Cryopreservation of macropodid spermatozoa: new insights from the cryomicroscope

• DOI: 10.1071/rd99076
• Publication Date: 2003-11-17T03:41:56Z
• Authors: W. V. Holt; L. M. Penfold; S. D. Johnston; P. Temple-Smith; J. Shaw; C. McCallum; W. Lindemans; D. Blyde
• Keywords: Glycerol; Male; Genetic Resource Banks; Cell Survival; Trichosurus-vulpecula; 300402 Animal Reproduction; Mammalian Sperm; Conservation; Frozen; Marsupials; 630; C1; Semen; Lipid Phase-transitions; Animals; Wallaby; Cryopreservation; Macropodidae; Reproductive Biology; Microscopy; 780105 Biological sciences; Cell Membrane; 0402 animal and dairy science; 04 agricultural and veterinary sciences; Spermatozoa; Sperm Plasma-membrane; Plasma Membrane; 060602 Animal Physiology - Cell; Brushtail Possum; Zoology; Kangaroo; Developmental Biology; Semen Preservation

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.