Since colonisation, Australia has witnessed widespread and devastating biodiversity losses and currently possesses the highest rate of mammalian extinctions globally. Marsupials are disproportionally impacted compared to other groups and remain particularly vulnerable to environmental threats. Over the past decade, assisted reproductive technologies (ARTs), including in vitro fertilisation (IVF), have been recognised as promising tools for conservation. However, progress in ART for Australian marsupials has been limited, and IVF protocols are yet to be established.
In this study, we used the fat-tailed dunnart (Sminthopsis crassicaudata) as a laboratory-based model to establish protocols for inducing and assessing sperm capacitation, a critical step towards achieving IVF. Epididymal spermatozoa were collected from adult dunnarts via swim-out and incubated in one of nine capacitation media. Sperm motility, longevity, and capacitation-related morphological changes were monitored. We adapted and optimised quantitative assays for motility, morphology, tyrosine phosphorylation, and the acrosome reaction for dunnart sperm.
Preliminary results indicated that a newly developed culture medium supports sustained progressive motility and morphological changes associated with capacitation in dunnart sperm under defined incubation conditions. The medium outperformed a standard comparative formulation, maintaining a significantly higher motility over a two hour period. By four hours, although overall motility declined in both media, the majority of motile sperm in the novel formulation exhibited morphological features consistent with capacitation. Further analyses are underway to expand replicates and quantify key capacitation-associated parameters across additional formulations.
These findings show that a high proportion of dunnart motility can be supported in simple media, and further challenges the notion that marsupial sperm requires complex media conditions to undergo capacitation. This work advances our understanding of marsupial sperm biology, and lays essential groundwork for optimisation of IVF protocols in marsupials, with the ultimate goal of developing next-generation conservation tools for threatened species.