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This study examined the effects of cooling andcryopreservation upon macropod spermatozoa (eastern grey kangaroo,Macropus giganteus and red-necked wallaby,Macropus rufogriseus). Sperm survival during and afterfreezing to -30˚C or -70˚C in minimum essential medium(MEM) + 5, 10, 20 or 30% (v/v) glycerol, MEM + 10 or20% (v/v) ethylene glycol and MEM containing a mixture of7.5% (v/v) glycerol + 10% (v/v) dimethylsulphoxidewas examined by cryomicroscopy. The MEM/glycerol mixtures permitted betterpost-thaw sperm recovery than the other cryoprotectants. After freezing to-30˚C at 10˚C min <emph type="7">-1 in 20%glycerol, then rewarming at 20˚C min <emph type="7">-1 ,flagellar activity resumed in more than 50% of spermatozoa when thetemperature increased into the range 5-10˚C. However, as thetemperature increased, into the range 20-25˚C, motility declinedrapidly so that less than 5% motile cells were seen at 35˚C.Spermatozoa in MEM without cryoprotectant were also examined by cryomicroscopyto evaluate changes in flagellar configuration, swimming behaviour andviability during cooling from 35˚C to approximately -7˚C, andrewarming to 35˚C. Cooling from 35 to 28˚C induced kangaroospermatozoa to exhibit rigid principal-piece bending and non-linear motility,which was reversed by further cooling and the spermatozoa resumed their normallinear movement. Rewarming induced principal-piece bending in the range of20-30˚C, but this effect was reversed by further warming. Althoughred-necked wallaby spermatozoa showed these effects, they also exhibited atendency to form rosette-like clusters during rewarming, especially when thetemperature reached approximately 14˚C. The clusters were induced whenthe flagellar end-pieces became anteriorly reflected, producing hook-likeflagellar conformations, which then became interlinked. |
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