Coomassie Blue Staining of Sperm Acrosome

The functional integrity of the sperm acrosome was assessed using Coomassie brilliant blue staining as described by Wang [30 (link)]. A Coomassie brilliant blue solution was prepared by first dissolving 0.1 g G-250 in 50 mL of 95% absolute ethanol. Secondly, 100 mL of 85% phosphoric acid was added. Finally, distilled water was added to make the total volume up to 1000 mL. After staining with Coomassie brilliant blue for 30 min at room temperature, each sample was examined under a phase-contrast microscope (OLYMPUS CX31, Tokyo, Japan) at a magnification of 1000× and at least 200 sperm were counted. The head of the intact acrosome sperm was stained blue.