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)
Abstract：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
