA) using a polyclonal anti-cortisol antibody R4866 protocol [39]. We demonstrated parallelism (between dilutions of pooled faecal extracts and the cortisol standard curve), recovery of exogenous cortisol added to extracts (y = 1.1026× − 0.8897, R 2 = 0.9929), sensitivity of the assay (2.04 ± 0.39 pg/well, n = 15) and degree of intra-assay variation (CV 4% for high binding internal control and CV 6% for low binding internal control) and inter-assay variation (CV 2% for high binding internal control, CV 12% for low binding internal control). ...
... Some felids have been housed singly, while others in groups due to space constraints commonly seen in leopard enclosures. (Wielebnowski et al. 2002a) and Sumatran tigers (Narayan et al., 2012(Narayan et al., , 2013a(Narayan et al., , b, 2014Evans et al., 2013). Samples were further dried in a lyophilizer and sieved to get a homogenised powdered hair-free sample. ...
Animals in captivity are confined to a small space, as compared to their peers in the wild, leading to many welfare problems. The main welfare problem appears to be stress, which affects the health physically, physiologically and psychologically. This study assessed the intensity of stereotypic behaviour and level of faecal corticosterone metabolite among the large felids— 42 Royal Bengal tigers (Panthera tigris tigris) and 21 Indian leopards (Panthera pardus fusca)—housed at six different Indian Zoos between April 2014 and March 2015. The stereotypic behaviour prevalence and its intensity assessed through behavioural observations on focal species revealed that tigers on an average spent 12% of the time on stereotypic behaviours during day light hours, while the leopards spent 7% of their time on stereotypic behaviours. Comparison of stereotypic behaviour individually with independent biological factors (animal origin, sex, age, reproductive history and health condition) and environmental factors (ambient temperature, enclosure size with enrichment, keepers‘ attitude and visitors influence) showed significant variation in categories found within each independent factors (like age-classes) both in tigers and leopards. However, comparison of all independent factors with the dependent factors (stereotypic behaviour) using a multiple regression model to find out the key factor driving stereotypic behaviours revealed that in case of tigers only the enclosure size showed negative relationship among environmental factors and age in case of biological factors showed positive correlation (as middle age individuals stereotyped more than young and old age individuals). These two factors, explained as responsible for 51% of stereotypic extent among the study tigers. In the case of leopards, unlike tigers, percent tree cover showed positive correlation among the environmental factors and age showed significant negative correlation with stereotypic behaviour and these two factors explained nearly 40% stereotypic extent in leopards. Using a non-invasive technique, faecal corticosterone, which is a faecal glucocorticoid metabolite, extracted from 18 tiger and 9 leopard scats revealed that leopards (27.2 ± 1.36 conc. ng/g) had marginally higher level of corticosterone as compared to tigers (23.6 ± 1.62 conc. ng/g). Though the comparison of individual animals stereotypic extent, with that of fecal corticosterone level did not correlate both in the case of tigers and leopards, comparison of fecal corticosterone with all the independent biological and environmental factors using multiple regression equation revealed that corticosterone level decreased significantly with size of enclosure, percent tree cover, temperature and among individuals with breeding signs (mating) than those without same in case of tigers. Similarly, in the case of leopards also, corticosterone level negatively correlated with % tree cover and positively with enclosure area. Interestingly, enclosure area that showed a significant negative correlation with stereotype also appeared significant with the same trend in comparison with corticosterone in case of tigers. In the case of leopards, among environmental factors, fecal corticosterone level appeared significant with % tree cover, as similar to that of stereotype. In conclusion, the study points out that large enclosure with dense tree cover are essential for reducing both stereotype as well as faecal corticosterone level among the tigers in captivity. While in the case of leopards, higher tree cover is the most important environmental factor to reduce the level of stereotypic behaviours and fecal corticosterone. The results observed from captive environment go in support of the behaviours exhibited by these species under natural habitats. Considering the results, the study suggests measure to reduce the stress level among tigers and leopards in Indian zoos.
... Conservation physiology has most recently been defined as 'an integrative scientific 28 discipline applying physiological concepts, tools, and knowledge to characterizing 29 biological diversity and its ecological implications; understanding and predicting how which is used to test whether the assay system is able to reliably measure the 67 metabolites of target hormone in excreta of the study species (Narayan et (Narayan et al. 2012). There is no information available 83 on FCMs levels in semi-wild populations of this marsupial. ...
Rapid and reliable physiological evaluation of stress is necessary for understanding the potential impacts of environmental changes on managed populations of threatened mammals. In-situ populations of Australia’s iconic marsupial, the greater bilby (Macrotis lagotis) are nearing extinction due to the impacts of competition and predation by feral animals and unpredictable climatic events (summer heat waves). In this study, we focussed our aim to identify a non-invasive method to measure adrenal activity in the species and also to identify potential factors that should be considered when comparing physiological stress in semi-free ranging populations of the species. We validated an enzyme-immunoassay (EIA) for detecting fecal cortisol metabolites (FCM) from fresh fecal pellets taken from bilbies within four captive sites and two semi-free ranging populations around Queensland and New South Wales, Australia. Our FCM EIA successfully detected the “raise and fall” pattern of FCMs levels within 3 days of exogenous adrenocorticotropic hormone (ACTH) challenge. Mean FCMs levels differed significantly between the captive sites and between sexes. All male bilbies grouped outdoor in captivity expressed the highest mean FCM level in comparison to all captive males that were housed individually or as groups indoors. Also, semi-free ranging bilbies expressed higher mean FCMs levels than the captive bilbies. Overall, our study successfully validated a non-invasive tool for monitoring physiological stress in the greater bilby. In future, it will be worthwhile to consider factors such as housing conditions, sex and location when comparing the adrenal sensitivity to environmental changes, to help evaluate the success of management interventions (such as predator free enclosures) and support the survival of the species.
... Non-invasive methods to quantify stress have been developed for many different taxa. The measurement of glucocorticoid metabolites (GCMs) found in faecal samples is now one of the most common of these methods (Miller et al., 1991;Kirkpatrick et al., 1996;Palme et al., 1996;Berkeley et al., 1997;Jurke et al., 1997;Wasser et al., 1997;Goymann et al., 1999;Harper and Austad, 2000;Dehnhard et al., 2001;Ganswindt et al., 2003;Turner et al., 2003;Hunt et al., 2004;Touma et al., 2004;Cyr and Romero, 2008;Chinnadurai et al., 2009;Sheriff et al., 2009;Ashley et al., 2011;Scarlata et al., 2011;Hogan et al., 2012;Howell-Stephens et al., 2012;Laver et al., 2012;Narayan et al., 2012Narayan et al., , 2013Shutt et al., 2012;Smith et al., 2012). Steroid hormones circulating in the body are metabolized by the liver, then excreted as metabolites into the gut (Taylor, 1971;Palme et al., 1996;Mostl and Palme, 2002), and GCMs can be detected in the excrement of birds and mammals. ...
