Studys On Modification Of Established Technique Without Assistance Of Piezo For Mouse Intracytoplasmic Sperm Injection And Its Applications

Since its short reproductive cycle and abundant genetic information, mice was an ideal animal model for ICSI research. Piezo-ICSI has its shortage in simulating human ICSI operation, such as:operating temperature, rupture model of membrane, etc. Our previous studies has established a mouse ICSI technique without assistance of Piezo(New-ICSI, N-ICSI), its operation was closer to human ICSI with a high survival rate of83%. But its survival rate was still lower than Piezo-ICSI. In this study, the step of deeply sucking and deeply insertion to mouse oocytes in the established method was modified to two steps, in which slightly sucking and slightly insertion was performed before deeply sucking and deeply insertion(two-step N-ICSI). The survival rate of oocytes and ICSI operating efficiency in two-step ICSI was significantly higher than that in N-ICSI(92.5%V.S.81.1%,18.9eggs/hour vs15.6eggs/hour,p<0.01); laid a solid foundation for the application and promotion of N-ICSI, and provided a more efficient method for the research of human ICSI security and weak activating ability sperm model.About2%-3%ICSI cycles failed in fertilization because of oocytes activation failure. Assisted oocytes activation(AO A) can improve this situation and has been widely used. But due to the lack of suitable research models, there were rare reports about factors influencing activation ability of sperm, the mechanism of activation failure, and the security of AOA. In this study, fertilization and early embryo development were examined following ICSI using sperm heads subjected to different conditions prior to ICSI. When sperm heads were incubated at4℃for4hour prior to injection, there was no significant difference in normal fertilization rate compared with control group(74.6%V.S.81.5%, p>0.05); but When sperm heads were incubated at37℃for4hour prior to injection, a significant decrease in normal fertilization rate was observed (35.3%V.S.81.5%,p<0.05). There was a significant time-dependent decrease in normal fertilization with the increase of incubating time at37℃. When sperm heads were incubated at37℃for2hour and4hour prior to injection, the normal fertilization rate was significant decreased compared with control group(56.0%,42.7%V.S.82.7%, p<0.05); incubated at37℃for8hour and12hour, a further decline in normal fertilization was observed (25.3%,13.5%V.S.82.7%, p<0.05). In addition, if the sperm heads were incubated at37℃for more than2hour, embryonic development was severely impaired, although the integrity of the sperm DNA was preserved. Using assisted oocyte activation, these defects in normal fertilization and blastocyst development were rescued(74.6%V.S.84.2%,42.0%V.S.48.2%, p>0.05), but the blastocysts was retarded and fragmental, the hatching rate was significantly decreased (4.0%V.S.27.1%, p<0.05). In summary, the sperm activating ability could be weakened and controlled by incubating at37℃for a certain time roughly. This could be an important model for researches about the mechanism of activation failure and the security of assisted oocytes activation.