Studies On Intracytoplasmic Sperm Injection (ICSI) In Bovine

These studies were conducted to optimize factors influencing ICSI in bovine. Bovine oocytes were matured in-vitro for 24h, bovine sperm was injected into an oocyte by assistance of a micromanipulation system. Four experiments were investigated factors affecting ICSI in bovine.In experiment l, Development of injected oocytes were compared after activated by three methods. No significant difference was found in cleavage and blastocyst and hatched blastocyst rates between A23187+(CHX+CD)+ CHX group and A23187+6-DMAP group(60.6%vs58.2% and 15.1%vs14.5% and 4.5%vs3.6%, P>0.05), Significantly higher cleavage and blastocyst and hatched blastocyst rates was found in Ionomycin+6-DMAP group than other two groups (79.3%vs60.6%vs58.2% and 31.0%vs15.1%vs14.5% and17.6%vs 4.5%vs3.6%, P<0.05). In experiment 2, Development of oocytes were compar -ed after injected dead or alive sperm. No significant difference was found in cleavage and blastocyst and hatched blastocyst rates(75.0%vs79.4% and 27.9%vs30.9% and13.2%vs16.2%, P>0.05)between dead sperm and alive sperm. In experiment 3, Development of bovine oocytes injected with various types of sperm were compared. No significant difference was found in cleavage and blastocyst and hatched blastocyst rates in testicular sperm between epididyma sperm(caput,corpus and cauda)(79.1%vs79.5%vs73.2% vs75.0% and23.3%vs20.5%vs19.5%vs18.2% and 14.0%vs13.4%vs12.2%vs 9.1%, P>0.05). In experiment 4, (1) Sperm treated with DTT for 1 h ,3h or 5h, with the time prolong, sperm with a decondensed nucleus rates was significa- ntly higher in bovine sperm treated with DTT for 3h group or 5h group than that of 1h group (43.2%vs49.8%vs0.0%, P<0.05), (2) Sperm treated with DTT for 1 h by ICSI, Significantly higher rate of sperm decondensation andmale pronuclear rates were found in oocyte injected with DTT-treated spem compared to the non-treated sperm (30.4%vs11.6% and 47.8%vs27.9%, P<0.05),There is no significant difference in cleavage between the two group (85.0%vs 80.0%, P>0.05),Significantly higher rate of blastocyst and hatched blastocyst rates was found in the DTT-treated group than the non-treated group(45.8%vs26.0% and 31.3% vs 14.0%, P<0.05).In conclusions, (1) After ICSI and activation,Culture of oocytes in 5μM/L Ionomycin for 5 min then 9F for 3 and then 2mM/L 6-DMAP for 3 hours can improve their subsequent embryonic development; (2) Dead sperm can also be used for ICSI in bovine; (3) Althouth, their is different on phase of sperm from obtained testicular to epididyma sperm, no significant affecting was found on embryonic development of ICSI; (4) DTT can promote sperm decondensation, and treatment of sperm with 5mM/L DTT for 1h can promote pronuclear and development of embryo.