The American pika (Ochotona princeps) is considered a sentinel species for detecting ecological effects of climate change. Pikas are declining within a large
portion of their range, but previous studies have focused only on local pika extinction as a metric of change. We designed
a procedure which can provide an earlier warning signal, based on non-invasive sampling and analysis of physiological stress
in living pikas. Pikas were sampled at several locations in the Rocky Mountains for the measurement of glucocorticoid metabolites
(GCMs) in faeces. Using a time series of faecal pellets from 12 individuals, we detected a significant increase in faecal
GCM level in response to capture, thus biologically validating the use of a corticosterone enzyme immunoassay. We also established
baseline, peak, and post-peak GCM concentrations for pikas in the Rocky Mountains, which varied according to gender and individual.
This is the first study to measure stress hormone metabolites in any species of pika. The methods developed and validated
in this study can be used to add non-invasive measurements of physiological stress to pika monitoring programmes and other
research designed to assess pika vulnerability to predicted changes in climate. Pika monitoring programmes currently in place
use a protocol that relates current site use by pikas with data on local habitat characteristics, such as elevation, to infer
potential effects of climate change. Data generated by these monitoring studies can be used to identify the trends in site
use by pikas in relationship to habitat covariates. However, this approach does not take into account the role of behavioural
thermoregulation and the pika's use of microhabitats to ameliorate variations in climate. Incorporating a stress metric, such
as GCM concentration, will provide relatively direct evidence for or against the hypothesis that pikas can be stressed by
climate regardless of behavioural adaptations.
... This short-term increase in corticosterone promotes key changes in behaviour and physiology that enable individuals to cope with stress [6][7][8][9][10]. Some of the key behaviours affected by corticosterone in amphibians and also in other vertebrates (small mammals and lizards) include initiation of hiding and defensive behaviours [11][12][13], increased locomotor activity [14,15], and increased thermoregulatory behaviour [16,17]. The sight of a predator can induce a corticosterone response in birds [18] and exposure to predator odour can induce corticosterone responses in male Sprague-Dawley rats [19]. ...
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.
... Other non-invasive biological samples , such as hair (Koren et al., 2002), saliva (Kirschbaum and Hellhammer, 1999), and aquatic medium, such as pond water (Gabor et al., 2013), have also been used successfully for monitoring the reproductive and stress endocrine functions in animals. Non-invasive endocrinology provides a direct measure of reproductive hormone cycles and physiological stress responses of wildlife in captivity as well as in situ populations (Ziegler et al., 1997; Dittami et al., 2008; Narayan et al., 2012f, 2013c). Thus, non-invasive endocrinology can provide crucial insight into the relationship between a population and its environment. ...
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 seminar will be based on the central theme of Conservation Physiology which shaped my early research career based on the reproductive and stress endocrinology and captive breeding of an endangered amphibian species. I will explore my postdoctoral research experiences where I have applied the Conservation Physiology theme to generate new knowledge on the comparative reproductive and stress endocrinology of numerous animal species that have high conservation and economical importance (e.g. native Australian small mammals, frogs and reptiles, birds and large mammals-big cats and bears). I will discuss the applications of non-invasive hormone monitoring techniques for evaluating animal responses to environmental stressors such as infectious diseases, climate change and feral animals. The conservation physiology research theme can make valuable contributions for fundamental animal biology and biodiversity conservation research.
YouTube video of presentation
https://www.youtube.com/watch?v=_fE5DTqdqC8
Updated tables, figures and references of Palme, 2019,
and the respective supplements (Date: 2nd January 2025)
Asiatic black bears face chronic stress in bile farms while Animals Asia rescues black bears from bile farms and rehabilitates them into sanctuaries that are supported with environmental enrichment and veterinary care to improve animal welfare. In this study, we investigated whether bile farmed bears show significantly high levels of stress at rescue and whether stress levels reduce over time in the bear sanctuary. We measured stress hormone levels using faecal cortisol metabolites (FCM) in 16 Asiatic black bears freshly rescued from bile farms in Vietnam. Fresh faeces were collected from each bear on the rescue truck and on a weekly basis for 22-week study period at a bear Sanctuary in Vietnam. Results showed that individual FCM levels of all 16 rescued bears, except one, from truck samples were above mean baseline FCMs of bears previously rehabilitated to a bear sanctuary. This result suggested that the majority of the rescued bears were still capable of showing a stress endocrine response during the rescue operation despite being exposed to conditions causing chronic stress in bears on bile farms. Results showed that mean FCM levels of the rescued bears were significantly different between time periods (higher at the rescue (on truck samples) compared to week 22 samples) and mean FCM levels showed an overall declining pattern over the first 22 weeks after they arrived at the bear sanctuary. The bears also demonstrated acute FCM stress responses to management interventions at the sanctuary such as veterinary health checks and transportation. In conclusion, rescued bears tend to modulate their stress endocrine response after rehoming at the bear sanctuary. This is an important result indicating that the rescue effort and rehabilitation of bile farm bears is effective. Whether this also coincides with behavioural adjustments in rehabilitating bears (e.g. lessening of stereotypic behaviour) warrants further investigation.
Evaluation of physiological stress in the tiger (Panthera tigris) using faecal cortisol metabolite (FCM) enzyme immunoassays (EIAs) provides a powerful conservation physiology tool for the
species. However, it is important to validate non-invasive endocrine sampling techniques in field conditions to ensure that
the method provides a reliable parameter of physiological stress in the species. This is because endocrine measurements are
highly species specific and FCM concentrations can be influenced by environmental factors. Here, we studied the impact of
the decay rate of FCMs and intra-sample variation of FCMs using a previously validated EIA. To determine the decay rate of
FCMs, we measured FCMs in freshly deposited tiger faeces (n = 8 tigers and 48 scats) that were randomly exposed to the natural environment (dry conditions with no rainfall) for up to
192 h. To determine intra-sample variation in FCMs, we used 10 scats from 10 tigers, divided each sample into four sections
and each section into four sub-sections and measured FCMs in each section and sub-section. The results of this decay-rate
experiment showed that FCMs in tiger faeces began to decay after 48 h exposure to the environmental conditions available.
Thus, FCMs within freshly deposited tiger faeces are influenced by available environmental conditions. Changes in weather
conditions (e.g. increased rainfall and humidity) could influence the stability of FCMs. The results of the intra-sample variation
study showed that inter-variation among scats accounted for 52% of the variations in FCMs, while intra-sample variation between
sections (32%) was greater than the sub-sample variation (16%). Intra-sample variation can be reduced by homogenizing the
entire lyophilized faecal sample prior to the EIA. In conclusion, careful evaluation of decay rate and complete homogenization
of faeces prior to EIA analysis will increase the reliability of FCMs as a non-invasive index of physiological stress in the
tiger.
