Maximizing the reliability of non-invasive endocrine sampling in the tiger (Panthera tigris): environmental decay and intra-sample variation in faecal glucocorticoid metabolites.

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.

Evaluating physiological stress in Sumatran tigers (Panthera tigris ssp. sumatrae) managed in Australian zoos.

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.

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

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

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

Koalas (Phascolarctos cinereus) are the only extant representatives of Australia's unique marsupial family Phascolarctidae and were listed as nationally Vulnerable in 2012. Causes of mortality are diverse, although the disease chlamydiosis, dog attacks, collisions with cars, and loss of habitat represent the principal reasons for the continued species decline. Koala breeding facilities in Queensland and New South Wales, Australia have been established for conservation and tourism. Non-invasive monitoring of physiological stress is important for determining the sub-lethal effects of environmental stressors on the well-being, reproduction and survival of Koalas in Zoos and also in the wild. In this study, we developed a faecal cortisol metabolite (FCM) enzyme-immunoassay (EIA) for monitoring physiological stress in Koalas from two established Zoos in Australia and also within a free-living sub-population from Queensland. Biological validation of the FCM EIA was done using an adrenocorticotropic hormone (ACTH) challenge. We discovered excretory lag-times of FCM of 24 h in females (n=2) and 48 h in male (n=2) Koalas in response to the ACTH challenge. FCM levels showed an episodic and delayed peak response lasting up to 9 days post ACTH challenge. This finding should be taken into consideration when designing future experiments to study the impacts of short-term (acute) and chronic stressors on the Koalas. Laboratory validations were done using parallelism and recovery checks (extraction efficiency) of the cortisol standard against pooled Koala faecal extracts. Greater than 99% recovery of the cortisol standard was obtained as well as a parallel displacement curve against Koala faecal extracts. FCM levels of the captive Koalas (n=10 males and 13 females) significantly differed by sex, reproductive condition (lactating versus non-lactating Koalas) and the handling groups. Handled male Koalas had 200% higher FCM levels than their non-handled counterparts, while females were not affected by handling as long they were not undergoing lactation. There was no significant difference in FCM levels between the captive and wild Koalas (n=9 males and 7 females). Overall, these results provide foundation knowledge on non-invasive FCM analysis in this iconic Australian marsupial. Non-invasive stress endocrinology opens up opportunities for evaluating the sub-lethal physiological effects of management activities (including caging, translocation) on the nutritional status, reproductive behaviors and disease status of captive and managed in situ Koala populations.