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 hig h percentage of acrosome reactions, but previously we have not been ab le to demonstrate the viability of these cells. Detection of the viabi lity 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 sativ um lectin (FITC-PSA) was used to assess acrosomal status by staining g lycoproteins in the acrosome of permeabilised spermatozoa. Diluted ram semen was incubated for 6 hours at 39 degrees C. At 2 hourly interval s, samples were taken and examined for evidence of a spontaneous acros ome reaction. In addition, calcium ionophore A23187 was used to induce the acrosome reaction and samples were examined at 10 minute interval s. PI was added and then washed out by filtration. Smears were made an d air-dried, permeabilised with absolute ethanol and then stained with FITC-PSA. The slides were later viewed under the fluorescence microsc ope 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 an d easily visualized. Results showed four categories of staining: PI-ve /PSA + ve (Live and acrosome-intact), PI + ve/PSA + ve (dead and acros ome-intact), PI - ve/PSA - ve (live and acrosome-reacted) and PI + ve/ PSA - ve (dead and acrosome-degenerated). About 75% spermatozoa that w ere acrosome-reacted were still viable after 4 h incubation in the abs ence of ionophore, and approximately 90% spermatozoa were acrosome-rea cted and still viable after 30 min incubation in the presence of ionop hore.