Natural weathering conditions can influence faecal cortisol metabolite (FCM) measurements in wildlife if fresh faeces cannot be collected immediately following defaecation. In this study, we evaluated this issue in a threatened Australian marsupial, the greater bilby (Macrotis lagotis). Fresh (<12h since defaecation) faecal samples (n=19 pellets per bilby) were collected one morning from seven adult bilbies kept in captivity. One control faecal sample (Day 1) from each bilby was immediately frozen. The remaining faecal pellets were randomly positioned outdoors. Subsequently, we froze one faecal pellet every 24h for 19 days. FCM levels in bilby faeces were quantified using an enzyme-immunoassay. Mean FCM levels showed variation (daily mean coefficients of variation [CV %]) of 56.83-171.65% over 19 days. Overall, FCM levels were affected by exposure time; however, multiple comparisons showed that no significant change in FCM occurred after environmental exposure (no significant difference in mean FCM between control (Day 1) with any of the exposure days (Days 2-19). Individuals and sex also affected FCM levels. We found no correlation between mean daily CVs with daily minimum-maximum temperatures or rainfall. Our results indicate that FCM in bilby faeces is fairly stable to long-term environmental exposure (19 days). In future, freshly excreted bilby faeces (where the sample maintains a distinct odour for 9-13 days) should be used to study FCM levels in wild bilbies.
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 andsurvival,warrants future investigation.
Koalas (Phascolarctos cinereus)arethe only extant representatives of Australia's unique marsupial familyPhascolarctidae and were listed as nationally Vulnerable in 2012. Causes ofmortalityare diverse, althoughthe diseasechlamydiosis, dog attacks, collisions with cars, and loss of habitat representtheprincipal 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,theseresults 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 (includingcaging, translocation) on the nutritional status, reproductive behaviors and disease status of captive and managed in-situ Koala populations.
Diet and food availability of the greater bilby
(Macrotis lagotis), was examined at two sites and over
two summer and two winter seasons in Astrebla Downs National Park in
south-western Queensland. The presence of both invertebrate and plant material
in almost all faecal pellets (99.6% and 98.5%, respectively)
indicated that bilbies in south-western Queensland are omnivorous. The plant
component of the bilby diet consisted predominantly of
Dactyloctenium radulans seeds (frequency of occurrence:
94.4%). The major invertebrate prey items were Isop-tera (termites,
73.1%) and Formicidae (ants, 77.6%). The frequency of occurrence
of invertebrates in faecal pellets was higher during summer than winter, but
the frequency of occurrence of plants was relatively constant regardless of
season. Within the invertebrate categories, Isoptera and Acrididae
(grasshoppers) displayed the greatest seasonal difference, with a
significantly higher proportion of both categories in the diet during summer
than winter at both study sites. The availability of invertebrate prey items
also varied with season, and for most taxa, frequency of oc-currence was
significantly higher in summer than winter. In contrast, the frequency of
occurrence of seeds and bulbs (Cyperus bulbosus) varied
only slightly with season. Seasonal changes in food availability were
reflected by variation in dietary composition; however, relative proportions
of some dietary items differed from their proportional availability.
Therefore, bilbies were considered to be qualitatively opportunistic;
selecting more invertebrates when their apparent availability increased, but
continually exploiting the available seeds and bulbs. Additionally, although
bilbies appeared to have some preferences for particular food items, several
food items were selected almost randomly, and therefore bilbies were also
considered to be dietary generalists. Such an opportunistic feeding strategy
is advantageous to an arid-zone species as it permits the continuous
exploitation of food resources that are unpredictable in their availability
both spatially and temporally.
Fecal glucocorticoid assays provide a potentially useful, noninvasive means to study physiological responses of wildlife to various stressors. Consequently we quantified fecal glucocorticoid concentrations among free-ranging elk (Cervus elaphus) by subherd, sex, and season and determined their relationship to various human activities and environmental conditions. Using a validated technique, we assayed 558 fecal samples collected from 30 radiocollared elk in the Black Hills, South Dakota, from June 1995 to September 1997. Fecal glucocorticoid measures were least in winter (x̄=17.41 ng/g, SE=2.97 for bull subherds and 18.9 ng/g, SE=2.85 for cow subherds) and increased to peak concentrations in summer (x̄=33.6 ng/g, SE=3.42 for bull subherds and 34.21 ng/g, SE=3.71 for cow subherds). Vehicle use along primary roads, primary road density, and mean temperature each independently explained a significant portion of the variation in fecal glucocorticoid concentrations ( , P<0.001). Annual glucocorticoid secretion also may be related to normal seasonal metabolic rhythms. Though more research is needed on fecal glucocorticoid concentrations of undisturbed elk, we hypothesize that human activities, high temperatures, or normal seasonal metabolic rhythms may have elevated summer glucocorticoid concentrations. Our findings suggested that fecal glucocorticoid assays, if coupled with population performance measures, could assess physiological effects of natural and human-induced disturbances on free-ranging elk. Also, our study illustrated the need for caution when interpreting fecal glucocorticoid measurements, because several confounding factors may influence interpretation.
This study compared the efficacy of plastic glitter, with a mean particulate size of 1.18 mm, as a faecal marker in group-housed captive wombats Lasiorhinus latifrons. The wombats voluntarily consumed the glitter through the use of appetizing food vehicles. Over 40 different food treats were tested as possible vehicles for the oral delivery of the faecal marker and of these six were deemed highly palatable: (1) golden syrup with horse pellets, (2) golden syrup with weetbix, (3) pitted dates, (4) honey with kangaroo pellets, (5) nutrigel with rolled-oats, and (6) strawberry sauce with rolled-oats. Mean transit time of glitter particulates through the alimentary tract of L. latifrons was 2.9 ± 0.5 d, with maximal output occurring 4.2 ± 0.3 d after administration. A marker dose of 1.6 g / 3 d was required to reach a steady and detectable state of marker output. Using this dosage > 2 particulates (i.e. flecks) of glitter were defaecated in > 90% of faecal pellets, allowing the accurate identification of individual samples. Reliable labeling was obtained using gold, silver, metallic red, metallic green, metallic blue and white glitter, i.e. digestion did not affect the integrity of these colours. There was no evidence that long-term feeding of glitter had any negative effects on the normal formation of faecal pellets, the clinical health, weight or appetite of the wombats.
There has been conflicting evidence about the impact of visitors on zoo animals. The present study was designed to increase understanding of visitor effect by assessing the relationship between visitor numbers and activity in the hypothalamic–pituitary–adrenal (HPA) axis. An enzyme-immunoassay was successfully developed and validated to quantify urinary cortisol in spider monkeys. The physiological impact of visitors on the monkeys was then investigated by assessing the levels of urinary cortisol in samples collected when no visitors were in the zoo and throughout the year when visitor density fluctuated widely. Our results suggest that an increase in the number of visitors was associated with an increase in cortisol. This study supports previous behavioural research that visitors have a meaningful impact on primates in zoos. It also increases our understanding of visitor effect by assessing the relationship between absolute visitor numbers and an aspect of the animals’ physiology.
