Cryopreservation of macropodid spermatozoa: new insights from the cryomicroscope

Abstract

This study examined the effects of cooling and cryopreservation upon macropod spermatozoa (eastern grey kangaroo, Macropus giganteus and red-necked wallaby, Macropus rufogriseus). Sperm survival during and after freezing to –30˚C or –70˚C in minimum essential medium (MEM) + 5, 10, 20 or 30% (v/v) glycerol, MEM + 10 or 20% (v/v) ethylene glycol and MEM containing a mixture of 7.5% (v/v) glycerol + 10% (v/v) dimethylsulphoxide was examined by cryomicroscopy. The MEM/glycerol mixtures permitted better post-thaw sperm recovery than the other cryoprotectants. After freezing to –30˚C at 10˚C min ^–1 in 20% glycerol, then rewarming at 20˚C min ^–1 , flagellar activity resumed in more than 50% of spermatozoa when the temperature increased into the range 5–10˚C. However, as the temperature increased, into the range 20–25˚C, motility declined rapidly so that less than 5% motile cells were seen at 35˚C. Spermatozoa in MEM without cryoprotectant were also examined by cryomicroscopy to evaluate changes in flagellar configuration, swimming behaviour and viability during cooling from 35˚C to approximately –7˚C, and rewarming to 35˚C. Cooling from 35 to 28˚C induced kangaroo spermatozoa to exhibit rigid principal-piece bending and non-linear motility, which was reversed by further cooling and the spermatozoa resumed their normal linear movement. Rewarming induced principal-piece bending in the range of 20–30˚C, but this effect was reversed by further warming. Although red-necked wallaby spermatozoa showed these effects, they also exhibited a tendency to form rosette-like clusters during rewarming, especially when the temperature reached approximately 14˚C. The clusters were induced when the flagellar end-pieces became anteriorly reflected, producing hook-like flagellar conformations, which then became interlinked.