The capacitation process was assessed using fluorescent microscopy and chlortetracycline (CTC) assay (Sigma-Aldrich, St. Louis, MO, USA), which allows more detailed and reliable identification of capacitated spermatozoa. The principle of this method is to identify a specific pattern following absorption of the fluorescent dye by the sperm cells. Based on the coloration three patterns were recognized. The „F”pattern indicated a uniform fluorescence of the entire sperm head, which was characteristic for uncapacitated cells. Pattern „B”was visible as a narrow band of fluorescence in the postacrosome region of the sperm cell, which detected capacitated cells. The „AR”pattern indicated spermatozoa that underwent acrosome reaction, which was visible as a band of fluorescence in the sperm flagellum [15 ].
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