The objective of this thesis was to investigate the factors controlling the reproductive cycle of the female koala and develop an oestrous synchronization technique to facilitate the further refinement and efficacy of artificial insemination (AI) in the koala. A component of this development was the establishment of less invasive strategies to accurately monitor the reproductive status of the female koala, without the need for physical restraint or serial venipuncture. Chapter 2 of this thesis evaluated the efficacy of faecal oestrogen analysis, combined with the detection of oestrous behaviour as a control, to monitor ovarian cycles. Whilst faecal oestrogens did not correlate well with plasma oestradiol-17β to allow for an accurate estimate of cycle length or indicate or predict the precise timing of oestrus, the individual mean faecal oestrogen concentrations did, however, show a strong relationship to the individual mean plasma oestradiol-17β concentrations for each koala; in addition total faecal oestrogens were significantly higher in cycling females (P = 0.007).
Chapters 3 and 4 of this thesis examined factors controlling reproductive cyclicity in the female koala. The pattern of prolactin (Prl) secretion and its relationship to oestrous behaviour and pouch young (PY) development throughout lactation in the koala was investigated in C]hapter 3. Oestrous behaviour was suppressed throughout the majority of lactation despite basal levels of Prl during early lactation. Koalas returned to oestrus some 102 days before PY had reached independence. The koala anterior pituitary also remained responsive in terms of luteinizing hormone (LH) secretion to injections of mammalian gonadotrophin-releasing hormone (mGnRH) during periods of high and low Prl secretion. Chapter 4 investigated the seasonality of oestrous behaviour (oestrous cycle activity) in a captive koala population in Southeast Queensland (SEQ). Although some individual koalas in the population showed signs of oestrous behaviour throughout the year, an obvious seasonality was apparent with significantly less females displaying oestrous behaviour in late autumn and winter (May - August), than September to April (P < 0.0001). While average monthly photoperiod (P < 0.0001) and average monthly temperature (P < 0.0001) were associated with oestrous behaviour, rainfall was not (P = 0.097).
Chapters 5 and 6 report studies which investigated techniques, designed to allow for the manipulation and planning of timed insemination of female koalas. The impact of the gonadotrophin-releasing hormone (GnRH) antagonist, azaline B on LH and ovarian steroid hormone secretion in response to stimulation with mGnRH and its potential application in an oestrous synchronization protocol in cycling koalas was examined in chapter 5. In experiment 1, single sub-cutaneous (SC) injections of azaline B successfully blocked the ability of the anterior pituitary (AP) to respond to exogenous mGnRH in a dose dependant manner: 0 mg (n = 4) did not suppress LH response, 1 mg (n = 6) suppressed LH response for 24h (P<0.05), 3.3 mg (n = 8) suppressed LH response for 3h (P<0.05) and 10 mg (n = 4) suppressed LH response for 7 d (P<0.05). In experiment 2, daily 1 mg SC injections of azaline B over a 10 d period during seasonal anoestrus (June – July) (n = 6), suppressed (P<0.01) the LH response to mGnRH and the LH response did not recover four days after cessation of treatment. Experiment 3 was designed to test the efficacy of a daily 1 mg SC dose of azaline B over 10 days to suppress plasma LH and oestradiol-17β concentrations and ultimately synchronize timed return to oestrus during the breeding season. Whilst treatment with azaline B did not suppress basal LH or oestradiol-17β, oestrus was delayed in all treated females by 24.2 ± 5.0 days, but this period was highly variable (range 9 - 39d). Overall, this study demonstrated the GnRH antagonist azaline B in koalas is able to inhibit the LH response to exogenous mGnRH and successfully delay the return to oestrus. However, whilst azaline B appears to clearly disrupt folliculogenesis, it was not able to effectively synchronise return to oestrus in the koala.
The ability of the synthetic progestogen implant levenorgestrel (LNG) to control ovarian activity for the purposes of oestrous synchronisation in the koala was investigated in chapter 6. Following implantation, LNG treated koalas immediately ceased displaying oestrous behaviour, showed reduced oestradiol-17β secretion to basal levels and no evidence of urogenital cytology consistent with a koala in oestrus. In contrast, plasma oestradiol-17β levels in control koalas showed evidence of cyclic activity associated with periods of behavioural oestrus and a coincident increase in the proportion of cornified epithelial cells in the urogenital smear at day 33 to 35 after saline injection. LNG treated koalas showed a subsequent oestrus 13, 14, 17 and 30 days after LNG implant removal and continued to produce PY in subsequent breeding seasons. This suggests that LNG implants can inhibit oestrous behaviour and elevated secretion of oestradiol-17β, most likely through preventing sufficient development of a pre-ovulatory follicle. While removal of the implant resulted in the synchronous return of oestrus in 3 of the 4 treated koalas, further studies on a larger population are required to validate this finding.
Whilst a reliable oestrous synchronization protocol was not achieved, this thesis has provided significant insight into the mechanisms controlling reproductive activity in the koala which can now form the basis for future research. These include: 1) Preliminary evidence for the importance of the suckling stimulus, not prolactin secretion, in the suppression of koala oestrous behaviour during early lactation, 2) Evidence for a clear seasonality in captive female koala reproductive activity, 3) The ability to temporarily disrupt the koala follicular cycle using repeated doses of azaline B, 4) Preliminary evidence for the use of synthetic progestogen as a mechanism for oestrous synchronization in the koala.