Combined use of fluorescence and phase contrast microscopy for the determination of sperm viability

Abstract

The aim of this study was to evaluate the combined use of fluorescenceand phase contrast microscopy as a new technique for the assessmentof sperm viability. For this, five pools of semen from three fertile boarswere frozen and thawed according to a standard protocol. Afterthawing, sperm samples were stained with 5 ll of propidium iodide(PI, 0.5 mg/ml) and incubated at 37°C in darkness 10 min. Subse-quently, samples were analyzed using a phase contrast microscopy(Nikon Eclipse E400, Tokyo, Japan) coupled with fluorescenceequipment (Nikon C-SHG1) with a mercury lamp (100 W) and aNikon G-2A filter with excitation/barrier filter of 510/590 which allowsa simultaneous excitation of blue and green. After displaying thesample under phase contrast microscopy the fluorescence wasconnected, non-viable sperm showed red color, and viable sperm werenot stained and visible. To determine the validity of this technique thesamples were also analyzed with a dual fluorescent staining (SYBR-14/PI) under fluorescence microscopy (Garner and Johnson, 1995; BiolReprod. 53, 276–284). The results did not show (57.6 vs. 57.2  1.5%live sperm; Garner and Johnson’s and described technique respec-tively) difference between techniques (p > 0.05). In conclusion, thecombined use of fluorescence and phase contrast microcopy using a PIstaining may be used for the determination of sperm viability, saving inthe use of fluorochromes and viable sperm are visible which wouldsimultaneously allow the acrosome state assessment.