We compared handmade cloned (HMC) buffalo blastocysts produced from oocytes stained with Brilliant Cresyl Blue (BCB) and classified into those with blue (BCB + ) or colorless cytoplasm (BCB -). The blastocyst rate was higher ( p < 0.001) for BCB + than for BCB -oocytes (43.41 -2.54 vs. 22.74 -1.76%). BCB + blastocysts had inner cell mass (ICM) cell number, ICM-to-trophectoderm ratio, global level of H3K18ac, apoptotic index, and expression level of BCL-XL, but not that of CASPASE-3, similar to that of blastocysts produced through in vitro fertilization (IVF), which was higher ( p < 0.05) than that of BCB -blastocysts. The global level of H3K9me2, which was similar in BCB + and BCB -blastocysts, was higher ( p < 0.01) than that in IVF blastocysts. The expression level of OCT4 and SOX2 was higher ( p < 0.05) and that of GATA2 was lower ( p < 0.05) in BCB + than that in BCB -blastocysts, whereas that of DNMT1, DNMT3a, NANOG, and CDX2 was not significantly different between the two groups. The expression level of DNMT1, OCT4, NANOG, and SOX2 was lower ( p < 0.05) and that of CDX2 was higher ( p < 0.05) in BCB + than in IVF blastocysts. In conclusion, because BCB + blastocysts have better developmental competence and are closer to IVF blastocysts in terms of quality, epigenetic status, and gene expression than BCB -blastocysts, BCB staining can be used effectively for selection of developmentally competent oocytes for HMC.
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