Evaluation of glycerol and dimethyl sulfoxide for the cryopreservation of spermatozoa from the wood frog (Rana sylvatica)

Conventional cryopreservation techniques were applied to spermatozoa from the freeze-tolerant wood frog as an initial step toward developing protocols for the frozen storage of amphibian gametes. In chilled, but unfrozen, samples, a high concentration (3.0 M) of glycerol or dimethyl sulfoxide (Me2SO) in the suspension medium was not harmful to spermatozoa. However, 2.0 M glucose promoted loss of viability as assessed by fluorescence vital dyes assays. Control suspensions prepared in isotonic saline contained 1.86 x 10(4) spermatozoa/mu L, of which 71.6 +/- 4.1% were viable. No cells survived freezing at -80 degrees C without cryoprotective additives, whereas samples containing 1.5 or 3.0 M glycerol or Me2SO yielded >1.2 x 10(4) intact spermatozoa/mu L upon thawing.