亮甲酚蓝染色筛选及曲古抑菌素A对东北野猪核移植胚发育的影响

体细胞克隆的效率一直较低,这可能与成熟前卵母细胞的质量有关。猪卵母细胞主要来源于屠宰场的废弃卵巢,这暗示,卵母细胞可能处于发情周期的各个阶段。因此,体细胞核转移之前有必要筛选优质卵母细胞。本研究旨在探讨亮甲酚蓝染色筛选及曲古抑菌素A(TSA)对东北野猪(Sus scrofa ussuricus)核移植胚发育的影响。卵丘卵母细胞复合体经亮甲酚蓝(BCB)染色后分三组:BCB+组(胞质着色,葡萄糖-6-磷酸脱氧酶(G6PDH)活性低)、BCB-组(胞质未着色,G6PDH活性高)和对照组(不使用亮甲酚蓝染色)。卵母细胞体外成熟培养后,对卵母细胞进行核移植操作,来源于BCB+卵母细胞的核移植胚使用50nmol/LTSA处理,未使用TSA处理的来源于BCB+卵母细胞的核移植胚作为对照组。结果表明,BCB+卵母细胞的直径显著大于BCB-组(P<0.05)。来源于BCB+卵母细胞的核移植胚的初次卵裂时间要早于BCB-组及对照组。来源于BCB+卵母细胞的核移植胚的囊胚形成率显著高于BCB-组及对照组(P<0.05)。且50nmol/LTSA处理可将来源于BCB+卵母细胞的核移植囊胚率提高到(29.6±2.8)%(P<0.05)。总之,来源于BCB+卵母细胞的核移植胚发育能力较强,且50nmol/L的TSA可促进重构胚的体外发育。对卵母细胞进行BCB染色筛选并使用50nmol/L的TSA处理重构胚,可提高东北野猪核移植重构胚的发育能力,为通过体细胞克隆法保护东北野猪资源创造条件。

Effect of Brilliant Cresyl Blue Staining and Trichostain A on the Development of Somatic Cell Nuclear Transfer Embryos in Northeastern Wild Boar(Sus scrofa ussuricus)

The efficiency of somatic cell nuclear transfer (SCNT) is low, which may be related to the poor quality of the oocyte at the beginning of maturation. Porcine oocytes are commonly recovered from ovaries of slaughtered animals, which indicates that oocytes may be at various stages of the estrous cycle. Therefore, selection of high quality oocytes is necessarry for SCNT. This study was conducted to examine the effect of brilliant cresyl blue(BCB) staining and trichostain A(TSA) on the development of SCNT embryos in Sus scrofa ussuricus. Cumulus-oocyte complex(COCs) were stained with BCB, then divided into three groups: Control(without BCB staining), BCB+ group(coloured cytoplasm, low glucose-6-phosphate dehydrogenase(G6PDH) activity) and BCB- group(colourless cytoplasm, high G6PDH activity). After in vitro maturation(IVM), oocytes were subjected to nuclear transfer. SCNT embryos derived from BCB+ oocytes without TSA treatment were used as control, SCNT embryos derived from BCB+ oocytes were treated with 50 nmol/L TSA for 24 h. The result showed that BCB+ oocytes had a significantly greater diameter than that of BCB- oocytes((119.2±1.4) μm vs (106.1±3.0) μm). This result indicated that BCB+ oocytes might have a bigger volume and contain more mRNA and protein needed for subsequent development. Oocyte diameter or oocyte volume has been shown to be associated with G6PDH activity. Timing of the first zygotic cleavage in SCNT embryos derived from BCB+ oocytes was earlier than that of BCB- oocytes and control oocytes. In our earlier study, selecting porcine oocytes for SCNT based solely on morphological criteria such as compaction of cumulus-corona investment and homogeneity of the ooplasm, it was possible to obtain blastocyst production rates of approximately (10.4±1.1)%. However, the results were showed better in the present study which attributed to the use of the BCB test to more competent oocytes. Namely, SCNT embryos derived from BCB+ oocytes achieved significantly higher blastocyst formation rate than other groups((20.0±1.4)% vs (6.6±0.9)% and (10.4±1.1)%). In addition, 50 nmol/L TSA could improve the blastocyst formation rate of reconstructed embryos up to (29.6±2.8)% and some blastocysts espanded and hatched with no blastocysts hatched in control group. SCNT embryos detrived from BCB+ oocytes might have higher developmental competent and 50 nmol/L TSA could improve the development of SCNT embryos. The result suggested that BCB staining selection and TSA treatment may improve the efficiency of SCNT in Sus scrofa ussuricus and provide a way for protecting Sus scrofa ussuricuss germplasm resources.