E 1020

For scanning electron microscopy (SEM) assay, the spermatozoa samples were fixed onto slides of 1 cm diameter using 2.5% glutaraldehyde for 4 h at 4 °C. After washing the slides with 1× PBS three times and post-fixing in 1% osmic acid for 1 h at 4 °C, dehydration was performed using 30, 50, 75, 95, and 100% ethanol sequentially, and the slides were dried using a CO₂ critical-point dryer (Eiko HCP-2, Hitachi). Finally, the slides were mounted on aluminum stubs, sputter-coated by an ionic sprayer meter (Eiko E-1020, Hitachi), and analyzed by SEM (Hitachi S3400) under an accelerating voltage of 10 kV.
For the transmission electron microscopy (TEM) assay, each semen sample was rinsed in Sperm Washing Medium. The semen samples were pre-fixed in 3% glutaraldehyde, post-fixed in 1% buffered OsO4, dehydrated through gradient acetone solutions, and embedded in Epon 812. Before ultrathin-sectioning, a half-thin section was made to enable sperm location under a light microscope. The ultrathin sections were double-stained with both lead citrate and uranyl acetate and then analyzed under a TEM (TECNAI G2 F20, Philips) with an accelerating voltage of 80 kV.

Scanning electron micrographs were obtained in a JSM-5900 SEM (JSM-5900, JEOL, Tokyo, Japan) operating at 20 KV. Magnifications ranged from 100 to 3000x. Samples were sputter-coated with platinum (ion sputter, E-1020, Hitachi) for observation of the surface morphology. The chemical composition of the surface coating was analyzed with an energy dispersive spectroscope (EDX, Oxford) incorporated into the scanning electron microscope with an acquisition time of 2 min.