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Eur J Wildl Res DOI 10.1007/s10344-014-0842-z ORIGINAL PAPER Monitoring physiological stress in semi-free ranging populations of an endangered Australian marsupial, the Greater Bilby (Macrotis lagotis) Edward J. Narayan & Nicole Evans & Jean-Marc Hero Received: 5 December 2013 / Revised: 10 June 2014 / Accepted: 3 July 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract 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 Communicated by A. W. Sainsbury E. J. Narayan (*) : N. Evans : J.<M. Hero Environmental Futures Research Institute, School of Environment, Griffith University, Gold Coast campus, Gold Coast QLD 4222, Australia e-mail: [email protected] success of management interventions (such as predator free enclosures) and support the survival of the species. Keywords Australian marsupial . Macrotis lagotis . Semi-wild populations . Predation . Conservation physiology . Physiological stress Introduction Conservation physiology has most recently been defined as ‘an integrative scientific discipline applying physiological concepts, tools, and knowledge to characterizing biological diversity and its ecological implications; understanding and predicting how organisms, populations, and ecosystems respond to environmental change and stressors; and solving conservation problems across the broad range of taxa (i.e., including microbes, plants, and animals) (Cooke et al. 2013, p. 1). Over the past few decades, there has been an increase in the use of non-invasive methods for the assessment of glucocorticoids or stress hormones (cortisol) in mammals. The stress endocrine system in mammals is the hypothalamic–pituitary–adrenal (HPA) axis. Through intrinsic negative feedback mechanisms between the brain and the adrenals, the HPA axis regulates the release of cortisol from the adrenal glands during exposure to environmental stress factors (Young et al. 2004). Cortisol secretion alters important biochemical processes, such as metabolism, which then leads to behavioral modifications that enable animals to cope with acute stimulus, such as predator presence (Wikelski and Cooke 2006; Narayan 2013; Narayan et al. 2013a). The process of physiological stress adaptation maintains the internal homeostatic balance of the animal’s HPA axis with the environment. If the environmental stimuli become prolonged, such as during extreme hot weather and drought, then the HPA axis enters into a disrupted state whereby the return of cortisol levels to baseline levels become difficult (Narayan and Hero 2014a). Eur J Wildl Res Consequently, sustained release of corticosteroids from the adrenal glands often also lead to disruptive effects on the reproductive hormones and immune systems, cognition, performance and behaviour, as shown in mammals (Muller and Wrangham 2004; Sheriff et al. 2009), birds (Ellenberg et al. 2007; Bonier et al. 2009) and amphibians (Narayan and Hero 2014a, b). Fecal cortisol metabolite (FCM) analysis is used frequently in conservation physiology programs for mammals (Dantzer et al. 2010; Marechal et al. 2011; Fanson et al. 2012),birds (Palme 2005; Wasser and Hunt 2005),and to a lesser extent, reptiles (Romero and Wikelski 2001),and fishes (Turner et al. 2003; Ellis et al. 2004). Non-invasive FCM analysis has contributed new knowledge in relation to wildlife management (Bosacker 2008; Thaker et al. 2010), population density (Kalz et al. 2006; Park et al. 2011), behaviour and social status (Muller and Wrangham 2004), reproduction (Hesterman et al. 2008), sociality (Bosacker 2008), territoriality (Barja et al. 2008), animal welfare (Owen et al. 2004), health status (Chapman et al. 2007), predation threat (Sheriff et al. 2009), and to predict population declines and mortality (McDonald et al. 1981; Ellenberg et al. 2007). As with any emerging quantitative physiological method, non-invasive analysis of glucocorticoids has pros and cons that need to be considered for each new study species for which this technique is being developed (Narayan 2013). Earlier, Sheriff et al. (2011) reviewed the technical considerations for standardizing the use of FCM analysis as a biomarker of stress in mammals. An important validation step includes biological challenge, using exogenous adrenocorticotrophic hormone (ACTH), which is used to test whether the assay system is able to reliably measure the metabolites of target hormone in