Cryopreservation of macropodid spermatozoa: New insights from the cryomicroscope

W. V. Holt, L. M. Penfold, S. D. Johnston, P. Temple-Smith, C. McCallum, J. Shaw, W. Lindemans, D. Blyde

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9 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)345-353
Number of pages9
JournalReproduction, Fertility and Development
Issue number6
Publication statusPublished - 1 Jan 1999
Externally publishedYes


  • Genetic resource banks
  • Kangaroo
  • Plasma membrane
  • Semen
  • Wallaby

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