Studies On Microinjection In Buffalo Oocytes

Sperm morphological change after ICSI and factors affecting the pronuclear formation and embryo development of buffalo oocytes after ICSI were investigated in this study. At 13h after ICSI, 59.4% of the sperms had a swell head and 18.8% was decondensed. Pronuclear formation was asynchronous, at 16h and 19h after ICSI 3.2% and 40.0% of oocytes formed male pronucleus, while 71.9% and 88.7% of oocytes formed female pronucleus at 13h and 16h. After ICSI and activation by ionomycin and 6-DMAP, 91.3% oocytes were activated and most of them shown 2PN + IPBi. More oocytes activated by ionomycin and 6-DMAP compared with ionomycin alone, the male pronuclus formation rate (33.3%) was higer in oocytes activated by ionomycin and 6-DMAP compared with ionomycin alone (20.6%, p<0.05). Treatment of sperm with 5mmol/L DTT increased the rate of sperm decondensation (64.2% vs 45.5%, p<0.05), but did not affect the male pronuclear formation (33.3% vs 32.7%, p>0.05). Similar pronuclear formation rate (29.8%vs33.3%), cleavage rate (86.9%vs 85.6%), blastocyst rate (30.3% vs 29.6%), hatched blastocyst rate(41.3%vs 43.2%) were obtained when dead sperm was used for ICSI. The cleavage rate (92.6% vs 85.5%, p<0.05) was increased by treatment of sperm with l.Ommol/L GSH and blastocyst rate (44.9% vs 28.0%, p<0.01) was also increased by treatment of sperm with 0.25 mmol/L GSH. Increase of haprin concentration from 50mg/L to 600mg/L did not affect the cleavage rate (90.7% vs 85.7%, p>0.05), blastocyst rate (27.1% vs 31.4%, p>0.05) and hatched blastocyst rate (40.6% vs 45.5%, p>0.05).In conclusions, (1) The pronuclear formation was asynchronous after ICSI in buffalo, the female pronuclear formed 3 hours earlier than male pronuclei; (2) After ICSI and activation, Culture of oocytes in 1.9mmol/L 6-DMAP for 3 hours can improve their subsequent embryonic development; (3) Treatment of sperm with 5mmol/L DTT can promote sperm decondensation; (4) Dead sperms can be used for ICSI in buffaloes; (5) Treatment of sperm with GSH can improve the efficiency of ICSI in buffaloes.