Effects Of Culture Medium On Development Of Transgenic Cloned Bovine Embryos

The technology of cloning works reliably, but the present status of somatic cloning is characterized by low efficiency, especially in transgene cloning. Factors affecting the success of cloning are poorly defined. Various protocols for augmenting embryonic development have been employed for improving cloning efficiency. But the efficiency of blastocyst formation upon SCNT in bovine embryos was remained poor. The search for combination of essential constituents in the culture media needed for the efficient blastocyst development is an ongoing process. Suboptimal culture conditions in vitro can led to the development of embryos are in poor-quality and incapable of establishing pregnancies. Supplementation of ruminant embryo culture media with serum or BSA is widely used. It has been shown that prolonged exposure to serum can greatly alter embryo morphology and biochemistry and it has been associated with the large offspring syndrome (LOS). The use of serum-free culture systems that support high rates of development in culture of high-quality embryos is thus desirable.The aim of this study was to assess the ability of the commercially available sequential media G1.3/G2.3 to support transgenic cloned bovine embryos development to the blastocyst stage and to examine the quality of the blastocysts produced, detect of the embryos quality and the development capability in vivo, compared with those produced in mSOF-serum and mSOF-BSA medium. At last, we desiged a defined culture medium to support transgenic cloned embryos develop in vitro. We undertook this study to determine an ideal culture condition that is conducible to blastocyst formation and that may help in increasing the efficiency of transgene cloning in farm animals and its popularization.1. There was no significantly difference in the cleavage rate, blastocyst formation rate and cell number among the three groups. But day 6 blastocyst formation rate of mSOF-serum group was higher than other groups (P<0.05); day 7 and day 8 hatching rate, and the 24 h, 48 h hatching rate after thawing of mSOF-BSA group were significantly lower than that of counterparts (P<0.05); the apoptosis rate of blastocyst in G1.3/G2.3 group was significantly lower than their counterparts. The relative expression levels of Oct4 and IFNtau in G1.3/G2.3 group was significant higher compared with counterparts, and relative expression levels of Hsp70 and Bax were the highest in mSOF-serum group. All results showed that transgenic cloned bovine embryos derived from G1.3/G2.3 group had high-quality, which indicates that G1.3/G2.3 was more suitable for culturing transgenic cloned bovine embryos.2. Although difference in pregnancy rate was observed at day 60 of gestation, the pregnancy rate of day 90 was significantly higher after embryo transfer in G1.3/G2.3 group than mSOF-serum and mSOF-BSA groups(P<0.05). However, 120 day after embryo transfer, the mSOF-serum group was almost all abortion, and the pregnancy rate of G1.3/G2.3 group was higher than mSOF-BSA group. We conclude that sequential media G1.3/G2.3 could support transgenic cloned bovine embryos in vivo development, and G1.3/G2.3 was the best choice for culturing transgenic embryos.3. Optimized defined culture medium could effectively support the in vitro development of transgenic bovine embryos. The total cell number, apoptosis rate and survival rate after thawing of the two culture groups were no difference (P>0.05). But 48 h after thawing, the hatching rate of defined culture group was higher than undefined culture group (P<0.05). The relative expression levels of Bax、Igf-2 in defined medium group was significant lower and Glut-1 was significant higher compared with that in the undefined group. Those results indicating that optimized defined culture medium can support transgenic cloned bovine embryos in vitro development, and embryos have high-quality than their counterparts.4. We detected the Igf-2 gene CpG island methylation of transgenic nuclear transfer bovine embryos derived from defined and undefined culture media by using bisulfite sequencing. Results indicated that methylation levels of the CpG island of Igf-2 gene in bovine embryos derived from defined medium group was significant higher compared with that in the undefined medium group (P<0.05).Results showed the abnormal DNA methylation reprogramme Igf-2 of gene occurred in undefined culture medium.