The Study On Boving Patrogenesis Technology

The objective of the present study was build a new method, patrogenesis, of cloning bovine embryos by injecting two sperms instead of somatic cells based on the mechanism of mammalian fertilization and principle of somatic cell nuclear transfer. In the present study, cloned bovine embryos were constructed by injecting capacited sperm in vitro into enucleated oocytes. This method successfully overcame the limitation of donor cell, from somatic cell to non-somatic cell, innovatively using two sperms instead of somatic cells. he following mainly discussed the factors influencing the production of bovine cloned embryos derived from sperms.Experiment 1. In order to improve the rate of maturation of bovine embryos, five combinations of FCS and BFF were tested in in vitro maturation medium. The results demonstrated that there was significant difference between the group supplemented with 10% FCS & 20% BFF and the group without FCS and BFF （65.8% and 42.42%, lowest, respectively）. There was no significant difference among the three groups supplemented with 10% FCS, 20% BFF, and 10% FCS and 10% BFF on the rate of maturation （52.5%, 53.4% and 58.6%, respectively, P<0.05）. The addition of 10% FCS and 20% BFF was benefit to increase the rate of maturation of bovine oocytes.Experiment 2. Since the whole process of micro-enucleation of oocytes and microinjection of two sperms were completed in microdrops, the microenviroment of microdrops played a pivatol role in the survival of ooplasm and sperm. The effect of two types of enucleation medium on the cloned embryos derived from sperms were analyzed in order to minimize the damage of manipulation on the cloned embryos. Since the process of enucelation and microinjection of sperms were done in the droplets, the environmental of the manipulation droplets played a pivotal role in the survival of oocytes and sperms. The results showed the manipulation medium II could significantly improved the development of cloned embryos compared with medium I （22.5% and 17.5%, P<0.05） and （3.0% and 6.0%, P>0.05）.Experiment 3. The efficacy of blind enucleation and chromosomal localization enucleation and their effect on the development of cloned embryos were studied. Matured oocytes with 1st polar body were enucleated by blind enucleation or chromosomal localization enucleation supplemented with 5μg/mL Hoechst33342 in microdrops. The experiments were repeated four times. The results showed that there was significant difference between the two groups on the rate of cleavage （15.6% and 20.3%, respectively）, while there was no sifnificant difference on the rate of formation of blastocyst （7.40% and 6.83%, respectively）. However, to simplify the manipulation and ensure 100% enucleation, we adopted the method of chromosomal localization enucleation.Experiment 4. To optimize the activation of cloned embryos derived from sperms, the effect of four types of chemical reagents on the development if cloned embryos were studied. I Ionomycin+6-DMAP group, II Ionomycin+CHX group, III 7% ethanol+6-DMAP group, IV 7% ethanol+6-DMAP group. Timing of activation: ionomycin and 7%, 5min,; 6-DMAP, 4h; CHX, 5h. The results showed that the rate of cleavage of the four groups were 20.5%, 12.1%, 7.60% and 7.0%, respectively, 2 days after in vitro culture. The rate of cleavage of group I significantly higher than that of the other three groups, while no significant difference existed among the other three groups （P<0.05）. The rate of blastocyst of group I was significantly higher than that of the other three groups （11.1%, 6.05%, 2.5% and 4.8%, respectively, P<0.05）, while no significant difference existed in the other three groups. It demonstrated that ionomycin and 6-DMAP was better for the development of bovine embryos after activation.Experiment 5. The timing of inomycin activation on the cloned embryos was study. The cloned embryos were divided into two groups, which were activated by Ionophore for 5min followed by 6-DMAP for 4h and Ionophore for 10min followed by 6-DMAP for 4h. The results demonstrated that the rate of cleavage of cloned embryos activated by inomycin for 10min was significantly higher than that of embryos activated for 5min （20.3% and 15.6%, respectively, P<0.05）. So did the rate of formation of blastocyst （7.40% and 4.26%, respectively, respectively）. It demonstrated that a relative longer time was beneficial to the development of cloned embryos.Experiment 6. The cloned bovine embryos were divided into two groups and cultured by CR₁aa and mSOF, respectively, to investigate the effect of type of medium on bovine sperm cloned embryos. The results demonstrated that the rate of cleavage of CRiaa group was significantly higher than that of mSOF group （20.3% and 17.8%, respectively, P <0.05）, while the rate of formation of blastocyst between the two groups showed no significant difference （7.40% and 8.10%, respectively; P>0.05）.