The Vitrification Of Transgenic Cloned Bovine Embryos

Transgenic and cloned technology is a major breakthrough in life science. At present, mostof the researchers produce transgenic animals by somatic cell nuclear transfer. But the presentefficiency of somatic cloning is low in transgenic cloning. There are a lot of factors affecting thesuccess of transgenic cloning, which still poorly understood. The technology of embryo andoocyte freezing provides a broader space for Transgenic and cloned technology. The scientistscould inspect the embryo quality, transplant across all time zones and preserve embryo for along-term by freezing the transgenic cloned embryos.The aim of this study was to assess the efficiency of the programmed freezing andvitrification. In addition, we also compared the different cryoprotectant, periods and carriers onthe efficiency of vitrification. In order to find out an appropriate way to freeze the transgeniccloned bovine embryos, the survival rate, hatching rate, cell number, apoptosis rate and theexpression level of several important genes after thawed embryos are detected. The presentstudy provides the basis for the vitrification of transgenic cloned embryos and lays thefoundation of producing transgenic cloned animals.1. In this part the efficiency between the programmed and vitrification frozen of transgeniccloned bovine embryos were assessed. The results showed that the survival rate of vitrificationgroup was higher than that of programmed freezing group (P﹤0.05), and the hatching rate wasalso significantly higher than that of programmed freezing group after thawed. Furtherexperiments revealed that the total cell number of blastocyst was no significant difference.However the apoptosis rate was significantly higher than programmed group. By the analysis ofRT-PCR, the expression of gene Bax was lower than programmed group (P﹤0.05), however,the expression of Oct4and Hsp70was higher (P﹥0.05).So the result showed that both theprogrammed and vitrification frozen could support the transgenic cloned bovine embryos frozen,but the second method got significant efficiency.2. By comparing the different cryoprotectant, periods and carriers on the efficiency ofvitrification, we aimed to further improve the efficiency of vitrification. There was no statisticalsignificant difference in rate of survival and rate of hatching in day1, when we used ethyleneglycol (EG), dimethyl sulfoxide (DMSO) and the mix as the cryoprotectant. But the hatchingrate of mix group in day2-3was significantly higher. This result showed that the mix of EG andDMSO was better suit for the development of transgenic cloned bovine embryos. The present study also compared the frozen effect of different stage such as2-cell,8-cell, morula, earlyblastocyst and blastocyst. By comparing the survival rate, hatching rate, cell number andapoptosis rate, we found that early blastocyst was the best stage for transgenic cloned bovineembryos frozen. Then though the contrast impact of different carrier used in the transgeniccloned bovine embryos frozen found that solid surface carrier was best for the vitrification thanglass straw carrier and plastic pulled straw carrier.In summary, the present study showed that it could get better result when used EG ascryoprotectant and solid surface as carrier to vitrify the early blastocyst in transgenic clonedbovine embryos.