This study investigated behavioral activities (resting, moving, aggressive, social, and reproductive behavior) and fecal cortisol levels in 8 individually identified adult male blackbucks during periods of varying levels of zoo visitors (zero, low, high, and extremely high zoo visitor density). This study also elucidated whether zoo visitor density could disturb nonhuman animal welfare. This study analyzed fecal cortisol from the samples of blackbuck by radioimmunoassay and found significant differences (p < .05) for time the animals devoted to moving, resting, aggressive, reproductive, and social behavior on days with high and extremely high levels of zoo visitors. The ANOVA with Duncan's Multiple Range Test test showed that the fecal cortisol concentration was higher (p < .05) during the extremely high (137.30 ± 5.88 ng/g dry feces) and high (113.51 ± 3.70 ng/g dry feces) levels of zoo visitor density. The results of the study suggest that zoo visitor density affected behavior and adrenocortical secretion in Indian Blackbuck, and this may indicate an animal welfare problem.
The introduction of threatened marsupials to islands affords a high degree of translocation success due to the lack of exotic species on islands, or the feasibility of eradicating them. The dibbler Parantechinus apicalis is a small marsupial endemic to the southwest of Australia. It is listed under international and national legislation as Endangered, and has been the focus of a successful conservation strategy to introduce captive-bred individuals to an island as a security measure, and as part of a formal Interim Recovery Plan. A total of 88 individuals were released in four groups on Escape Island from 1998 to 2000. The population was monitored using radiotelemetry and trapping techniques from 1998 to 2001. Breeding and dispersal of young occurred within the first year of release. Three years after the initial release, the third generation of wild-born dibblers had entered into the population. The total cost of this translocation exercise approximated $AUS 0.6 million. The conservation effort to give additional security to dibblers has been successful, at least in the short term, due to the collaboration between four organisations and a commitment to support a monitoring program of the released population over time.
The greater bilby, Macrotis lagotis, is a species of conservation significance in the arid and semiarid zones of Australia. A species recovery program has been underway since the mid-1990s but the incorporation of molecular genetic data within the program has been difficult due to the problems of obtaining regular, population-wide samples of this trap-shy and sparsely distributed species. In this study, we demonstrate that faecal pellets collected from around burrows in the dry, arid habitat of western Queensland provide a viable source for DNA extraction and analysis. Faecal DNA was used to generate population-level estimates of microsatellite and mtDNA diversity for comparison with previous estimates for the natural population derived from tissue samples. Data were used to assess both the reliability of faecal-derived genotypes and the extent of any diversity loss since the previous study. Microsatellite diversity recorded from eight polymorphic markers for the natural population (A = 4.31 ± 0.30, HE = 0.76 ± 0.03) was comparable with the previous study, indicating little change in genetic diversity for the natural population in the 10-year interim. Faecal genotypes generated for the recently reintroduced population matched the known number of founders as well as a known genotype, providing support for the reliability of the faecal DNA approach. The captive and reintroduced populations had significantly lower diversity levels than the natural population (A = 3.59 ± 0.28, HE = 0.68 ± 0.03; A = 3.57 ± 0.20, HE = 0.65 ± 0.03 respectively). Mitochondrial control region analysis, incorporating nested clade phylogeographic analysis (NCPA), agrees with earlier findings that populations of bilbies across the arid zone in Australia have only recently become fragmented, but the case for Queensland bilbies being strongly differentiated from other regions is diminished. Implications from this study include the need to further supplement the captive and reintroduced populations with additional out-bred individuals and that faecal DNA can be used effectively for ongoing monitoring and management of this species. Yes Yes
Gilbert's potoroo (Potorous gilbertii) was rediscovered in 1994 after having been presumed extinct for 120 years. Estimates indicate fewer than 40 individuals remain at Two Peoples Bay Nature Reserve on the south coast of Western Australia although a translocated population of approximately 20 animals has recently been established on nearby Bald Island. A captive breeding facility has been established adjacent to the mainland population but few young have been produced (8 since 1995). Faecal levels of oestradiol-17beta (E(2)) were monitored over a 2-year period in an effort to determine cyclic reproductive activity, and faecal cortisol levels were also monitored to gauge whether chronic stress may be a factor limiting breeding in captivity. Faecal steroids were monitored in six captive females, and four captive male potoroos, and four wild females. The only captive births recorded after 1998 were one in August 1999 and one in February 2001, both to the same female. Peaks in E(2) concentration, up to 10 ng g(-1) of dried faecal mass were measured and results to date suggest the main breeding period to be November-December based on elevated E(2) levels at this time. Clear patterns of reproductive activity in the captive females, however, were not evident. Analysis of epithelial cell counts from urinogenital swabs and faecal E(2) and progestagen (PM) levels from a single female kept at the Perth Zoo, suggest that Gilbert's potoroo has an oestrous cycle of approximately 39 days. Faecal cortisol levels in captive females were significantly lower than those in wild-caught individuals and thus there is no indication that elevated cortisol levels per se inhibited reproduction in captive females.
Stress responses play a critical role in the ecology and demography of wild animals, and the analysis of fecal hormone metabolites is a powerful noninvasive method to assess the role of stress. We characterized the metabolites of injected radiolabeled cortisol in the urine and feces of Columbian ground squirrels and validated an enzyme immunoassay for measuring fecal cortisol metabolites (FCM) with a 5 alpha-3beta,11 beta-diol structure by stimulation and suppression of adrenocortical activity and by evaluation of the circadian pattern of FCM excretion. In addition, we also evaluated the impact of capture, handling, and acclimation to the laboratory on FCM. Cortisol is highly metabolized, with virtually none being excreted, and of the radiolabeled cortisol injected, 31% was recovered in urine and 6.5% in feces. The lag time between cortisol injection and its appearance in urine and feces was 4.5 +/- 0.82 (SE) h and 7.0 +/- 0.53 (SE) h, respectively. FCM levels varied over the day, reflecting circadian variation in endogenous cortisol. Dexamethasone decreased FCM levels by 33%, and ACTH increased them by 255%. Trapping and housing initially increased FCM levels and decreased body mass, but these reversed within 3-7 d, indicating acclimation. Finally, FCM levels were modestly repeatable over time (r=0.57) in wild, live trapped, nonbreeding animals, indicating that FCMs provide a measure of the squirrel's stress-axis state. This assay provides a robust noninvasive assessment of the stress response of the Columbian ground squirrel and will facilitate an integration of its life history and physiology.
