143. THE FIRST EVIDENCE OF HIGH SUSCEPTIBILITY TO COLD SHOCK BY THE SPERMATOZOA OF A MARSUPIAL, THE FAT TAILED DUNNART (SMINTHOPSIS CRASSICAUDATA)

Abstract

Carnivorous marsupials are native Australian predators including the highly threatened northern quoll (Dasyurus hallucatus) and Tasmanian devil (Sarcophilus harrisii). These species are currently actively managed in captive populations but assisted reproductive techniques such as gamete banking may also contribute to their conservation. Previous studies on a model dasyurid, the fat tailed dunnart (Sminthopsis crassicaudata), have found that spermatozoa do not survive freezing and thawing using a variety of freezing protocols and cryoprotectants. We have re-examined cold shock to investigate problems with sperm cryopreservation in S. crassicaudata. Epididymal spermatozoa were rapidly cooled to 0.5ºC in a pre-cooled tube held in an iced water slurry and upon re-warming the spermatozoa were non-motile (n=6). The addition of up to 20% egg yolk, which is considered protective to the spermatozoa of cold shock sensitive eutherians, did not improve the outcome (n=6). Similarly when S. crassicaudata spermatozoa were rapidly cooled to 4ºC, just 2% remained motile upon re-warming (n=10). However when spermatozoa were combined with at least 10% egg yolk and rapidly cooled to 4ºC only small reductions in motility were observed upon rewarming (n≥8). In order to achieve motile spermatozoa at 0ºC, controlled rate cooling at 0.5ºC/minute was examined. In the absence of egg yolk there was a decline in the percentage of motile spermatozoa below 4ºC (n=6). However if spermatozoa were combined with at least 10% egg yolk there was no significant loss of motility at temperatures as low as 0ºC (n=6). This study has revealed that at least one species of marsupial is highly susceptible to cold shock. These paradigm shifting findings give direction to future experiments aiming to develop a robust technique for sperm preservation in endangered dasyurids.