大菱鲆胚胎玻璃化方法研究

Study on vitrification method of turbot, Scophthalmus maximus, embryos

用大菱鲆(Scophthalmusmaximus)神经期胚对6种抗冻剂的毒性进行检测,发现其毒性排列为:1,2-丙二醇(PG)<甲醇(MeOH)<甘油(Gly)<二甲基甲酰胺(DMF)<乙二醇(EG)<二甲亚砜(DMSO)。以DMF为主因子配制和筛选出11种玻璃化程度较好的玻璃化液,并用大菱鲆肌节期胚对其中5种玻璃化程度最好的玻璃化液进行检测,结果显示,A4(DMSO20%+DMF25%)、A7(DMSO25%+DMF20%)较适合于大菱鲆胚胎的平衡处理,利用A4和A7平衡处理神经期胚、肌节期胚、心跳期胚、出膜前期胚,结果显示大菱鲆神经胚和肌节胚对玻璃化液的适应能力较强。实验测试出不同时期胚胎在两种玻璃化液中的成活率,为大菱鲆各期胚胎在玻璃化液中的平衡处理提供了依据。利用大菱鲆肌节胚对不同的平衡步骤进行了筛选,发现五步法的平衡效果较好。

The toxicity of six different cryoprotectants on turbot Scophthalmus maximus neurula was examined. The results showed that the toxicity of the six cryoprotectants sequenced as follows: PG<MeOH<Gly<DMF<EG<DMSO. Eleven better vitrificable solutions mainly composed of DMF were compared, of which 5 kinds were tested using turbot somite embryos. The results showed that A4 (DMSO20%+ DMF25%) and A7 (DMSO25%+ DMF20%) were two vitrification solutions suitable for equilibrating treatment of turbot embryos. Neurula, somite embryos, heart-beating embryos and pre-hatching embryos were treated with A4 and A7 and the experiment results indicted that neurula and somite embryos are more resistant to vitrification solution. The survival rate of turbot embryos at different stages in A4 and A7 was also determined, which could offer evidence for determining the treatment time of different stage embryos in vitrification solutions. The effect of different equilibration procedures on turbot embryos was examined and the five-step equilibration method was found to be most effective.