超排山羊卵巢卵母细胞体外成熟和体外受精的研究

Studies on IVM and IVF of Goat Oocytes in Superovulated Ovary

以ＴＣＭ１９９＋１０ｍｍｏｌ／ＬＨＥＰＥＳ＋青霉素（６ｍｇ／Ｌ）＋链霉素（５ｍｇ／Ｌ）为基础培养液（ＢＭ），再分别加入不同成分，配成５种卵母细胞成熟液：（Ａ）ＢＭ＋１０％ＥＧＳ；（Ｂ）ＢＭ＋１０％ＥＧＳ＋ＨＣＧ（２．５ｍｇ／Ｌ）；（Ｃ）ＢＭ＋１０％ＥＧＳ＋ＨＣＧ＋Ｅ２（１ｍｇ／Ｌ）；（Ｄ）ＢＭ＋１０％ＦＣＳ＋ＨＣＧ＋Ｅ２；（Ｅ）ＢＭ＋１０％ＥＧＳ＋ＨＣＧ＋Ｅ２＋颗粒细胞（１．５×１０６～３．０×１０６个／ｍＬ）。在３８．５℃、５％ＣＯ２下培养２６ｈ，排卵后第１天卵巢卵母细胞体外成熟率分别为６７．６７％（２０／３０），６０．４２％（２９／４８），８３．６７％（４１／４９）和８０．８２％（５９／７３），排卵后第５天卵巢卵母细胞，在培液Ｄ中体外成熟率为７１．４３％（４５／６３）。排卵后第１天的９８枚卵巢卵母细胞，体外成熟体外受精卵裂率为２１．４３％，２１枚２细胞胚移植５头受体获２头羔羊。研究表明，超排山羊卵巢卵母细胞经体外成熟可获得大量廉价的成熟卵母细胞。

Studies on in vitro maturation (IVM) and in vitro fertilization (IVF) of goat oocytes in superovulated ovary had been made. TCM199 with 10 mmol/L HEPES, penicillin (6 mg/L), and streptomycin (5 mg/L) used as a basic medium (BM), which supplemented with other composition to form the following mediums: (A)BM+10% estrus goat serum(EGS), (B)BM+10%EGS+HCG(2 5 mg/L), (C)BM+10%EGS+HCG+E 2(1 mg/L), (D)BM+10%FCS+HCG+E 2, (E)BM+10%EGS+HCG+E 2+granulosa cells (1 5×10 6 to 3 0×10 6/mL). The OOC were maintained in 100 μL droplets of medium under silicon oil and a humidified 5% CO 2 at 38.5℃ for 26 h, IVM rate of goat OOC from superovulated ovary for 24 h, were 20/30 (67.67%), 29/48(60.42%), 41/49(83.67%), 40/59(67.79%) and 59/73(80.82%) respectively, but for 120 to 144 h, IVM rate was 45/63 (71.43%) in medium D. After the goat OOC from superovulated ovary for 24 h was to be IVM and IVF, the cleaved rate of IVF egg was 42.86% and 2 lambs from 21 cleaved embryos were got. Evidences showed above indicated that a large number of maturation oocytes can be got from goat oocytes in superovulated ovary by IVM, and IVF goats can be got from oocytes by IVM and IVF.