The use of bright-field microscopy in evaluating bovine acrosome reaction

A novel stain for evaluating the acrosomes of bovine spermatozoa was investigated. Acrosome reactions were induced by incubating spermatozoa enriched by swim-up with 1 mumol/l calcium ionophore A23187 for 1 or 1.5 h, while control samples were incubated in modified Tyrode's medium alone. After fixing in formaldehyde, spermatozoa were stained either with naphthol yellow S plus erythrosin B (NE) or with the novel stain, naphthol yellow S plus aniline blue (NA). The number of spermatozoa that had undergone acrosome reaction was counted and compared with results obtained using differential interference contrast microscopy (DIC). The correlation between the 2 staining methods was high (r=0.99), as was the correlation between NA staining and DIC (r=0.97). Slides stained with NA showed little background staining and the preparations were permanent. The results indicate that NA may be a useful stain for the bright-field evaluation of the bovine acrosome.