实验兔胞内单精子注射技术

Fertilization by Intractoplasmic Sperm Injection in Rabbit Oocytes

目的建立实验兔胞内单精子注射技术(intracytoplasmic sperminjection,ICSI)。方法实验1比较了hCG注射后不同取卵时间对ICSI胚体外发育的影响。实验2比较了不同的激活方式对ICSI胚体外发育的影响。实验3比较了不同状态的兔精子ICSI胚胎体外发育结果。结果(1)hCG注射后14 h取卵,其卵裂率、桑椹胚率和囊胚率(82.2%、72.9%和62.2%)都比16 h(75.9%、70.0%和53.3%)的高,但是差异无显著性(P>0.05);18 h取的卵注射后不能卵裂。(2)机械刺激组和离子霉素+6-DMAP组,ICSI后其卵裂率分别为82.2%和81.1%(P>0.05),桑椹胚率分别为72.9%和66.2%(P>0.05),囊胚率分别为51.3%和62.3%(P<0.05),机械刺激组和离子霉素组之间卵裂率和桑椹胚率差异无显著性,但是囊胚率差异有显著性。(3)新鲜精子组和冻融活精子组卵裂率(81.1%和68.8%)和囊胚率(62.3%和40.4%)差异有显著性(P<0.05),而桑椹胚率(66.2%和61.9%)差异无显著性(P>0.05)。结论精子冷冻前后,通过ICSI所得的桑椹胚均能孵化,表明已初步建立了实验兔的ICSI技术。

Objective This study was conducted to investigate the factors influencing the fertilization by intracytoplasmic sperm injection （ICSI） in rabbit oocytes. Methods In experiment 1, the effects of the time of oocyte collection after superovulation on in vitro development of intracytoplasmic sperm injection rabbit embryos were compared. In experiment 2, the different ways to activate oocytes after sperm injection were examined. In experiment 3, the effects of different state of sperm on in vitro development of intracytoplasmic sperm injection rabbit embryos were compared. Results （ 1 ） The cleavage rate, morula rate, and blastocyst rate after sperm injection into oocytes collected at 14 h after hCG administration were higher than that at 16 h, but the difference was statistically not significant （ P 〉 0.05）. Oocytes collected at 18 h after hCG administration were not cleaved after sperm injection. （2） the ICSI cleavage rates were 82.2% and 81.1% （P 〉 0.05）, the morula rates were 72.9% and 66,2% （ P 〉 0.05）, and the blastocyst rates were 51.3 % and 62.3 % （ P 〈 0.05 ） between machinery stimulation group and ionomycin ＋ 6-dimethylaminopurine （DMAP） group, respectively. （3） the cleavage rate （ 81.1% and 68.8 % ） and blastocyst rate （62.3% and 40.4%） were significantly different （P 〈 0.05）, the morula rate （66.2% and 61.9） was not significantly different （P 〉 0.05） between the fresh sperm and frozen-thawed live sperm. Conclusion With these fresh and frozen-thawed sperm, ICSI morulaes can be hatched, showing that the technique of intractoplasmic sperm injection in rabbit oocytes has been established.