An investigation was made into the role of increased adrenocortical activity in the causation of the total postmating mortality of male Antechinus stuartii—a small shrew-like marsupial. A progressive rise in plasma androgen concentration of the males up to the time of mating was associated with increasingly aggressive behaviour and a progressive fall in plasma CBG concentration. During the mating period, the rate of fall in plasma CBG concentration increased markedly and plasma total corticosteroid concentration rose above the MCBC, so that there was a sharp rise in plasma free corticosteroid concentration just before the males all died. Although the plasma CBG concentration of females also fell during the mating period, with a moderate increase in plasma total glucocorticoid concentration, there was only a slight increase in plasma-free corticosteroid concentration at that time and it did not change significantly during the postmating, lactation period. Castration of captive males caused a marked increase in their plasma CBG concentration, and injection of testosterone or ACTH depressed it. There was a marked reduction in immunological competence and serum immunoglobulin concentration of the males just before their death. Postmortem examination revealed evidence of increased invasiveness of parasites and microorganisms, as well as severe haemorrhagic ulceration of the digestive tract. These effects could all be reproduced in captive animals by injection of cortisol acetate. It is concluded that a state of stress, induced by aggressive interactions at the time of mating and exacerbated by the fall in plasma CBG, results in suppression of the immune and inflammatory systems of the males, causing their death from gastrointestinal haemorrhage as well as invasion by parasites and microorganisms.
This study examined the relationships between a number of stockperson and cow variables at 66 commercial dairy farms. Variables such as the attitudes and behavior of stockpeople toward their cows and the behavioral response to humans and productivity of cows were studied over one lactation. There were consistent and significant correlations between some of these stockperson and cow variables. For example, a positive attitude by stockpeople toward the behavior of dairy cows was negatively correlated with the number of forceful, negative, tactile interactions used by stockpeople in handling cows (r = -0.27, df = 127, P < 0.01). Furthermore, based on farm averages, the number of forceful, negative, tactile interactions used by stockpeople was negatively correlated with the percentage of cows approaching within 1 m of an experimenter in a standard test (r = -0.27, df= 64, P< 0.05). Although not confirming a fear-productivity relationship, a moderate but nonsignificant correlation was found between flight distance of cows to an experimenter in a standard test and milk yield (r = -0.27, df = 33, P > 0.05). Support for the existence of a negative fear-productivity relationship was the finding that the use of negative interactions by stockpeople was significantly and negatively correlated with milk yield, protein, and fat at the farm (r = -0.36, -0.35 and -0.33, respectively, df = 64, P < 0.01) and was significantly and positively correlated with milk cortisol concentrations at the farm (r = 0.34, df= 64, P < 0.01). Furthermore, the percentage of cows approaching within 3 m of an experimenter in a standard test was positively correlated with conception rate to the first insemination (r = 0.38, df = 46, P < 0.01). The significant correlations found in the present study between stockperson attitudes and behavior and cow behavior and productivity, although not evidence of causal relationships, indicate the possibility of targeting these human characteristics to reduce fear responses of dairy cows to humans and improve the cows' productivity.
We used captive European starlings (Sturnus vulgaris) to test whether corticosterone responses differed in birds held under normal laboratory conditions or conditions of chronic stress. Surprisingly, both basal corticosterone concentrations and corticosterone responses to acute stress were significantly reduced when birds were chronically stressed. To determine the mechanism underlying this reduced response, animals under both conditions were injected with lactated Ringer's solution (control), adrenocorticotropin (ACTH), arginine vasotocin (AVT), or dexamethasone (DEX). ACTH increased corticosterone concentrations above stress-induced levels in both cases, although maximum responses were lower in chronically stressed birds. AVT did not augment the corticosterone response under nonchronically stressed conditions, but it did under chronically stressed conditions. DEX reduced maximal corticosterone concentrations in both cases. Neither ovine nor rat corticotropin-releasing factor (CRF) altered normal stress responses. These data indicate that changes in responsiveness of the hypothalamic-pituitary-adrenal axis to ACTH and AVT serve to downregulate corticosterone responses during chronic stress. Furthermore, these data lead to the following hypothesis: ACTH output from the pituitary limits maximum corticosterone concentrations under normal conditions, but reduced AVT release from the hypothalamus regulates lower corticosterone concentrations under chronic stress conditions.
The reproductive physiology of the polyoestrous Honey possum (Tarsipes rostratus) is virtually unknown except that it shares with the kangaroos and wallabies the phenomenon of embryonic diapause. Its tiny size necessitates an alternate approach to study their reproductive cycle. We have accordingly utilised faecal steroid analysis. Baseline faecal cortisol levels in the Honey possum, at 4.1+/-0.3 mug g-1, are approximately 100-fold those of other mammals and are associated with adrenal glands that, on a mass-specific basis, are almost 10 times larger than the adrenals of other mammalian, including marsupial, species. Histological examination of the adrenal glands revealed no abnormalities, however, but their hypertrophy and the peaks recorded in faecal levels following disturbance suggest that the Honey possum is vulnerable to chronic stressors in the captive situation. Mean faecal progestagens (124.4+/-107.3 ng g-1) and oestradiol-17beta (4.1+/-1.1 ng g-1) in 4 non-pregnant females maintained long term were not different from those of 5 pregnant females (101.4+/-61.0 ng g-1 and 4.3+/-1.5 ng g-1, respectively) and, on analysis, revealed a cyclicity of 24+/-1.2 days. We would predict from this evidence that the gestation period, in the absence of lactation, is approximately 23 days. Four of the pregnant females, monitored from July to November under conditions of 10:14 L:D photoperiod, showed a fall in levels of progestagens from 175.9+/-10.8 ng g-1 in July and August to 30.9+/-9.4 ng g-1 in October, while mean faecal levels of oestradiol-17beta increased from 3.8+/-0.4 ng g-1 in July to 5.7+/-0.3 ng g-1 in October. September and October are months of peak reproductive activity in the wild and we suggest that these hormonal modulations may represent an entrained reproductive rhythm. Blastocysts appear to develop at varying rates, both within the one uterus, and between the two uteri of a single female. In addition, the time taken to reach the blastocyst stage may be longer than in any other marsupial studied to date. An association of the age of the pouch young with the stage reached by the developing blastocyst does not support the conclusion that blastocysts, once formed, grow slowly during lactation or diapause. Contrary to previous reports, we have documented what appears to be a lactational inhibition on blastocysts in diapause and have estimated the length of the 'delayed' reproductive cycle in two females as less than 2 weeks. Reactivation of blastocysts in Tarsipes has been shown to be stimulated by shortening day lengths after the summer solstice, a response similar to the annual breeding period of macropodid marsupials. Results from studying Honey possums in captive conditions suggest that the control of diapause in Tarsipes appears to be three-fold; lactational, photoperiodic and an entrained rhythm.
Greater Bilbies (Macrotis lagotis) once occupied 70% of Australia but are now an endangered species under the Environment Protection and Biodiversity Conservation Act (C'wlth) 1999. A dedicated 29 km 2 enclosure to protect reintroduced bilbies from predators was built in Currawinya National Park in southwest Queensland in 2003. Ten bilbies (three male and seven female) were released in the enclosure during the period of December 2005 to September 2006. The objective of this research was to develop a method to identify suitable Greater Bilby habitat from remote sensing imagery. A related objective was to spatially characterize how bilbies used their environment for feeding and resting. Aerial photographs (1:40,000) were used to classify the vegetation and land cover. Soil samples were used to construct a detailed soils map. Radio tracking (2005-06) and field tracking data (2008) were used to identify spatial associations between bilby activities and land cover and soils features in order to spatially characterize bilby micro-habitats. These results formed the basis of a Weighted Sum model that accurately identified potential bilby micro-habitats within the enclosure.
