Cryopreservation of macropodid spermatozoa: new insights from the cryomicroscope

This study examined the effects of cooling and cryopreservation upon macrop od spermatozoa (eastern grey kangaroo, Macropus giganteus and red-necked wa llaby, Macropus rufogriseus). Sperm survival during and after freezing to - 30 degrees C or -70 degrees C in minimum essential medium (MEM) + 5, 10, 20 or 30% (v/v) glycerol, MEM + 10 or 20% (v/v) ethylene glycol and MEM conta ining a mixture of 7.5% (v/v) glycerol + 10% (v/v) dimethylsulphoxide was e xamined by cryomicroscopy. The MEM/glycerol mixtures permitted better post- thaw sperm recovery than the other cryoprotectants. After freezing to -30 d egrees C at 10 degrees C min(-1) in 20% glycerol, then rewarming at 20 degr ees C min(-1), flagellar activity resumed in more than 50% of spermatozoa w hen the temperature increased into the range 5-10 degrees C. However, as th e temperature increased into the range 20-25 degrees C, motility declined r apidly so that less than 5% motile cells were seen at 35 degrees C. Spermat ozoa in MEM without cryoprotectant were also examined by cryomicroscopy to evaluate changes in flagellar configuration, swimming behaviour and viabili ty during cooling from 35 degrees C to approximately -7 degrees C, and rewa rming to 35 degrees C. Cooling from 35 to 28 degrees C induced kangaroo spe rmatozoa to exhibit rigid principal-piece bending and non-linear motility, which was reversed by further cooling and the spermatozoa resumed their nor mal linear movement. Rewarming induced principal-piece bending in the range of 20-30 degrees C, but this effect was reversed by further warming. Altho ugh red-necked wallaby spermatozoa showed these effects,, they also exhibit ed a tendency to form rosette-like clusters during rewarming, especially wh en the temperature reached approximately 14 degrees C. The clusters were in duced when the flagellar end-pieces became anteriorly reflected, producing hook-like flagellar conformations, which then became interlinked.