不同因素对牛卵母细胞胞质内精子注射效果的影响

Influence of Different Factors on Intractoplasmic Sperm Injection in Bovine Oocytes

采用胞质内精子注射法(ICSI)构建牛显微受精胚胎,探讨卵母细胞成熟培养时间、精子状态及精子操作液中聚乙烯吡咯烷酮(PVP)浓度对牛卵母细胞胞质内显微受精(ICSI)卵体外发育的影响。结果显示:成熟培养20 h、24 h和28 h的卵母细胞用于ICSI时,ICSI卵的形态完整率分别为94.6%(175/185)、97.2%(173/178)和96.4%(189/196)(P>0.05);成熟培养24 h和28 h的卵母细胞,其ICSI卵的卵裂率(73.4%和70.9%)和囊胚发育率(31.8%和29.6%)显著高于成熟培养20 h的卵母细胞(61.1%和20.6%)(P<0.05)。对成熟培养24 h的卵母细胞注射获能精子时,ICSI卵的卵裂率和囊胚发育率(72.7%和31.4%)高于未获能精子组(69.4%和29.3%)和不运动精子组(71.5%和30.4%),但无统计学差异(P>0.05)。采用含5%,10%和15%PVP的精子操作液处理获能精子后对成熟培养24 h的卵母细胞进行ICSI操作,PVP浓度为5%和10%时,ICSI卵的卵裂率(73.2%和66.9%)和囊胚发育率(32.7%和29.7%)差异不显著(P>0.05),但二者均显著高于15%PVP浓度组的卵裂率和囊胚发育率(51.0%和16.3%)(P<0.01)。结论认为,选用获能精子,并使用含5%PVP的精子操作液进行处理,对成熟培养24 h的牛卵母细胞进行ICSI操作,有利于ICSI卵的体外发育。

This study investigated the effects of in vitro maturation time of oocyte, sperm state and the polyvinylpyrrolidone （PVP） concentration in the spermic manipulation medium on in vitro develop- ment of the intracytoplasmic sperm injection （ICSI） zygotes in bovine. The oocytes matured in vitro for 20, 24 h and 28 h were used in ICSI, there was no effect of the maturation time of oocyte on the modality integrity rates of ICSI zygotes （ 94.6%, 97.2%,96.4% P）〉 0.05 ）, the cleavage rates and blastocyst rates of ICSI zygotes were significantly higher with 24 h （ 73.4%, 31. 8% ） and 28 h （ 70.9%, 29.6% ） maturation than those with 20 h （ 61.1%, 20.6% ） （P〈 0.05 ）. Three different sperm states （capacitated sperm, without capacitated sperm and unmovable sperm ） and the oocytes were matured in vitro for 24 h were used in ICSI , the higher cleavage and blastocyst rate of ICSI zy-pacitated sperm were used in ICSI, there was no different between 5% and 10% PVP concentration in the cleavage rates （ 73.2% vs. 66.9% ） and blastocyst rates （ 32.7% vs. 29.7%, P〈0.05 ） of ICSI zygotes, but the cleavage rates and blastocyst rates of ICSI zygotes were significantly higher than those with 15% PVP concentration （ 51.0 % and 16.3% ） （ P〈0.01 ）. The result indicated that the oocytes with in vitro maturation for 24 h and the capacitated sperm be used in ICSI, and the sperm was treated in the manipulation medium with 5% PVP, can improve the in vitro development of the ICSI zygotes in bovine.