excreta of the study species (Narayan et al. 2010, 2013b, c). Australia is a biodiversity-rich continent; however, it also has recent high record of mammalian extinctions and species endangerment, with 21 species already extinct and 31 species listed as endangered or critically endangered (IUCN 2006; James and Eldridge 2007). The greater bilby (Macrotis lagotis) is one of Australia’s most threatened animals and is currently listed as Vulnerable (C1 ver 3.1) by the IUCN Red List (IUCN 2006). Once widespread across 70 % of mainland Australia, this burrowing marsupial has disappeared from 90 % of its historical range and now exists only in fragmented populations in the driest and least fertile parts of its former range (Gibson 2001; Smith et al. 2009). 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. The aim of the current study was to develop a non-invasive tool that could be used to assess adrenal activity in the semifree ranging populations of the bilby. The goal is that the methods developed could be used in future studies to assess physiological sensitivity to environmental change in this vulnerable endemic Australian marsupial — aiming to make suggested in situ management changes — protecting the species. Material and methods Study sites: captivity Table 1 provides detailed information regarding the four captive study sites. The bilby husbandry and veterinary databases from each site provided information about age, sex and health status along with daily activities of individual bilby. Study sites: semi-free ranging Currawinya National Park (semi-wild population 1; SW-1), located in south-western Queensland (28.6817 S, 144.6978 E) contains a 25-km2 predator-proof enclosure that was built in the National Park in 2003 to protect the reintroduced population of the greater bilby from ground dwelling predators (Dunwoody et al. 2009; DEHP 2012). Unfortunately, during the time of sampling the predator-proof exclusion fence had been compromised by recurrent rains that caused corrosion and enabled the infiltration of feral cats (F. catus) into the enclosure. Consequently, there was a dramatic decline (the population was reported as critically low) of the bilby population residing within the fence (Williams and Manthey 2012). Scotia Wildlife Sanctuary (SWS) (Semi-wild population 2; SW2) (64,653 ha; 141.100 E, 33.100 S) is located 150 km south of Broken Hill, on the boundary of the arid and semiarid climatic zones of New South Wales. Greater bilbies were reintroduced within a fenced area (8,000 ha) between 2004 and 2005.A total of 2,103 individual bilbies were residing in the fenced area at the Sanctuary (Finlayson et al. 2008; AWC 2012). Bilby fecal samples were collected within 210 ha area of the fenced area, which also contained bridled nail-tail wallaby (Onychogalea fraenata). Fecal sampling: captive At all four captive locations (Table 1), the entire enclosures were racked in the evening to remove old feces. Fecal samples were then collected daily early morning (0600 hours) by husbandry staff over a period of 14 consecutive days during March and April of 2012. Fresh fecal samples (defecated within the previous 12 h) were collected from each enclosure Both individuals were born in captivity at the Charleville Breeding Centre. Fed a diet of dog kibble and bird seed. 2 male bilbies Charleville Breeding Centre (CBC), located in Charleville, south-western Queensland (C4) in a single zip-lock bag (labelled with the date and the animal’s ID) and stored in a −20 °C freezer prior to processing. Identification of fecal samples in grouped housing: a fecal marker technique Each site is coded (C1, C2, C3 or C4) and discussions are made with reference to these codes None reported. Fed a diet of seed, dog kibble, vegetables and various live insects daily. 2 male bilbies Currumbin Wildlife Sanctuary (CWS), Gold Coast, Queensland (C3) All individuals were housed separately. The female’s enclosure consisted of a 10×2.5 m area bedded with coarse red sand, vegetation and a permanent artificial burrow site. The male enclosure consisted of a 3×5 m fenced area bedded with dry fine sand with two enclosed sleeping dens (0.6×0.5 m) bedded with blankets and towels. Housed together on permanent display. 