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.

As an integral part of the development of an artificial insemination programme in the captive koala, female reproductive physiology and behaviour were studied. The oestrous cycle in non-mated and mated koalas was characterized by means of behavioural oestrus, morphology of external genitalia and changes in the peripheral plasma concentrations of oestradiol and progestogen. The mean (+/- SEM) duration of the non-mated oestrous cycle and duration of oestrus in 12 koalas was 32.9 +/- 1.1 (n = 22) and 10.3 +/- 0.9 (n = 24) days, respectively. Although the commencement of oestrous behaviour was associated with increasing or high concentrations of oestradiol, there were no consistent changes in the morphology or appearance of the clitoris, pericloacal region, pouch or mammary teats that could be used to characterize the non-mated cycle. As progestogen concentrations remained at basal values throughout the interoestrous period, non-mated cycles were considered non-luteal and presumed anovulatory. After mating of the 12 koalas, six females gave birth with a mean (+/- SEM) gestation of 34.8 +/- 0.3 days, whereas the remaining six non-parturient females returned to oestrus 49.5 +/- 1. 0 days later. After mating, oestrous behaviour ceased and the progestogen profile showed a significant increase in both pregnant and non-parturient females, indicating that a luteal phase had been induced by the physical act of mating. Progestogen concentrations throughout the luteal phase of the pregnant females were significantly higher than those of non-parturient females. Parturition was associated with a decreasing concentration of progestogen, which was increased above that of basal concentrations until 7 days post partum.

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Captive breeding programs are increasingly being used as a management option for threatened mammals. The greater bilby Macrotis lagotis, for example, is a vulnerable species which is maintained in captivity at several facilities in Australia. Non-invasive evaluation of stress hormones (cortisol in mammals) via excretory metabolites can be used to monitor physiological stress responses of captive individuals. In this study, we validated an enzyme-immunoassay (EIA) to measure cortisol metabolites in fresh faecal samples of adult male and female bilbies (n = 7) held in captivity at the Dreamworld Theme Park, Queensland, Australia. The faecal cortisol EIA was validated via parallelism and the recovery of exogenous cortisol added to pooled faecal extracts (> 99% recovery). Female bilbies had higher average faecal cortisol metabolite concentrations and higher day-today variation than male bilbies; however, there was no relationship with bilby age. Cortisol metabolites for most individuals varied widely through time, with numerous peaks and troughs in response to long-term stressors (illnesses, injury and reproductive issues) and 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 cortisol metabolite concentrations of individuals suffering long-term stress was related to a greater response to short-term stressors. This suggests an interaction between responses to short-term and long-term stressors which is perhaps due to habituation and/or facilitation of long-term stressors. Non-invasive faecal monitoring of stress hormones could provide further information on the implications of captive breeding programs and the release of animals reared in captivity.

Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of intermolecular disulfide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulfide bonds within their chromatin, but have been successfully cryopreserved. The present study examined the hypothesis that the cryoprotectants used for fish sperm cryopreservation would confer a similar degree of protection on koala spermatozoa. Three concentrations each of five cryoprotectants (dimethyl sulfoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide (DMA)) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Post-thaw assessment of progressive motility, plasma membrane integrity and mitochondrial membrane potential (MMP) revealed that protocols using 15% DMA achieved 62.2 +/- 3.6% (P < 0.05) sperm survival, of which 79% (P < 0.05) had high MMP, an improvement of 32% and 40%, respectively, over sperm frozen in 14% glycerol. The percentage of spermatozoa with swollen nuclei was also lowest when frozen in 15% DMA, both immediately after thawing (18.0 +/- 3.5%; P < 0.05) and after 2 h incubation at 35 degrees C (35.8 +/- 4.4%; P < 0.05). A second study was conducted to determine the optimal concentration of DMA for use in the cryopreservation of koala spermatozoa. High DMA concentrations (17.5% and 20%) resulted in significantly lower proportions of live spermatozoa showing high MMP immediately after thawing compared with spermatozoa frozen in the lower concentrations. The percentage of koala spermatozoa with swollen chromatin following cryopreservation was not affected by DMA concentration.