Study On The Technology Of Intracytoplasmic Sperm Injection (ICSI) In Bovine

This study investigated the effect of direction of in vitro maturation time of oocyte, the first polar body (PB), the polyvinylpyrrolidone (PVP) concentration in the spermic manipulation medium, sperm state and different activation method on the development of the bovine intracytoplasmic sperm injection (ICSI) zygote in vitro.The oocytes were treated by ICSI under different conditions, the ICSI zygotes cleavage rate and blastocyst rate shows the effectiveness of the experimental conditions. 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 %, 28.6 %) maturation than those with 20 h (61.1 %, 21.7 %) ( P < 0.05 ).Oocytes after ICSI were treated with three different chemical motheds for activation as followings, the ICSI zygotes were activated by 7 % ethanol, Ionomycin and Ionomycin combine with 6-Dimethylaminopurine(6-DMAP), or without activating treat (the control group), the ICSI zygotes cleavage rate and blastocyst rate were higher with activated by 7 % ethanol (51.9 %, 14.5 %) and Ionomycin(56.6 %, 17.9 %) than that with the control group (15.8 %, 0) (P < 0.05), the ICSI zygotes cleavage rate and blastocyst rate were significantly higher with activated by Ionomycin combine with 6-DMAP (70.8 %, 28.5 %) than those with other groups (P < 0.05).Two directions (6 and 12 o'clock) of the first polar body modulated in oocytes at ICSI were examined, there were no significant differences in the ICSI zygotes modality integrity rate (95.1 % vs. 94.0 %, P > 0. 05), cleavage rate (39.3 % vs. 67.8 %, P > 0. 05), blastocyst rate ( 30.7 % vs. 28.8 % , P > 0. 05 ) between the two directions of the PBâ… .Three different PVP concentrations (5 %, 10 % and 15 %) in spermic manipulation medium were compared when the oocytes of in vitro maturation for 24 h and the 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).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 zygotes were obtained from the capacitated sperm (72.7 %, 31.4 %) than those from the without capacitated sperm and the unmovable sperm (69.4 %, 29.3 % and 71.5 %, 30.4 %), but there was no significant different (P>0.05).In conclusion, the oocytes with in vitro maturation for 24 h and the positions (6 o'clock or 12 o'clock) of the first polar body modulated in oocyte, the capacitated sperm be used in ICSI, and the sperm was treated in the manipulation medium with 5 % PVP, activated with Ionomycin followed by 6-DMAP could improve the development of ICSI zygotes.