罗非鱼卵子的显微受精分析

Analysis of Microscopic Fertilization of Tilapia Eggs

为探讨一个全新的罗非鱼成熟卵母细胞胞质内单精子注射的方法以及注射后不同处理方法对罗非鱼卵母细胞激活以及早期胚胎发育的影响,本实验共设定了3组,第一组用含浓度为0.1%氯化钙的L-15培养基处理成熟卵母细胞,注射后直接放入纯水内培养;第二组用含浓度为0.1%氯化钙的L-15培养基处理卵子,注射后30 s,放入7%乙醇激活处理5 min后转移至纯水内培养;第三组用L-15培养基处理卵子,注射后10 min,转移至含0.1%氯化钙和7%乙醇的溶液内激活处理5 min后放入纯水内培养。结果显示,第二组的卵裂率和囊胚发育率最高(均为1.74%),第一组的次之(卵裂率为0.99%,囊胚发育率为0.66%),第三组最低(卵裂率为0.45%,囊胚发育率为0)。综上所述,本实验应用氮气瓶、注射泵、常规解剖镜、LED外光源组成一套简易且高效的显微注射装置。应用这套装置能够快速注射稀释后的精子并且确保每次注射液滴内精子数不超过一个。本实验为具有巨大卵子的鱼类的显微受精研究提供了一个全新的解决方案。

In this study,a new method of intracellular single sperm injection of mature oocytes of tilapia and the effects of different treatments on the activation of oocytes and early embryonic development of tilapia were discussed.In this experiment,three groups were set.The first group treated mature oocytes with the L-15 medium containing 0.1%calcium chloride,and then injected directly into the pure water.In the second group,the eggs were treated with L-15 medium containing 0.1%calcium chloride,and 30 s after injection,and then transferred to the pure water after 5 min of 7%ethanol activation.In the third group,the eggs were treated with L-15 medium,10 min after injection,and were transferred to the solution containing 0.1%calcium chloride and 7%ethanol.After 5 min,they were cultured in pure water.The results showed that the cleavage rate and blastocyst development rate were the highest(1.74%)in the second group,followed by the first group(0.99%of the cleavage rate,0.66%of the blastocyst development rate),and the lowest(0.45%)in the third group(0.45%,and the blastocyst development rate was 0).In conclusion,in this experiment,a simple and efficient microinjection device was constructed using a nitrogen bottle,a syringe pump,a conventional dissecting microscope,and an external LED light source.This device can be used to quickly inject diluted sperm and ensure that no more than one sperm is contained in each drop.This experiment provides a completely new solution for microscopic fertilization of fish with huge eggs.