Cryopreservation Of Japanese Flounder Embryos By Vitrification

This article studies several factors affecting the cryopreservation of Japanese flounder embryos by vitrification such as straw diameter, equilibrating temperature, embryo numbers in a straw and et al.. The methods and conditions of cryopreservation Japanese flounder embryos are opotimized; especially, the vitrification solution component are improved. This study selects the embryo stage and equilibrating time and also argue on both the embryo sensitivity to chill and the damage mechenism of cryopreservation.Results are as follows. Vitrification solution PMDD (2% PVP) containing many cryoprotectants could vitrify stably and its devitrification rate was also, so it is suitable for embryo vitrification. Embryos at tail bud stage are more tolerant to vitrification solutions. Equlibrating time is related to embryo quality, and usually vitrification solutions can completely penetrate into Japanese flounder embryos when equilibrating for 40 min in vitrification solution. While equilibrating time exceeds 40 min, the survival rate of embryos decrease significantly (P<0.05). The survival rate equilibrating at 15℃ is significantly lower than that at 0 ℃ and 4 ℃ . Nonpermeabable cryoprotectants (PVP, Ficoll) are benifitable for vitrification. Japanese flounder embryos have high sensitivity to chill and when temperature is below zero, the survival rate decreases significantly (P<0.05). Among three type straws, the one of 2.5 mm diameter has the lowest devitrification rate and is suitabale for vitrification. There is no significant changes in the rate of devitrification when 2-5 embryos are put in a straw. The egg sac is the barrier when cryoprotectants penetrate into embryos and is easy to damage during cryopreservation.Based on the optimized conditions, vitrification experiments of Japanese flounder embryos were conducted. From four successful experiments, 8 viable embryos were obtained from 26 cryopreserved embryos and 7 larvae with normal morphology successfully hatched. The survival time of 7 larvae varied from 2 days to 13 days and the hatching rate varied from 12.5% to 60%. The study supplies the basic data for fish embryo vitrification and demomstrates the possibility of cryopreserving Japanese flounder embryos by vitrification.