Cryopreservation of Bufotes viridis embryos by vitrification

Loss of biodiversity among amphibians is a current concern. Our hypothesis is that the embryos of amphibian species at risk of extinction could be cryopreserved by vitrification, using methods which have proved successful with fish oocyte. To test this hypothesis, samples of four cryoprotectants - methanol (MeOH), dimethyl sulphoxide (Me2SO), propylene glycol (PG) and polyethylene glycol (PEG), some singly, some in combination, were plunged in liquid nitrogen for 5 min to find the best solution for vitrification. To find the least toxic of these solutions, blastulae and stage G17 embryos of Bufotes Viridis, a typical amphibian, were exposed to solutions at different concentrations (0.5-10 M) for different lengths of time (15-30 min), with and without their normal protective jelly coats. In each case the number of survivors, which reached stage G25 was counted. Finally a series of embryos was vitrified in liquid nitrogen using the most efficient and least toxic cryoprotectants.Propylene glycol had the best vitrification characteristics, but MeOH vitrified at higher concentrations. The optimum regime, with the least toxic ctyoprotectants, consisted of 1M Me2SO for 15 min and a combination of 15% PEG(w/v) + 3M PG + 2M Me2SO for 3 min, with the jelly coat intact, followed by vitrification. This gave a survival percentage of 87.6% immediately after vitrification. Methods designed for cryopreservation of fish embryos make a good starting point for cryopreservation of the embryos of amphibian.