After culture for in vitro fertilization, the oocytes were fixed in 25% (v/v) acidic alcohol at room temperature for at least 3 days, stained with 1% (w/v) orcein in 45% (v/v) acetic acid for 3 min and mounted on a glass slide, and sperm penetration and pronuclear formation were observed under a phase-contrast microscope (x400) [27].