The response of animals to handling by humans has been extensively evaluated in domesticated livestock, but rarely examined in wildlife species. Twelve captive wombats (Lasiorhinus latifrons) were subjected to two treatments in a replicated design: (1) daily handling, involving 15min of tactile contact 5 d/wk for 12 wk and (2) no-handling, involving no contact apart from that received during routine husbandry. The effect of handling was assessed via overt responses to human approach and touch, a stressor, and a novel stimulus. Daily handling reduced the wombat's flight distance in response to human approach; more in the first handling replicate (−0.16±0.02m/wk) than in the second (−0.06±0.02m/wk). A behavioural reactivity score also declined faster in the first than second handling replicate. Synthetic ACTH was used to validate the measurement of faecal cortisol metabolites in L. latifrons by EIA. Faecal cortisol metabolite secretion consistently increased in reaction to a handling procedure involving forced human contact (indicating a lack of habituation) but the magnitude of this response was not reduced by regular handling. Regular handling therefore changed the human–wombat relationship by lowering reactivity to and avoidance of the human handler, but did reduce the stress response, suggesting that the wombats entered into a state of learned helplessness.
The breeding biology and growth of Macrotis lagotis was
investigated using captive stock in Alice Springs and Dubbo and a reintroduced
population at Watarrka National Park. Individuals of
M. lagotis reached sexual maturity at about six months
of age and continued growing until about 18 months old. Pouch life and weaning
took approximately 90 days and females produced up to four litters per year.
Litters comprised 1–3 young. Average litter size at pouch exit ranged
from 1.0 to 1.88, depending on whether animals were studied in captivity or
under reintroduced free-range conditions. Females commonly continued breeding
past the age of four years and longevity extended to 10 years in captivity. In
comparison, the most animals caught in the wild were estimated to be less than
12 months old. Animals in the reintroduced population exhibited a greater
reproductive output than the captive populations examined. However,
survivorship was far greater for animals in captivity. An intensively managed
captive population may achieve a three-fold increase in size in a 12-month
period.
In order to assess potential sex differences in fear reactions, 40 rams and 40 ewes were studied using a battery of tests previously designed and validated. Animals were individually confronted with situations classically reported to induce fear in sheep: i.e. isolation from conspecifics, surprise effect and presence of a human. Eight groups were studied, each comprising ten animals of the same sex, breed (Romanov or Ile de-France) and rearing condition (artificially or dam-reared). Males and females of the same breed and rearing condition were tested alternately during the same week (4 days of habituation plus 3 days of tests) in order to prevent fear reactions from being biased by differences in the environment (such as noises, ambient light, etc.) which could influence fear reactions. Twenty-six behavioural items related to fear were recorded. Furthermore, an overall score, based upon those items and reflecting the fear level of each subject in each of the test situations, was calculated. All the significant differences between sexes (P < 0.05 ) consistently indicate that rams are less fearful than ewes. Their overall scores were respectively, 37.0 +/- 10.5 vs. 44.0 +/- 11.9 (P < 0.01) in the isolation test, 36.1 +/- 11.0 vs. 44.9 +/- 8.4 (P < 0.001) in the surprise test, 38.5 +/- 12.5 vs. 42.5 +/- 13.6 (P = 0.06) in the human test (higher scores reflect heigthened fearfulness). When subjected to non-social fear-eliciting situations, rams were seen to be less fearful than ewes. This conclusion could serve as an impetus for more general behavioural studies of factors influencing fearfulness and thus adaptability of farm animals to intensive housing conditions.
Fifty-one silver fox vixens, subjected to three different handling treatments as cubs (no-handling, gentle or forced handling), were studied for long-term effects on behavioural, physiological and production-related parameters in a one and a half year period following the last handling session. As juveniles, the animals were exposed to three different behavioural tests at 18, 22, 28, and 32 weeks of age. Both forcibly and gently handled animals showed reduced fear responses compared with nonhandled controls in test situations involving close contact with humans (P<0.05). The foxes were tested again as adults at 10, 13, 15, and 18 months of age with the same behavioural tests. It was revealed that forcibly handled animals persistently showed reduced fear responses compared with control animals, both in close contact with humans and when exposed to a novel object, whereas gently handled animals only differed from control animals in one of the tests involving some human contact and when exposed to a novel object (P<0.05). Significant differences between adult gently handled and forcibly handled animals were found in the 'confront' test involving close human contact. In this test more flight responses were observed in the gently handled group than in the forcibly handled group (P<0.01). Control animals had significantly larger adrenal weights compared with both forcibly handled animals and gently handled animals at 22 months of age (total mean adrenal weight: controls, 0.62 g; gentle, 0.54 g; forced, 0.54 g, P<0.05, general linear models). Other physiological measures and production-related parameters such as body weight, body size, gastric ulceration and pelt qualities did not differ between treatments (P>0.05). Early post-weaning handling made foxes less fearful towards humans. Forced handling seemed somewhat superior to gentle handling as a means to produce animals which, in the long term, adapted better to the farm environment both behaviourally and physiologically. Non-handled control animals suffered from long-term stress as reflected by high levels of fear responses and enlarged adrenals. Thus post-weaning handling may be beneficial in the long term for the wellbeing of farmed foxes. When applied as a management routine, handling had no significant influence on later production-related parameters and was not considered an economic risk.
The North American clouded leopard (Neofelis nebulosa) population is far from self-sustaining. Breeding success is poor and behavioral problems (i.e., fur-plucking, tail-chewing, excessive hiding or pacing, and intersexual aggression that results in mate killing) are common. This study was undertaken to investigate whether some of these problems may be indicators of chronic stress (as reflected by persistently elevated glucocorticoid levels) and whether they are associated with specific management factors. A fecal corticoid metabolite assay was validated to monitor adrenal activity in clouded leopards. Adrenocorticotropic hormone (ACTH) challenges conducted in four clouded leopards established the biological relevance of the assay system. Fecal corticoid concentrations increased 14-fold above baseline within 24 hours after ACTH administration. Adrenal activity then was monitored in 72 (36 males; 36 females) clouded leopards (65% of the North American Species Survival Plan population) during a 6-week period and compared to husbandry and behavior data. There was a significant (P < 0.01) gender difference in fecal corticoid concentrations, with females producing higher concentrations than males. Multiple regression analyses revealed negative associations (P < 0.01) between enclosure height, number of hours keepers spent with each animal per week, and corticoid concentrations. A positive correlation (P < 0.001) was found between the number of keepers caring for an individual and corticoid concentrations. Higher fecal corticoid concentrations (P ≤ 0.05) were measured in clouded leopards kept on public display or near potential predators compared to individuals maintained off exhibit or in the absence of predators. Individuals that performed self-injuring behaviors also had elevated fecal corticoids (P < 0.01). Spearman-rank correlation analysis of keeper ratings and hormone data revealed positive associations (P ≤ 0.05) between some behaviors (pace, sleep, hide, and fearful/tense) and fecal corticoid concentrations. Overall these results indicate that noninvasive fecal corticoid monitoring has enormous potential for investigating how management and behavioral problems are related to animal well-being. If conducted under carefully controlled experimental paradigms, this technique could allow researchers and managers to identify problem areas of captive management for clouded leopards (e.g., enclosure height, keeper time) and evaluate the efficacy of strategies designed to promote animal welfare and increased reproductive success. Zoo Biol 21:77–98, 2002. © 2002 Wiley-Liss, Inc.
