Combined use of fluorescence and phase contrast microscopy for the determination of sperm viability
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Cita bibliográficaTomas, C., Gomez-Fernandez, J., Gomez-Izquierdo, E., De Mercado, E. (2014). Combined use of fluorescence and phase contrast microscopy for the determination of sperm viability. Reproduction in Domestic Animals, 49, 102-102.
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.