Cryopreservation of Spermatozoa from Freeze-Tolerant and -Intolerant Anurans

Author： Beesley S.G. Costanzo J.P. Lee R.E.

ISSN： 0011-2240

Source： Cryobiology, Vol.37, Iss.2, 1998-09, pp. : 155-162

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Abstract

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°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.