CRYOPRESERVATION OF SPERMATOZOA FROM FREEZE-TOLERANT AND FREEZE-INTOLERANT ANURANS

Spermatozoa of the freeze-tolerant wood frog (Rana sylvatica) were use d to develop a general protocol for the frozen storage of amphibian sp ermatozoa. Tolerance of spermatozoa to cryoprotective agents and freez ing in suspension (-80 degrees C) was determined from rates of sperm l ysis 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 combination s of these cryoprotectants and supplements. Me2SO and fetal bovine ser um 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 seru m. Thus, this combination was used to cryopreserve spermatozoa from th e freeze-intolerant anurans, Rana pipiens and Bufo americanus. Recover y of viable spermatozoa was significantly greater for R. sylvatica (me an +/- 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 promotin g its freeze tolerance. Nonetheless, our results support the feasibili ty of using gamete cryopreservation techniques in programs aimed at th e captive propagation of amphibians. (C) 1998 Academic Press.