A SIMPLE GLYCEROL-BASED FREEZING PROTOCOL FOR THE SEMEN OF A MARSUPIAL TRICHOSURUS-VULPECULA, THE COMMON BRUSHTAIL POSSUM

Frozen storage of semen and embryos is now a well established part of the breeding of many eutherian mammals but it has not been applied to marsupials. This paper reports the first successful technique for the frozen preservation of marsupial spermatozoa. Semen was collected by electroejaculation under anaesthesia from a pool of five brushtail possums. The ejaculated semen was diluted 1:1 with Krebs Henseleit Ringer, centrifuged at 800 g for 5 min, resuspended in the test cryoprotectant media at 1, 2 and 5 x 10(6) spermatozoa mL-1 and 7, 10.5, 14 and 17.5% glycerol and then drawn up into 0.25 mL plastic straws. The spermatozoa were rapidly frozen in the vapour phase, 6 cm above liquid nitrogen, for 30 min before the straws were plunged into the liquid. Sperm motility was assessed blind for coded straws by phase-contrast microscopy on a warmed stage (35-degrees-C), before freezing and after rapid thawing in a water bath at 37-degrees-C (10 s). The highest recovery of both percentage motility (around 50-60%) and progressive motility (around 0.5-1 unit lower than prefreeze) occurred when spermatozoa were frozen and thawed in the presence of 17.5% glycerol. Recovery of motility was greater at the higher sperm concentrations (2 and 5 x 10(6) mL-1). There was no evidence of acrosomal damage or loss after freezing and thawing in high concentrations of glycerol. The only defect detected in spermatozoa subjected to the protocol was a variable tendency to bending of the neck region. This ranged from heads inclined at a slight angle to the tail through to complete flexure. Head bending was found to be due not to freezing but to incubation of spermatozoa in high glycerol medium at RT. If post-thawing assessments were made at 35-degrees-C head bending was abolished. The fertility of the possum sperm frozen by this method has yet to be established.