Studies On The Embryos Production Completely In Vitro And The Sampling Methods In Embryo-sex-identification In Bovine

Successful embryo production in vitro of mammalian oocytes would provide a steady source of materials for embryo transfer and research. The embryo production in vitro include oocytes in vitro maturation (IVM), in vitro fertilization (1VF), parthenogenetic activation (PA), in vitro culture (IVC) and sex identification. The aim of this study was to perfect the technology of embryos in vitro production through the research in the key factor of each important part.1. in vitro maturation (IVM)The experiment was carried out to study effects of different follicle size, hormones, sera, big follicular fluid(BFF) and maturation time on the in vitro maturation of bovine oocytes.The result shows that follicle size directly affected maturation rate of oocytes after IVM. The maturation rate ofoocytes from follicles which diameters were 2⁵mm, 5⁸mm and 8¹1mm were significantly higher thanfrom follicles which diameters were less than 2 mm (40.91%, 41.82% and 39.09% VS 16.36%, P<0.01), butthe IVM oocytes from follicles which diameters were 8 1 lmm was low quality.Mammalian oocyte maturation and cumulus expansion are induced by hormones, LH, FSH and 17(3-E2 areimportant hormones to promote oocytes maturation.In order to learn the interacts among the three hormones,we carried cross combination experiments and selected the best combination concentrations, the concentrationsof 17β-E₂, LH and FSH were 2.55μg/mL, 25.08μg/mL and 15.03μg/mL, respectively.Serum supplementation is a kind of important material during bovine oocyte maturation in vitro, the maturationrates of oocytes cultured in the maturation media added with fetal bovine serum (FBS) and oestrus cow serum(OCS) were significantly higher than its of oocytes matured in medium with neonatal calf serum (NCS)(48.97%, 47.59% VS 22.76%, P<0.01). So FBS and OCS are effective Serum supplementations in bovineoocyte maturation.Without Serum and hormones, BFF can improve the maturation of oocytes obviously, the maturation rates ofoocytes cultured in the maturation media added with 10% BFF and 5% BFF ware 29.63% and 16.30%respectively, but the maturation rate was rather low. With Serum and hormones, the effect of BFF isunconspicuous.Suitable maturation time of oocytes is important. Oocytes maturing From 18h to 30h, the maturationrates of different maturation time were no difference (52.27%, 54.55%, 55.30%, P>0.05), but if thematuration time over 24h, the IVM oocytes become low quality.2 in vitro fertilization (IVF)The experiment was carried out mainly to study effects of different component in fertilization medium, structure of Cumulus oocyte complexes (COCs) and oocyte-sperm interact time on the in vitro fertilization of bovine oocytes.Heparin and caffine are important materials during bovine oocyte fertilization in vitro. A 3 (heparin, 15, 20, 25 mg/L)×3 (caffine, 2, 4, 6 mmol/L) factorial experiment was designed to determined the effect of different concentration of heparin and caffeine in fertilization medium on cleavage rate of bovine oocytes and their subsequent development to blastocyst stage. The result shows that the optimal concentrations of heparin and caffeine ware 20 mg/L and 2 mmol/L, respectively.Oocyte maturation is accompanied by changes in protein synthesis of the cumulus oocyte complex (COC), the differentially expressed proteins are relative to equipping the oocyte with further developmental competence. This study was mainly to investigate the effect of cumulus cells surrounding oocytes on in vitro fertilization (1VF) and in vitro culture (IVC). The result shows that the cleavage rate and blastocyst rate of the oocytes with 4⁵ or over 6 layers of cumulus cells were significantly higher than that of less than 4 layers of cunmulus cells (P<0.01).Oocyte-sperm interaction time directly affected fertilization rate of oocytes. The experiment was conducted to determine the effect of different oocyte-sperm interaction time on in vitro fertilization and the subsequent embryo development. The result shows that the group of oocyte-sperm interaction time less than 6h resulted in lower cleavage rate (44.79%) than those for 12h, 18h or 24h treatments (54.17%, 57.29% or 56.25%, P<0.01). And also, the group of oocyte-sperm interaction time in 12h had significant higher blastocyst rate (33.33%) than that of other groups (P<0.01).3 parthenogenetic activation (PA)Parthenogenetic activation of oocytes was another important technology of embyos production in vitro. Thisexperiment was undertaken to improve development of parthenogenetic embryos following various combinedtreatments of ethanol, ionomycin, and 6-DMAP.In Experiment 1, in vitro matured oocytes were treated with 5fiM ionomycin or 7% ethanol for 5min followedby incubation in 6-DMAP for 4h, which concentration was 0.5 mM, 1.0 mM, 2.0 mM and 3.0 mM, The resultshows that the cleavage rates of the 2.0 mM and 3.0 mM 6-DMAP groups were significantly higher than theother groups (37.50% VS 27.08% and 19.79%, P<0.01).In Experiment 2, in vitro matured oocytes were treated with S\iM ionomycin for 5min followed by incubationin 2.0 mM 6-DMAP for Jh, 3h, 5h, 7h or 9h, The result shows that the blastoeyst rates of the 5h and 7h groupswere significantly higher than the other groups (12.38% and 10.48 VS less than7.62%, P<0.01).4 in vitro culture (IVC)The experiment was carried out to study effects of culture media volume, granulosa cell and epidermal growth factor (EGF) on the in vitro maturation, in vitro fertilization of bovine oocytes and in vitro culture of oosperms. Oocytes or embryos were cultured in 50jiL droplet and 500^iL U-shaped Petri dish respectively, the cleavge and blastocyst rates of oosperms in 50p.L droplet were significantly higher than those in 500|iL U-shaped Petri dish(50.67% VS 47.33%, 30.67% VS 28.67, P<0.05).Granulosa cell monolayers cultured with oocytes or oosperms, the result shows that there were no significant differences in cleavage rates between the co-culture group and the no granulosa cell group (67.11% VS 65.78%, P>0.05), but the blastocyst rate of oosperms of the co-culture group was significantly higher than that of the no granulosa cell group (67.11 % VS 19.11 %, P<0.01).EGF as a kind of growth factor play an important role in embryo culture in vitro. In this experiment EGF was added to IVC medium of oosperms, which concentration was 0 ng/mL, 20 ng/mL, 40 ng/mL, 60 ng/mL, 80 ng/mL and 100 ng/mL. The blastocyst rates of the 60 ng/mL and 80 ng/mL groups was significantly higher than those of the other groups (27.21%, 28.68% VS less than 22.79%, P<0.01).5 sampling methods in embryo-sex-identificationIn order to perfect a series of techniques of sex determination of IVF bovine embryos, realize the production and commerce of female embryos of milch cow, The study compared two sampling methods in embryos, incision method and stiletto method, and also, we gathered embryo cells by the two methods separately. The result showed that the veracity of sexing result by the two method was no difference, but the stiletto method excel the incision method in pregnancy rate of morula in evidence (35.29% VS 25.00%), however, the incision...