Study On ICSI Save GFP Rabbits And Mediated Transgenic

Purpose:We investigated the effect of electrical stimulation on rabbit oocyte activation using intracytoplasmic sperm injection (ICSI) to determine whether viable offspring can be produced from deceased rabbit sperm. Furthermore, the alkali treatment to rabbit sperm to mediated genetically modified for a preliminary study using ICSI. Methods:Sperm were collected from a heterozygote GFP male rabbit5hours after sacrifice and cryopreserved in liquid nitrogen. Mature oocytes were fertilized using ICSI. A series of electrical pulse procedures were used to activate oocytes before and/or after ICSI. Following ICSI, zygotes were cultured in B2medium for4days or transferred into the oviducts of recipient rabbits at the2-or4-cell stage. Moreover, in the study, the rabbit sperm was treated with1OmM NaOH to make its membranes ruptured and incubated with the plasmid, and then used to produce transgenic embryos through ICSI technology.Results:The blastocyst formation rate was significantly greater in oocytes that received one or two pulses prior to ICSI compared to controls and other electrically stimulated groups. In the single pulse before ICSI group,23%of the blastocysts expressed GFP, which was significantly greater than all other groups. In two sham injection groups, twice pulses before ICSI resulted in a higher rate of parthenogenetic development (40%VS0%, respectively P<0.05). However, those that received treatment before and after, or just following ICSI, showed a significant decrease in embryo survival. Finally,93embryos from the single pulse before ICSI group were transferred into recipient female rabbits and a full-term kit was successfully delivered. In addition, the results show that the sperm plasmid incubation and alkali treatment had no significant difference effect on embryonic development (P>0.05). After ICSI. green fluorescent was observed in blastocysts, accounting for34.78%of the total number of blastocyst, and the exogenous DNA transfection rate is19.51%.Conclusions:One pulse of electrical stimulation prior to sperm injection was an effective method to activate rabbit oocytes for fertilization. Sperm collected from a deceased rabbit is able to produce viable embryos through ICSI that are capable of normal fetal and kit development. Furthermore, in the research preliminary method is established for producing transgenic rabbits with alkali treatment sperm before ICSI.