Research And Application Of Bovine Embryo Production In Vitro And A Novel Method For Embryo Sex Determination

This dissertation studied how to effectively product the bovine IVF embryos and improved a novel and simple method for bovine sex determination-Loop-mediated isothermal amplification, including the following conclusions.1.In the study, effects of different factors, including maturation time, grades of cumulus-oocyte complexes (COCs), the addition of E₂ concentration and origin of oocyte, were evaluated on oocyte maturation efficiency in bovine. Cleavage rate of oocyte maturation for 21h and 27h were significantly lower than for 24h. The blastocyst production rate of maturated oocyte for 21h was lower than for 24h and 27h(P<0.05); Oocyte of grade A and B had a significantly higher cleavage and blastocyst rate than of grade C and D(P<0.01); The supplementation of 1μL/mL E₂ in the maturation medium had more Cleavage and blastocyst rate than of 10μL/mL E₂(P<0.05), but it had no difference with control group; Cleavage rate of oocyte from 2-8 mm follicles was significantly greater than that from small follicles(<2 mm) and competent follicles(>8 mm), blastocyst rate was higher in the oocyte from 2-8 mm follicles and competent follicles than that of oocyte from small follicles. The result showed that oocytes maturated for 24 h were optimized to the cytoplasmic and nucleus maturation at the same time; oocyte of grade A and B had more developmental potential for in vitro embryo production; addition of 1μL/mL E₂ in the maturation medium increased nucleus maturation rate, but high concentration of E₂ inhibited the embryo development; oocyte from 2-8 mm follicles maturated for 24 h had more competence to develop blastocyst stages.2.The present study was conducted to assess the effect of bovine follicular fluid (bFF) from the different diameter follicles (small <3mm, middle 3-6mm and large 6-12 mm) on the oocyte maturation by parthenogenesis and in vitro fertilization. The first polar rate with addition of mbFF and lbFF in the maturation medium was significantly higher than addition of sbFF(P<0.05) and control group (P<0.01). Cleavage and blastocyst rate were improved with addition of 50% lbFF for parthenogenetic embryo, but for IVF embryo, addition of 50% lbFF decreased the cleavage. The result showed higher concentration of lbFF in the maturation medium was powerful for oocyte cytoplasmic maturation, but was against the fertilization. In order to overcome the disadvantage, two-step method for oocyte maturation was used to judge the maturation efficiency, which oocytes were incubated in the 50% lbFF for 6 h, then oocytes were transferred to M-199+10% FBS to culture another 24 h. The result...