3.6×2.5 m area bedded with coarse red sand. Vegetation provided along with a permanent artificial burrow site. Large separate outdoor enclosures (30×20 m) located away from tourist activities, large enough to facilitate selfforaging and natural burrowing behaviours. Ipswich Nature Centre (INC), Ipswich, Queensland (C2) 2 male and 2 male bilbies All but two female bilbies participated in educational shows. Male individuals participated in a school educational visit on day 10 of sampling involving transport and manual handling. Fed whole grains and carnivore dental chews supplemented occasionally with whole insects. Fed a diet of whole oats, feline bites, dog kibble and a range of insects daily. All individuals were housed separately. 3 male and 4 female bilbies Dreamworld Theme Park, Gold Coast, Queensland (C1) Housing and enclosure description Animals Captive study sites Table 1 Detailed information regarding the four captive study sites Husbandry regime Activities Eur J Wildl Res A fecal marker was used to identify the feces of bilbies present in grouped housing at the Ipswich Nature Centre (INC) and Currumbin Wildlife Sanctuary (CWS). Powdered bakers green food colouring (Baking Pleasures; ABN: 23 717 428 010; Queensland, Australia) was used as the fecal marker. Similar products have been used previously to aid fecal identification in a range of mammal species, such as the numbat (Myrmecobius fasciatus) (Hogan et al. 2013) and domestic cats (Griffin 2002). At each grouped housing location, one bilby (Individual A) received a 2-g dose of powdered bakers food colouring mixed through regular daily food for the duration of the 14-day sampling period. Individuals were separated for feeding to ensure that only one bilby was exposed to the food colouring. All feces showing bright green colouration were identified as belonging to Individual A. All fecal samples that were absent of any artificial colour were identified as belonging to Individual B and were analysed separately. Individuals exposed to the bakers powered food colouring showed no aversion to taste or consistency of the powdered food colouring when combined with daily food. The individuals consumed all food as usual and no adverse gastrointestinal effects were observed. Visual inspection of fecal samples confirmed the successful application of bakers’ powdered food colouring for marking an individual’s feces. Coloured feces (green) were present within 48 h of initial administration of food colouring and colour intensity was sufficient to enable the identification of feces containing colour and those that did not. Colour saturation was consistent in all feces throughout the 14-day sampling period. Serial dilutions of XGreen bakers powdered food colouring did not show parallelism with serial dilutions of cortisol standard (R4866) and there was significant difference between bakers powdered food colouring serial dilution and cortisol standard serial dilutions (t=−2.12, p=0.040). This indicated that the XGreen bakers powdered food colouring does not contain a significant amount of cortisol hormone and as such did not influence the assay of cortisol metabolites in bilby feces. Fecal sampling: semi-free ranging Fecal samples were collected from the semi-free ranging populations’ using a minimum of ten 250 m×60 cm strip transects. Transects were positioned at a minimum of 1 km apart in order to ensure independence of samples. Samples were collected over 14 days at CNP during March 2012, and over 7 days at the SWS during July of 2012. Transects were raked to clear old feces and leaf litter in the evening, and Eur J Wildl Res subsequently visited daily for up to 14 days. All fresh (<12 h) fecal samples found within each transect were collected in ziplock bags (labelled with date, time and transect) and stored on ice packs until returning from the field site. Fresh feces were characterised by a layer of mucus, a strong smell, and no signs of dehydration. Only a single fecal pellet was collected from each transect to avoid pseudo-replication within and between transects. Samples were then stored in a −20 °C freezer prior to assay. Over the 7-day sampling period, a total of 28 fresh bilby fecal samples were collected from SWS. Due to the extremely low density of the bilby population at CNP during the time of sampling, only five fresh fecal samples were collected. Fecal cortisol metabolite enzyme immunoassay: laboratory and physiological validations 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. Recovery of cortisol standard that was added to pooled bilby fecal extracts was y= 0.98x+1.26, r2 =0.997 (n=7), where y is the concentration observed and x is the concentration expected resulting in an extraction efficiency of >99 %. Assay sensitivity was calculated as 1.2±0.2 ng/well (n=10). Intra- (within) and inter(between) assay coefficients of variation (CV) were 2.3 % and 6.4 % for the high-binding internal control and 1.9 % and 14.3 % for the low-binding internal control, respectively (n= 15). Serial dilutions of bilby fecal extracts yielded displacement curves parallel to those of the cortisol standard. FCM concentrations were presented as ng/g FCM net dry feces. Physiological validation was done using administration of adrenocorticotropic hormone (ACTH) to four captive individuals (one male and one female from Dreamworld and two males from CWS). Individuals were removed from their enclosure and manually restrained whilst 250 μl of Synacthen (Provet Pty Ltd, Australia). Synacthen was available in a glass ampoule containing 1 ml liquid. Each ampoule contains 250 μg of tetracosactide (as the hexa-acetate salt). Each bilby received a quarter of the 1-ml dose that was administered as a single intramuscular injection (Dose = 62.5 μg of tetracosactide per total body weight of an adult bilby). Daily fecal samples were collected from 2 to 3 days prior to and 4 to 5 days after injection based on sampling methods described above. This dose and similar daily sampling design was previously used for studies on the southern hairy nosed wombat (Lasiorhinus latifrons) and in the roe deer (Capreolus capreolus) (Dehnhard et al. 2001; Hogan et al. 2011). Each individual then acted as its own control during a saline challenge that was performed 3 weeks after the ACTH challenge. The procedure followed the same protocol as for the ACTH challenge; however, this time, the animals received an injection of 250 μl sterile isotonic saline solution (0.9 % NaCl sol.), with a 7-day interval between the ACTH and saline injections. Animals were individually housed during the experiment and so all samples collected were of known origin. We calculated the percentage raise in FCM post injection of either ACTH or saline through time (days). Mean (±SE) FCM plotted for the ACTH and saline challenge data for visual interpretation of the results. FCM concentrations increased to the highest levels within 24 h of administration of the ACTH in male 1 and female 1 (Fig. 1). For male 2, the highest FCM level was noted on day 3 post ACTH challenge (Fig. 1). Male 3 showed a decrease in FCM level 1 day post ACTH challenge suggesting that the ACTH dose may have not been sufficient to elicit an adrenocortical response for this individual (Fig. 1). Individual peak responses were 14.49 ng/g net dry weight (first highest Female 1), 5.84 ng/g net dry weight (second highest Male 1) and 4.80 ng/g net dry weight (Male 2) (Fig. 1). Highest combined sexes mean concentration of FCM was detected at 24 h post ACTH injection at 6.25 ng/g (±2.8) FCM net dry weight (Fig. 2). Mean FCM concentrations increased by 280.31 % within 24 h of the ACTH injection, began to decline at day 2 and returned to near pre-treatment FCM levels by day 4 following ACTH injection (Fig. 2). There were no appreciable changes were recorded in FCM concentrations in any of the four bilbies subjected to the saline challenge with a 5 % decline in mean FCMs recorded on day 1 following injection (Fig. 2). Statistical analysis Statistical analysis were performed using and SYSTAT Version 13.0. All data were tested for normality using the Shapiro–Wilk normality test (applied to the residuals) and data were log-transformed where necessary to meet the assumptions of equal variances. For all analyses, cortisol was treated as the response variable and significance was assessed at the p<0.05 level. A Generalised Linear Model (GLM) ANOVA was used to compare the mean FCM levels between the captive groups (study sites), including location and sex as the factors, and sex×location interaction terms. Post-hoc comparisons between sexes for each study site were analysed using Tukey's honestly significant difference test. For the two semi-free ranging populations, FCM data are presented as individual points; one data point represents one individual sample per day. The statistical differences in mean FCM between the semi-free ranging sites and comparisons with the captive study groups were not analysed because of the fact that there were limited samples from