Swelling of the apical ridge and anterior acrosome of motile bovine sperm was observed using differential interference contrast optics. Transmission electron microscopy indicated that the acrosomal matrix was extended into complex folds and/or projections. Outer acrosomal and plasma membrane integrity was retained. Anterior acrosomal swelling (AS) was first observed after neat-semen had been slowly cooled to 4°C and stored for 1 day. With subsequent dilution (25 X 10⁶ sperm/ml) and incubation of sperm in a Tris fructose medium at 37°C, a majority of the motile sperm had AS. Of these unique conditions, storing sperm (1500 X 10⁶ /ml) in seminal plasma (SP) at 4°C was very conducive to AS. Replacing SP with egg yolk-citrate (EYC) inhibited AS. Using a Tris buffer with 54 mM fructose as an incubation medium eliminated the necessity of storing sperm in SP provided storage in EYC at 4°C was ≥3 days. AS occurred after storage at 37 or 4°C but not at 21°C. Storing cauda epididymal sperm at 4°C with and without 30.8 mM fructose or glucose in EYC or egg yolk-Tris (EYT) media demonstrated that AS formation required the presence of either sugar. Storing cauda sperm in EYT at 4°C with 0, 3.9, 7.7, 15.4, 30.8, and 61.7 mM fructose indicated that a minimum of 7.7 mM fructose was necessary for a strong AS response. Storage media pH was measured at the end of storage. Media pH was 6.7 with 0 mM fructose and had decreased during storage to 5.7-6.0 in media giving a strong AS response. Further experimentation with cauda sperm demonstrated that storage at reduced media pH would induce AS with or without the presence of fructose. Conversely, storage at normal pH's (6.6-6.8) inhibited AS even with fructose in the media.

The proportion of motile sperm with AS was estimated from wet smears initially. Evaluation of acrosomal morphology from dry smears of vitally stained sperm was developed as a means of quantifying the proportion of viable sperm with AS. This technique also allowed closer scrutiny of the acrosomal morphology of viable sperm.

The physiological importance of AS is unclear. It may be a unique form of sperm deterioration or a prelude to the true acrosome reaction since AS occurs without loss of sperm viability.