Since European colonisation, Australia has faced devastating losses in biodiversity, with native marsupials particularly vulnerable to immediate threats. Assisted reproductive technologies (ART) such as in vitro fertilisation (IVF) offer potential to transform conservation efforts for some species. While ART have been used to support conservation for numerous eutherian mammals, conventional IVF has not yet been optimised to produce live young in any Australian marsupial. It has been suggested that this is, in part, because marsupial sperm are unable to undergo capacitation in vitro. Using the fat-tailed dunnart (Sminthopsis crassicaudata, family: Dasyuridae) as a model, this research aims to improve in vitro conditions for sperm motility and survival, and further optimise to induce capacitation.
Adult male dunnarts between 250-400 days old were used for sperm and tissue collection. Testis and epididymal tissues were analysed using routine histological techniques, to give context to the environment in which spermatozoa develop and mature. Sperm were collected from cauda epididymides by swim-out in one of nine base media commonly used in capacitation or IVF research. Media composition, temperature and pH were adjusted to assess effects on motility, survival and capacitation-associated changes. Sperm responses were determined using manual motility scoring, head orientation, acrosome reaction and visualisation of tyrosine phosphorylation.
Preliminary results indicate that sperm show improved and sustained progressive motility in HEPES-buffered Human Tubal Fluid with addition of 6mM fructose, and incubated at 35C under atmospheric oxygen. After two hours, these conditions sustain a high proportion of progressively motile sperm with T-shaped head orientation, indicative of capacitation in marsupials. This work demonstrates the highest proportion of sustained progressive motility seen in dasyurid sperm to date, with promising indications of capacitation, facilitating the next steps in IVF research for conservation.