Studies On Factors Influencing On In Vitro Production Of Bovine Embryos And Rapid Sexing By Lamp Methods

Cumulus oocytes complexes(COCs) were recovered fromΦ2～6 superficial follicles of slaughter discarded ovaries, then followed with IVM, IVF and IVC, the purpurse is to obtain blastulas. After that we detected the sex of blastulas by LAMP method, the method time-saving, simple, accurate rate is high, concrete content including below several parts:1. we studied the effect of EGF concentration on the IVM of bovine ooocytes, effect of heparin on the fertilization and embryo development, effect of different culture system on in vitro fertilization of bovine oocytes(1) The addition of EGF increased the frequency of in vitro maturaion of bovine oocytes in a concentration-dependent manner. Along with concentration increase, the mature rate also along with it enhancement. The oocytes mature rate is 64.74% when adding 30 ng/mL EGF, and it had no difference with control group(P>0.05);there were no significant differences between the effect of adding 50ng/mL and 70ng/mL EGF (P>0.05), but both all had a significant differences with the control group and 30 ng/mLEGF. The optimal concentration of EGF in maturation medium is 50ng/mL.(2) There were significant effects of differents concentration heparin in the fertilization medium. Especially, when the concentration heparin was 5μg/mL, the No.of embryos cleaved was 53.93%,had a significant lower than others(P<0.05).The No.of embryos cleaved of other three had a grow along with concentration increase, about 67.22%, 73.08%,76.50%,and had no differences; the blastocysts rate was 22.22% with the 80μg/mL heparin, had a sinficant differences than others(P<0.05),the others blastocysts rates were 33.33%, 32.23%,35.96%respectively.(3) There were no significant differences between the effect of different culture systems (TCM199+granulosa cells, mSOF+granulosa cells) on the in vitro development of bovine oocytes (P>0.05), but had significant differences on the in vitro development of bovine embryos(P<0.05), the morular rate of TCM199+granulosa cells was 25.49%, the morular rate of mSOF+granulosa cells was 36.24%.So the best in vitro bovine embryo culture system is mSOF co-cultured with granulosa cell. 2. Basing on the LAMP method, we sexed the embryos that were produced by ourselves.The study systemically researched the LAMP method based on the bovine genome as amplification templets. The study designed the bovine common primers and male-special primers and optimized the LAMP reaction condition for the bovine sex judgment. At last, we did not use GEL as the detected method, and then adding EB, CuSO₄ to reaction system, in order to form color change or to judge by precipitation, so we can check up with the naked eyes, the whole manipulation process is about 1h. We detected 56 stylebooks (bovine embryos) by this method and the sensitivity and accuracy of sex prediction was 100% (when the blastomere(s) dissociated from a morula exceed three). Thus, this study showed for the first time that the present method can be applied in bovine breeding programs to facilitate manipulation of the sex ratio of offspring.