the semi-free ranging sites, and due to the potential differences that were Eur J Wildl Res Fig. 1 Individual fecal cortisol metabolite concentrations (ng/g net dry weight) in bilby subjected to adrenocorticotropic hormone (ACTH) challenge. Day of injection is depicted using vertical dashed line unaccounted for such as sampling time of the year, reproductive state and diet. FCM levels in semi-wild bilby populations The average FCM concentration for samples from SWS was higher than that of Currawinya National Park (CNP) (2.78 ng/ g net dry weight cf. 2.26 ng/g FCM net dry weight). Results FCM levels in captive bilby populations: comparison between sites and between sexes Discussion There was a significant difference in mean FCM concentrations between the four captive populations (GLM ANOVA; F=16.53, p<0.001, Fig. 3). Male bilbies grouped in outdoor enclosure at the Charleville Breeding Centre (CBC) recorded the highest mean baseline FCM concentration at 0.89 ng/g net dry weight. Female bilbies at the INC recorded the second highest mean FCM at 0.74 ng/g net dry weight, followed by female bilbies at Dreamworld at 0.36 ng/g net dry weight (Fig. 3). Male bilbies at the INC recorded the third lowest level of mean FCM at 0.22 ng/g net dry weight. Male bilbies grouped in indoor enclosure at the CWS recorded the second lowest mean baseline FCM level at 0.132 ng/g net dry weight. Male bilbies at Dreamworld recorded the lowest mean baseline FCM level at 0.10 ng/g net dry weight (Fig. 3). As shown in Fig. 3, there was a significant difference in FCM levels between male (n=3) and female (n=4) greater bilbies housed at Dreamworld (t = −0.249, p < 0.001). Likewise, there was a significant difference in mean FCM levels between male and female bilbies housed at the INC (t=−0.481, p<0.001). We validated a fecal cortisol EIA for evaluating physiological stress in the Greater Bilby (M. lagotis). ACTH challenge causes a maximal cortisol response in mammals (Benhaiem et al. 2012). Our ACTH challenge results verify that the FCMs excreted after the ACTH injection were related to the stimulation of the HPA axis of the individual bilbies in response to the ACTH injections. The ‘rise and fall’ pattern showed the levels of metabolic end-products of free cortisol, denoted as FCMs. Our results showed that on average FCM levels should appear in bilby feces in response to acute stressor within 24 h. This approximation fits well with estimates of 8–12 h for average FCM expression for the snowshoe hare (Lepus americanus) (Sheriff et al. 2009), and 12 h in the red-backed vole (Clethrionoours gapperii) (Harper and Austad 2000). Actually, this period provides a critical window for obtaining baseline FCM data from semi-free ranging populations. For bilbies, if fecal samples can be collected from individuals that have defecated within less than 24 h after capture then prestimulus baseline FCM level can easily be obtained. Furthermore, we have demonstrated that fresh (<12–24 h Eur J Wildl Res Fig. 2 Mean (±SEM) fecal cortisol metabolite concentrations (ng/g net dry weight) in bilby subjected to adrenocorticotropic hormone (ACTH) (n=4) and saline (n=4) challenges on day 0. Fecal samples were collected 2 days prior to and 4 days post administration of ACTH and saline injections. Day of injection is depicted using vertical dashed line old) bilby feces are fairly stable to natural weathering conditions up to 13 days; that is, the FCMs in these feces are not deteriorated (Evans et al. 2013). Therefore, our sampling methods using transects provide a reliable way of obtaining fresh bilby fecal samples that will eventually be used for indicating the physiological sensitivity of semi-free ranging populations against environmental threats such as stochastic weather events (storms and droughts) and predation by feral pests. In some situations, identification of individual feces is usually problematic in situations such as group housing in captivity (Fuller et al. 2011). Our data confirms the success of bakers powdered food colouring (XGreen) as a fecal marker for the bilby. Administration of approximately 2 g of powdered food colouring once daily produced colour saturated feces consistently. Food colouring was easily mixed into food Fig. 3 Fecal cortisol metabolite concentrations (ng/g net dry weight) in bilby from all captive sampling locations. C1 Dreamworld Theme Park, C2 Ipswich Nature Centre, C3 Currumbin