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鱼类胚胎冷冻保存技术还远没有成熟, 为了寻找最佳的鱼类胚胎玻璃化冷冻保存条件, 我们以牙鲆(Paralichthys olivaceus) 胚胎为例, 研究了影响鱼类胚胎玻璃化冷冻保存的几个主要因子: 玻璃化液、麦管直径、胚胎阶段、平衡时间及平衡温度、洗脱浓度和洗脱时间。发现: (1) 含有多种抗冻剂的玻璃化液PMDD(2% PVP), 玻璃化稳定, 脱玻璃化率较低, 适宜进行玻璃化冷冻; (2) 尾芽期胚胎较其他时期耐受力强, 平衡40 min就足以使玻璃化液渗透完全, 时间延长, 成活率显著降低, 各个时期的胚胎对温度都比较敏感, 0°C与4°C下平衡的成活率显著高于15°C; (3) 洗脱浓度和洗脱时间对胚胎成活率影响不大; (4) 根据优化的条件, 对牙鲆两个时期的胚胎进行超低温冷冻保存实验, 共成活4次, 获得成活胚胎8粒, 其中7粒孵化出健康的鱼苗。本文为鱼类胚胎冷冻保存技术的建立提供基础资料, 并显示了牙鲆胚胎玻璃化冷冻保存是可行的。
Fish embryo cryopreservation technology is far from mature, in order to find the best fish embryos vitrification conditions, we Paralichthys olivaceus embryos, for example, studied the impact of fish embryos vitrification of a few Main factors: vitrification, diameter of the straw, embryonic stage, equilibration time and equilibration temperature, elution concentration and elution time. The results showed that: (1) Vitrified liquid PMDD (2% PVP) containing a variety of cryoprotectants showed stable vitrification with a low rate of de-vitrification and was suitable for vitrification; (2) Forced force, the balance of 40min is enough to vitrification complete penetration, prolonged survival rate was significantly reduced, the embryos at all times are more sensitive to temperature, 0 ° C and 4 ° C under the equilibrium survival rate was significantly higher than 15 ° C; (3) Elution concentration and elution time have little effect on the survival rate of embryos; (4) According to the optimized conditions, cryopreservation of embryos in both periods of Paralichthys olivaceus survived 4 times to obtain survival Eight embryos, of which seven hatched healthy fry. This article provides the basic information for the establishment of fish embryo cryopreservation technology and shows that it is feasible to vitrify frozen embryo of Japanese flounder.