THE FIX VITAL STAIN METHOD : SIMULTANEOUS DETERMINATION OF VIABILITY AND ACROSOMAL STATUS OF BOVINE SPERMATOZOA

: A rapid, accurate and precise method for simultaneous determination of acrosomal status and viability of bull spermatozoa is introduced and evaluated. The method involves fixation of semen with glutaraldehyde and subsequent addition of the fluorescent dye Hoechst bisbenzimide 33258 (H33258). Wet mounts were examined using a combination of phase-contrast and fluorescence microscopy (×500) for simultaneous visualization of the acrosomal apical ridge, which is indicative of the presence of an intact acrosome, and H33258-labeled nuclei, which is indicative of membrane-damaged cells. This fix-vital stain method allows differentiation between true acrosome reactions and degenerative postmortem loss of acrosomal membranes. Incubation of frozen-thawed spermatozoa for 60 minutes at 37°C in the presence of calcium ionophore A23187 resulted in an increase in the percentage of true acrosome-reacted spermatozoa. The fix-vital stain method does not contain any processing steps that result in loss, selection, or damage of spermatozoa and therefore allows evaluation of representative semen samples.