Study On The Safe System And Method Of Mouse Germplasm Conservation

Objective to establish a comprehensive system for mouse strain conservation through natural mating, sperm cryopreservation and assisted reproductive technology. Mouse fresh epididymal sperm were collected by vasectomy of unilateral testis and the vasectomized mice were naturally mated for breeding. If no satisfactory results obtained from natural breeding, then the cryopreserved sperm were thawed for In Vitro Fertilization (IVF). If no results obtained, Intracytoplasmic Sperm Injection (ICSI) was used to reconstructed embryos and which were finally transferred into pseudopregnant recipient female mouse. The results show that parts of mice vasectomized unilateral testis such as BDF1and C57BL/6J mice could be used for natural breeding. Among the remaining sterile males, only ICR mice could be preserved through IVF while ICSI technology was the last approach to save GFP mice with C57BL/6J background. The present study confirmed that the genetic background of mouse sperm cryopreservation was a key factor in IVF and setup a comprehensive system, which could achieve the purpose of strain conservation while mouse strains could be utilized at maximum extent.To investigate the effect of cryopreservation on oocytes at different times after ICSI and parthenogenetic activation. This study was also performed in mouse oocytes fertilized by ICSI, or in artificially activated oocytes, which were cryopreserved immediately,1hour or five hours later through slow-freezing. After thawing, the rates of survival, fertilization/activation, embryonic development of oocytes/zygotes and changes in the cytoskeleton and ploidy were observed. Our results reveal a significant difference in survival rates of0,1and5hour group cryopreserved oocytes following ICSI and artificial activation. Moreover, significant differences in two pronuclei (PN) development existed between the0,1and5hour groups of oocytes frozen after ICSI, while the rates of two PN development of activated oocytes was different between the1hour and5hour group. Despite these initial differences, there was no difference in the rate of blastocyst fonnation from two PN zygotes following ICSI or artificial activation. However, compared with ICSI or artificially activated oocytes cryopreserved at5hour, lots of oocytes from0or1hour cryopreservation groups developed to zygotes with abnormal ploidy, therefore it suggests that too little time before cryopreservation can result in some activated oocytes forming abnormal ploidy. However, our results also demonstrate that sperm can maintain normal fertilization capacity in frozen ICSI oocytes and the procedure of freeze-thawing did not affect the later development of zygotes.