Mineral oil is usually used to cover the microdrops of medium in oocytes or embryos culture system here designated as oil method. A large number of oocytes are needed for the production of porcine embryos for in vitro fertilization or somatic cell nuclear transfer (SCNT). The oil method not only wastes a lot of mineral oil, but needs tedious steps in the transferring of embryos. Here we designed a new method called nest dish, which need not mineral oil, to replace the oil method and improve the development rates of porcine eggs in vitro. The oocyte maturation rate with the mTCM199 (83.2%) was significantly higher than with the NCSU23 (75.5%, P﹤0.05), although the parthenogenetic cleavage rates with two media were not significantly different (77.7 and 72.4%, P﹤0.05 ). Chosing mTCM199 as base medium, the rate of maturation with concave dish (90.1%) was significantly higher than with the flat dish (82.6%, P﹥0.05) in nest method, although no significant differences in the oocyte maturation were found between flat dish (82.6%) in nest method and oil method (80.0%). Parthenogenetic cleavage from nest method (80.1% for concave dish, 78.0% for flat dish) did not show any decrease compared to oil method (76.2%), but the developmental rate to blastocysts in the nest groups(17.9 and 19.5%) were significantly higher than the oil method (12.3%, P﹤0.05). These results showed that mTCM199 presented higher maturation rate than that NCSU-23 did, and the nest method with concave dish significantly improved the maturation rate of porcine oocytes in vitro and can replace the conventional oil method.
Key words: Porcine oocytes, in vitro maturation (IVM), microdrop method, nest dish method.
IVM, In vitro maturation; IVF, in vitro fertilisation; BME, β-mercapthonal; SCNT,somatic cell nuclear transfer; PMSG, pregnant mare's serum gonadotropin; hCG,human chorionic gonadotropin; DPBS, Dulbecco's phosphate buffered saline; pFF,porcine follicular fluid; COCs, cumulus-oocyte-complexes; AC, alternating current;BSA, bovine serum albumin.
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