Simultaneous detection of the acrosomal status and viability of incubated ram spermatozoa using fluorescent markers

Incubation of diluted ram spermatozoa at 39 degrees C results in a high percentage of acrosome reactions, but previously we have not been able to demonstrate the viability of these cells. Detection of the viability and stages of acrosomal exocytosis, either spontaneous or induced, was carried out using fluorescent probes. Propidium iodide (PI) was used to determine cell viability and, simultaneously, FITC-Pisum sativum lectin (FITC-PSA) was used to assess acrosomal status by staining glycoproteins in the acrosome of permeabilised spermatozoa. Diluted ram semen was incubated for 6 hours at 39 degrees C. At 2 hourly intervals, samples were taken and examined for evidence of a spontaneous acrosome reaction. In addition, calcium ionophore A23187 was used to induce the acrosome reaction and samples were examined at 10 minute intervals. PI was added and then washed out by filtration. Smears were made and air-dried, permeabilised with absolute ethanol and then stained with FITC-PSA. The slides were later viewed under the fluorescence microscope with a peak excitation wavelength of 488 nm. With this combination of two fluorescent probes using a single excitation wavelength, both the cell viability and the acrosomal status could be simultaneously and easily visualized. Results showed four categories of staining: PI-ve/PSA + ve (Live and acrosome-intact), PI + ve/PSA + ve (dead and acrosome-intact), PI - ve/PSA - ve (live and acrosome-reacted) and PI + ve/PSA - ve (dead and acrosome-degenerated). About 75% spermatozoa that were acrosome-reacted were still viable after 4 h incubation in the absence of ionophore, and approximately 90% spermatozoa were acrosome-reacted and still viable after 30 min incubation in the presence of ionophore.