Individual study: Cryopreservation of spermatozoa from freeze-tolerant and intolerant anurans
Beesley S.G., Costanzo J.P. & Lee R.E. (1998) Cryopreservation of spermatozoa from freeze-tolerant and intolerant anurans. Cryobiology, 37, 155–162
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Freeze sperm or eggs for future use
A replicated study in 1997–1998 of captive amphibians in the USA (Beesley, Costanzo & Lee 1998) found that recovery of viable sperm following freezing was significantly lower for leopard frogs Rana pipiens and American toads Bufo americanus compared to freeze-tolerant wood frogs Rana sylvatica. Sperm recovery was 59%, 48% and 81% respectively. Survival and viability of wood frog sperm was significantly greater using the cryoprotectant dimethyl sulfoxide and supplement of fetal bovine serum (survival: 96%; viability: 45%) than the other three protectants with glutathione (survival: 34–54%; viability with methanol: 10%) or without protectants (survival: 44–54%; viability with methanol: 16%). Testes from wild or commercially obtained males were macerated in a buffer solution. Sperm solutions from wood frogs were mixed with 0.5 M cryoprotectant (dimethyl sulfoxide, methanol, glycerol or ethylene glycol), a supplement (fetal bovine serum or glutathione) or a combination of these. Using the most successful cryopreservation treatment, sperm from each species was incubated on ice for 15 minutes, then frozen to -80°C for 1 hour (rate: 130°C/minute). Thawing was in warm water.