Cryopreservation of spermatozoa from freeze-tolerant and -intolerant anurans.

Spermatozoa of the freeze-tolerant wood frog (Rana sylvatica) were used to develop a general protocol for the frozen storage of amphibian spermatozoa. Tolerance of spermatozoa to cryoprotective agents and freezing in suspension (-80 degrees C) was determined from rates of sperm lysis and dual-fluorochrome vital dye assays. We tested the efficacy of four cryoprotectants (Me2SO, methanol, glycerol, and ethylene glycol), two supplements (fetal bovine serum or glutathione), and combinations of these cryoprotectants and supplements. Me2SO and fetal bovine serum were the most effective cryoprotectant and supplement, respectively, in reducing sperm lysis. Vital dye assays showed that viability was greatest for spermatozoa treated with both Me2SO and fetal bovine serum. Thus, this combination was used to cryopreserve spermatozoa from the freeze-intolerant anurans, Rana pipiens and Bufo americanus. Recovery of viable spermatozoa was significantly greater for R. sylvatica (mean +/- SE = 81.2 +/- 9.6%) than for R. pipiens (59.0 +/- 2.8%) and B. americanus (47.8 +/- 4.1%), perhaps owing to inherent factors promoting its freeze tolerance. Nonetheless, our results support the feasibility of using gamete cryopreservation techniques in programs aimed at the captive propagation of amphibians.