Efficiency Analysis Of Several Techniques Of Embryo Manipulation In Cynomolgus Monkeys

As human diseases model,experimental animals provide a large amount of research data for the understanding the pathogenesis of diseases and for exploring new drugs and treatment methods.Animal models can effectively replace human experimental risks and ethical restrictions;make an indelible contribution to the development of human biomedicine.Human and non-human primates have similar genetic structure and also have highly similar in physiological and biochemical aspects.Therefore,non-human primates can be used as potential animal model for human diseases by using transgenic and assisted reproductive technology.Microinjection techniques such as ICSI have been widely used for injecting the sperm into oocyte for in-vitro embryo production which further used to obtain genetically modified progeny.Application of cynomolgus monkeys as human diseases model has been reported.In this study,the oocytes of superovulated female monkey were collected by laparoscopic surgery.While,sperm of male monkey was collected by electrical stimulation,embryo fertilized by intracytoplasmic sperm injection(ICSI),experimental processes such as cryopreservation of embryos or embryo injection,gene modification vectors,analyzing the possibilities of MI oocytes' fertilization and embryos' growing;analyze genetically modified cryopreserved sperm and normal sperm on embryo developmental differences;analyze carrier cytoplasmic injection and pronuclear injection at pronuclear stage on embryo developmental differences;analyze 4?8 cell embryo zona pellucida's incision on blastocysts;analyze resuscitation rate and implantation rate of cryopreservated embryos after blastocyst biopsy.Further,we systematically assessed the effectiveness and safety of assisted reproductive technologies of cynomolgus monkey.The research results are as follows:(1)Comparing embryos' pronuclear rate,cleavage rate and blastocyst rate of MI group,vitro maturation MI group and MII group which were not significantly different.The in vitro maturation MI oocytes also have developmental potential,but direct ICSI fertilized have a lower embryo potential development than other groups.(2)Comparing embryos' pronuclear rate,cleavage rate and blastocyst rate that fertilized by SHANK3 gene modified cryopreserved sperm and normal fresh sperm through ICSI in vitro.Which shows frozen sperm's rates slightly lower than fresh sperm,but the difference is not significant.Newborn monkeys have been obtained by embryos that fertilized by SHANK3 gene modified monkey's cryopreserved sperm.(3)Cryopreserved sperm or fresh sperm ICSI fertilized embryos in the pronuclear cytoplasmic injection of gene editing vectors.Cryopreserved sperm ICSI fertilized embryos blastocyst rate was significantly lower after injected vector,the cleavage rate and blastocyst rate of other fertilized embryos were not significantly different from those of the control group(no carrier).(4)The zona pellucida incision at 4?8 cells stage embryo has no adverse effect on embryo blastocyst development,and has the effect of promoting blastocyst hatching.(5)Newborn monkeys were obtained from transplanted cryopreserved blastocyst embryos.The pregnancy success rate of embryo transplantation in cryopreserved and resuscitated embryos was not significantly different compared to fresh embryos,but the pregnancy success rate of cryopreserved PGD embryo was significantly lower than that of normal embryos.