This chapter discusses the significance of the stress concept in gaining a better understanding of social mechanisms in nonhuman mammals. The development of this concept during the past years and the resulting present understanding of different stress reactions are described in the chapter. The triggers of stress reactions are mainly psychical processes resulting from the assessment of a situation by an individual. Dependent on the coping behavior of the individual, these processes lead to different physiological response patterns, which can result in a number of pathophysiological effects. The chapter introduces the most important currently applied methods in assessing stress levels in animals. Particular attention is paid to methodological problems, as well as to the limits of interpretation. The focal points of the chapter are the sympathetico–adrenomedullary and pituitary–adrenocortical systems, the pituitary–gonadal axis, and the immune system. An overview of the relationships between social situations and stress responses is provided, in which the research focuses on the monogamous and territorial tree shrews and the polygamous and territorial European wild rabbits. In these cases, the social rank of an individual, its sociopositive interactions with conspecifics, and the stability of the social system are determinants in the effects of a social situation on the individual's vitality and fertility.
A spatial analysis of greater bilby (macrotis lagotis) habitat in south-west queensland. In: Ostendorf, ABSTRACT Greater Bilbies (Macrotis lagotis) once occupied 70% of Australia but are now an endangered species under the Environment Protection and Biodiversity Conservation Act (C'wlth) 1999. A dedicated 29 km 2 enclosure to protect reintroduced bilbies from predators was built in Currawinya National Park in south-west Queensland in 2003. Ten bilbies (three male and seven female) were released in the enclosure during the period of December 2005 to September 2006.
The purpose of this study was to investigate the relationship between stres and cortisol levels in captive animals. Stress is defined as a physiologic response to events perceived as potentially or actually threatening the integrity of the body. Serum, saliva or urine cortisol has been extensively used as a stress indicator in many mammalian species. Stereotypic behavior is exhibited by a wide range of captive animals. Its association with hormones, especially elevated cortisol level and lack of naturalistic stimuli in the environment, has been little studied. Therefore, Cortisol levels were reviewed after short-term stressors and compared them to cortisol levels in captive animals in this article. It was found some significant important the relationships among the stess and cortisol level in captive animals. Recently, fecal cortisol metabolites have been identified as an index of stress in animals. Since many captive and wild populations of endangered species are intensively managed, measuring fecal cortisol metabolites will be useful to identify factors affecting animal well-being in captivity.
The sections in this article are:
Physiological stress assessment is important for in-situ conservation and captive management of threatened wildlife. Leukocyte (white blood cell) evaluation, especially the neutrophil : lymphocyte (N : L) ratio, provides a logical representation of experimentally elevated corticosterone (stress hormone) in amphibians. Urinary corticosterone enzyme-immunoassay (EIA) is a rapid non-invasive tool for assessing stress responses in amphibians. To our knowledge, no one has explored the relationship between N : L ratio and urinary corticosterone in wild amphibians in a non-experimental way. This study provides a comparative assessment of relative leukocyte numbers, N : L ratios and urinary corticosterone responses of the endangered Fijian ground frog (Platymantis vitiana) during transportation and captivity. Adult frogs (n = 40) were collected from Viwa, Fiji Island for captive breeding. Frogs showed significant changes in leukocyte proportions during transportation and captivity. N : L ratios were higher 6 h after transportation and over 5 and 15 days in captivity. Urinary corticosterone responses of the frogs were also higher 6 h after transportation and after 5 and 15 days in captivity. All leukocyte proportions, N : L ratios and urinary corticosterone concentrations of the frogs returned near baseline levels after the frogs were kept in an environmentally enriched outdoor enclosure for over 25 days. These results highlight the value of leukocyte evaluation and urinary corticosterone EIAs as physiological tools for evaluating stress in amphibians.
We present research and management implications for captive propagation of the endangered Fijian Ground Frog Platymantis vitiana to develop methods for supplementing populations in the wild. In 2004, a captive propagation program was instituted at Kula Ecopark, Sigatoka, Fiji. However, there was little success with only a single froglet reared after three years. In 2006, a more intensive programme was undertaken between the University of the South Pacific (USP), Kula Ecopark and the community on Viwa Island. The aim of this programme was to create an outdoor enclosure to mimic natural habitat conditions so the frogs could exhibit natural breeding behaviour. A total of 39 froglets was reared after one year of this programme. We provide information on the methods of captive management, reproductive biology, captive diet varieties, and on problems of rearing froglets in captivity. This research is useful baseline information in guiding captive propagation techniques in zoological parks like the Kula Ecopark and for organizations such as the National Trust of Fiji.
We reviewed 180 case studies and a number of theoretical papers on animal relocations published in 12 major international scientific journals over the last 20 years. The study focused on re-introductions, supplementations and translocations (sensu IUCN, 1996. IUCN/SSC Guidelines for Re-introductions. 41st Meeting of the IUCN Council, Gland, Switzerland, May 1995. Http://iucn.org/themes/ssc/pubs/policy/hinte.htm.). We did not assess introductions. Re-introductions were the most common type of relocation (116/180); three quarters of these were conducted for conservation purposes. Supplementations (48/180) and translocations (36/180) occurred less frequently, and both were commonly carried out for reasons other than conservation. Simple descriptive statistics were used to analyse factors influencing relocation success. Translocations that aimed to solve human–animal conflicts generally failed. Re-introduction success was not found to have changed over the last two decades, but re-introductions appeared to be more successful when the source population was wild, a large number of animals was released (n>100), and the cause of original decline was removed. More complex trends were found for the effect of predation and the use of supportive measures such as provision of food or shelter, or predator control prior to release. The success of 47% of re-introductions was uncertain at the time case studies were published in journals. This was partly due to the lack of generally accepted and widely applied criteria to assess success. Very few case studies (3%) reported the cost of the relocation attempt. We conclude that there were three primary aims for animal relocations. These were to solve human–animal conflicts, to restock game populations, and conservation. Our extensive review of the present literature leads us to conclude that the value of animal relocations as a conservation tool could be enhanced through (1) more rigorous testing for the appropriateness of the approach in a given case, (2) the establishment of widely used and generally accepted criteria for judging the success or failure of relocations, (3) better monitoring after a relocation, (4) better financial accountability, and (5) greater effort to publish the results of relocations, even ones that are unsuccessful.
