Assisted breeding technology (ART), including artificial insemination (AI), has the potential to advance the conservation and welfare of marsupials. Many of the challenges facing AI and ART for marsupials are shared with other wild species. However, the marsupial mode of reproduction and development also poses unique challenges and opportunities. For the vast majority of marsupials, there is a dearth of knowledge regarding basic reproductive biology to guide an AI strategy. For threatened or endangered species, only the most basic reproductive information is available in most cases, if at all. Artificial insemination has been used to produce viable young in two marsupial species, the koala and tammar wallaby. However, in these species the timing of ovulation can be predicted with considerably more confidence than in any other marsupial. In a limited number of other marsupials, such precise timing of ovulation has only been achieved using hormonal treatment leading to conception but not live young. A unique marsupial ART strategy which has been shown to have promise is cross-fostering; the transfer of pouch young of a threatened species to the pouches of foster mothers of a common related species as a means to increase productivity. For the foreseeable future, except for a few highly iconic or well studied species, there is unlikely to be sufficient reproductive information on which to base AI. However, if more generic approaches can be developed; such as ICSI (to generate embryos) and female synchronization (to provide oocyte donors or embryo recipients), then the prospects for broader application of AI/ART to marsupials are promising. #

The mammalian ovary contains numerous immature preantral follicles that are not dependent on endocrine support, unlike the more mature hormone-dependent antral follicles. Preantral follicles can be enzymatically dissociated to yield immature oocytes that survive sub-zero preservation better as they lack a temperature-sensitive meiotic spindle. These techniques are highly applicable to gamete banking, which is an urgent requirement for Australian carnivorous marsupials as several species have rapidly declining populations and risk extinction. The present study developed protocols for the transport, dissociation, preservation and culture of granulosa cell-oocyte complexes (GOC) from the ovaries of dasyurid marsupials. High viability of GOC following enzymatic dissociation is reported and it was demonstrated that GOC are of significantly better quality following refrigerated storage for 24 h compared with storage at room temperature. Oocytes from primary follicles were not damaged by cold shock or the toxicity of vitrification media and following vitrification in liquid nitrogen 69.42+/-2.44% of oocytes were viable. However, the surrounding granulosa cells demonstrated significant damage post-thaw. These granulosa cells proliferated during a 48-h culture period resulting in significant improvements in GOC quality. The present study is a valuable step towards cryostorage of dasyurid gametes and represents fundamentally important methods by which we can contribute to the conservation of Australia's native predators.

Non-invasive techniques were used to investigate the reproductive biology of captive squirrel gliders (Petaurus norfolcensis) for 3 months during the breeding season. The squirrel glider is a medium-sized marsupial glider of eastern Australia and is currently listed as a threatened species as a result of habitat destruction and fragmentation. Urinary cytology was used to determine the timing of oestrus, and the presence of sperm confirmed mating. Progesterone and oestradiol-17β were identified in faecal samples via thin-layer chromatography, and were used to characterise the reproductive cycle. Reproductive activity was observed in three of four females, with births occurring during June and July. A preoestrus increase in faecal oestradiol-17β was detected in a single female, whilst significant increases occurred post partum (±2 days) in two of four females, suggesting that the squirrel glider may undergo a postpartum oestrus. Faecal progesterone profiles showed low concentrations before oestrus and significantly elevated concentrations after oestrus, which were maintained throughout pregnancy. Parturition coincided with a decrease in progesterone concentrations (±1 day). This study successfully used non-invasive monitoring of urinary cytology and faecal steroids to define luteal and gestational length as 16–17 days, a previously unpublished detail.

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