Wildlife Sanctuary, C4 Charleville Breeding Centre. C3 males were grouped indoor. C4 males were grouped outdoor. Sample sizes range from 2 to 4 bilbies per site. Level of significant difference between the sites and sexes are denoted by asterisks: ***p<0.001 vehicles and was largely recoverable in the feces. Earlier only one other study has successfully utilised food colourant to detect feces of individuals within group housed numbat (Myrmacobius fasciatus) (Hogan et al. 2013). Both semi-wild populations sampled in this study (SW1and SW2) were re-introduced within predator-proof fenced reserves, and play critical roles in the National Recovery Plan for the bilby (Pavey 2006; Finlayson et al. 2008). Unfortunately, prior to sampling at the SW1, the predator-proof fence had been compromised by recurrent rains that caused corrosion to the fencing. Furthermore, a substantial feral cat (F. catus) population was able to establish within the Currawinya National Park reserve (SW1) and has been singled out as the main reason for the decimation of this bilby population (Williams and Manthey 2012). Predation is a fundamental feature of the natural environment and is known to cause elevations of up to 89 % in glucocorticoid levels in prey animals (Cavigelli 1999; Sheriff et al. 2009; Thaker et al. 2010). It is known that the bilby is highly sensitive to the risk of predation (Pavey 2006) and even a slight increase in predator numbers may lead to elevations in FCM concentrations. In future, fecal hormone monitoring could be used to monitor the vulnerability of semi-wild bilby populations against feral prey species in situ. Ground-based predation was not a factor influencing the average baseline FCM concentrations recorded at the SWS (SW2). Intense management has ensured that the sanctuary remains free from feral animal species. The only issue is that the bilby population sampled at SW2 exists at high density (~1 individual per 3 ha) and co-inhabits the fenced compound with a particularly high-density population of bridled nail-tail wallaby (Onychogalea fraenata; ~2 wallabies/ha) (AWC 2012). It is evident that within closed populations high population densities lead to high levels of both intra- and inter-specific competition (Rogovin et al. 2003). At the SW2, as in any closed population, the issue of available space is pertinent and when combined with the particularly high density of bridled nail-tail wallabies with which bilbies share the area, the effects of competition are likely to be significant. Inter-specific competition has been known to change circadian activity rhythms, habitat preferences, and foraging sites and has been shown to cause significant elevations in FCM levels in mammals Buzzard 2006; Watts and Holekamp 2008; Forristal et al. 2012). Furthermore, when space becomes limited then access to food often also becomes limited (Creel and Creel 1998). The population of bridled nail-tail wallabies within the breeding compound at the SW2 has reached to a point where supplementary food is required (and is provided every other day) (AWC 2012). Whilst very little is known of the effects of supplementary feeding on FCM levels in mammals (Krebs 1996), one study reported that wild supplementary fed elk (Cervus elaphus) had FCM concentrations 43 % higher than non-supplementary fed elk during times of food scarcity and also documented strong Eur J Wildl Res correlations between FCM levels and animal density. It is believed that the artificial aggregation around feeding sites and the associated social interactions may be the primary cause behind these results (Forristal et al. 2012). Overall, it is clearly evident from our results that bilbies at both SW1 and SW2were experiencing stress to some degree due to the unpredictable environmental stimuli and factors discussed above. Furthermore, the mean FCM levels were pretty close to the highest mean level of FCM levels recorded in the bilbies after the ACTH challenge. Thus, comparatively it is likely that the HPA axis of bilbies in the semi-wild environment will express higher circulatory levels of cortisol. It will be interesting to investigate potential modulation of the adrenocortical response to ACTH in semi-wild bilbies in order to confirm whether these populations are indeed stressed. We strongly recommend that the data should be treated with caution when using them to explain whether the bilbies are facing distress (pain) or eustress (i.e., good stress). This is because there is no ‘gold standard’ level of bilby FCM level above which the animal is negatively stressed. The comparison of average FCM concentrations in captive populations revealed that individuals housed at the C4 had an appreciably higher average FCM level than the other three captive populations sampled. The most apparent difference between this population and the other captive populations is that these two individuals were housed in separate large, unroofed, outdoor enclosures. Bilbies housed at the C1, C2 and C3 were all housed in entirely indoor or roofed enclosures with access to an indoor area. It is possible that the elevated FCM levels recorded in the individuals housed at C4are related to their relative exposure to environmental conditions and aerial predation from birds of prey. Furthermore, individuals housed at C1, C2 and C3 were provided artificial burrows and ‘hides’ for resting and are fed within a daily routine, whereas the two males housed at C3 engage in natural behaviours such as foraging during the night and have to dig their own burrows for shelter. The environment in which an animal lives can greatly influence its biology (Schwarzenberger 2007). Past studies have also reported significant differences in average baseline FCM levels between captive and free-ranging populations of the same species. Some of which found that captive populations had significantly lower averages than their wild conspecifics (e.g., Gilbert’s potoroo (Potorous gilbertii); SteadRichardson et al. 2010) whereas other studies have demonstrated the opposite pattern. (Terio et al. 2004) reported that concentrations of baseline FCM were significantly higher in captive cheetahs (Acinonyx jubatus) than in free ranging cheetahs. Based on our findings of difference in mean FCM between the semi-wild and captive populations, we suggest that the wide range of unpredictable environment within the two fenced reserves (SW1 and SW2) sampled for this study are generally more challenging than the predictable stimuli (such as adequate security and environmental enrichment) offered in captivity. Similar results have also been reported in the Tuco-tuco (Ctenoours sociabilis) (Woodruff et al. 2010), and yellow-bellied marmot (Marmota flaviventris) (Smith et al. 2012). In conclusion, by determining average baseline FCM levels for the bilby in both semi-wild and captive populations, future research can now focus on investigating the causes of short term change and/or long-term elevations in FCM levels. The identification of sources of high FCM in semi-wild populations would provide an opportunity for wildlife managers to intervene and, for example, change social dynamics, housing or enrichment or implement feral animal control programs in order to minimise the effects on the animal’s well-being. Thus these findings have important implications for future management of threatened mammalian species by providing a simple quantification tool for monitoring the effects of environmental change on semi-free ranging populations. Acknowledgments This project was completed in accordance with approval from Griffith University’s Animal Ethics Committee (ENV/ 17/11/AEC). Field work was conducted under Queensland Department of Environment and Heritage Protection scientific permit number WITK10064911. This research was undertaken as an Honours Research Project by NE that was co-supervised jointly by EJN and J-MH. We would like to thank the Australian Wildlife Conservancy and Queensland National Parks and Wildlife Service for providing field equipment, onsite accommodation and other facilities whilst sampling in the field. Special thanks to numerous volunteers assisted with the fieldwork and Greg Lollback provided feedback on earlier version of this manuscript. We also thank the staff of Dreamworld, Ipswich Nature Centre, Currumbin Wildlife Sanctuary and Charleville Breeding centre for all their help and cooperation. We also thank the veterinarians Vere Nicolson and Michael Pyne who assisted with applying the ACTH and saline treatments. Funding was provided by Save the Bilby Fund and Griffith University. We are grateful to the editor and two anonymous referees for helpful reviews and comments. References AWC (2012) Scotia Sanctuary. http://www.australianwildlife.org/AWCSanctuaries/Scotia-Sanctuary.aspx. 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