The aim of this study was to validate a method for measuring glucocorticoids noninvasively in feces of spotted hyenas (Crocuta crocuta). Three established enzyme immunoassays (EIA) for cortisol, corticosterone, and 11-oxoetiocholanolone were tested, but proved unsatisfactory. A new EIA using another corticosterone antibody was established and was used for all subsequent analyses; this EIA was validated by demonstrating parallelism between serial dilutions of spotted hyena fecal extracts and dilutions of standard corticosterone and by the recovery of corticosterone added to fecal extracts. High-performance liquid chromatography (HPLC) fractions analyzed by EIA showed various immunoreactive substances with polarities of unconjugated steroids. The physiological relevance of fecal glucocorticoid metabolites was further validated by demonstrating that (1) injection of exogenous ACTH to four males and two females led to a significant increase in fecal glucocorticoid metabolites within 24–50 h, (2) the translocation of a male spotted hyena to a new enclosure resulted in a fivefold increase compared to baseline concentrations, and (3) agonistic social interactions and physical conflict resulted in large increases of fecal glucocorticoid metabolites in both protagonists. Fecal steroid assessment is therefore of use in monitoring adrenal activity in spotted hyenas.
In physiological studies of free-living species, it is essential to consider the context of the life history stage at which an individual was observed in order to link measures of physiology with ecological parameters. One such measure that is important to consider is the age of an individual. We tested whether baseline or stress-induced corticosterone levels vary with age in free-living Florida scrub-jays (Aphelocoma coerulescens) during the pre-breeding period. Corticosterone (CORT), the primary avian stress hormone, is released in response to stressful stimuli, and stimulates gluconeogenesis; however, it also serves as a chemical messenger that can influence other physiological processes, reproduction, and behavior. We monitored both baseline CORT levels longitudinally throughout a five-year period and stress-induced CORT responses over a shorter two-year period. We predicted that older jays would have lower baseline CORT levels and a dampened stress response compared to younger birds, as has been shown in other avian species. We found no significant differences in baseline CORT levels with age. We found a decrease in total corticosterone responses to a stressor with age, however, the oldest birds in the population showed greater total corticosterone responses to a stressor. These results may be a product of age-related changes in physiological processes related to the stress response or a result of selection acting on the population, resulting in only the most responsive individuals surviving to old age.
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 thefirst 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 mgg–1 bodyweight), indicating that the corticosterone enzyme-immunoassay could detect changes in circulating orticosterone 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. Additional keywords: adrenocorticotropic hormone, capture, stress, urine Yes Yes
Prolonged or frequent secretion of adrenal glucocorticoids in response to aversive stimuli can negatively impact reproduction. Because female southern white rhinoceros (Ceratotherium simum simum) reproduce poorly in captivity, we compared fecal corticosterone metabolite concentrations among parous, nulliparous, and adolescent females and examined social and physical aspects of the captive environment that might be related to differences in corticosterone metabolite concentrations. Aggression, dominance, sexual and play interactions, social group size and composition, enclosure size, and other housing characteristics were assessed though behavioral observations and review of historical and institution records. Concentrations of metabolized corticosterone in fecal samples were analyzed by enzyme immunoassay. The proportion of nulliparous females did not differ (p>0.05) between subordinate and dominant animals, and subordinates did not have a higher mean fecal corticosterone concentration than dominants (p>0.05). Of the behaviors examined, only the frequency of sexual play behaviors differed (p<0.05) between dominants and subordinates. Average corticosterone concentrations differed (p<0.05) across housing institutions but were not consistently elevated (p>0.05) for females housed in most of the environmental conditions assessed. Housing with a female companion known from adolescence, however, tended to be associated (p=0.06) with a lower mean corticosterone concentration than that when housing with a female companion introduced during adulthood or no female companion. Wild-caught females had a higher (p<0.05) average corticosterone concentration than captive-born females. Average corticosterone concentration did not differ (p>0.05) between acyclic and cycling, or nulliparous and parous females.
Bandicoots are opportunistic omnivores that feed on invertebrates, fungi and both epigeal and hypogeal plant parts. We examined the performance of the digestive tract of the long-nosed bandicoot (Perameles nasuta) in terms of intake and total digestibility, patterns of excretion of inert digesta markers, and likely sites of digesta retention, on two diets designed to mimic part of their natural plant and insect diets. On the insect diet (mealworm larvae), bandicoots virtually maintained body mass at a digestible energy intake of 511 kJ.kg-0.75.day-1 and were in strongly positive nitrogen balance. In contrast, on the plant diet (shredded sweet potato), bandicoots ate only one-third as much digestible energy, lost 7% body mass, and were in negative nitrogen balance. Mean retention times of two particle markers on the plant diet (27.5 and 27.0 h) were more than double those on the insect diet (12.4 and 11.2 h), and on both diets the mean retention time of the fluid digesta marker was greater than those of the particle markers, indicating consistent selective retention of fluid digesta in the gut. It was seen radiographically than in mealworm-fed bandicoots major sites of digesta retention were the distal colon and rectum, whereas in the sweet potato-fed animals the caecum and proximal colon were principal sites.(ABSTRACT TRUNCATED AT 250 WORDS)
The ACTH response to CRF and the role of glucocorticoids on the pituitary-adrenal responsiveness to acute stressors after a period of chronic stress were assessed in Sprague-Dawley rats. After chronic immobilization (IMO) an enhanced ACTH response to CRF administration was observed. In another experiment, control and chronic IMO rats were adrenalectomized (ADX) or sham-adrenalectomized (SHAM) and 2 days later killed in resting conditions or after having been subjected to acute IMO or tail-shock for 30 min. Chronic IMO increased basal corticosterone but not adrenocorticotropin (ACTH) levels in SHAM rats. As expected, ADX increased ACTH levels in all conditions. In response to the novel acute stressor (shock), ACTH levels were drastically dependent on the presence of corticosterone: thus, whereas in SHAM rats chronic IMO reduced the ACTH response to shock, in ADX rats a clear enhancement of the ACTH response to shock was observed in chronic IMO rats. In order to demonstrate that, in our experimental conditions, chronic stress also induces down-regulation of glucocorticoid receptors in the hippocampus, an additional experiment was done: rats subjected chronically to IMO or administered 5 mg corticosterone (B) were adrenalectomized and killed 20 h later under basal conditions. Both chronic IMO and chronic B administration decreased glucocorticoid type II binding in the hippocampus. From these results, it is concluded that chronic IMO induces facilitation of the ACTH response to novel acute stressors which is uncovered after corticosterone removal.