Lectins Histochemistry: Wheat germ agglutinin (WGA), peanut agglutinin (PNA), and concanavalin A (ConA) (all from Sigma, USA) were used to detect non-capacitated, acrosome intact, and acrosome-reacted spermatozoa, respectively. The smears of the thawed samples were fixed with 2% paraformaldehyde for 20 min. After washing with phosphate-buffered saline (PBS), the samples were incubated with fluorescein isothiocyanate (FITC)-conjugated lectins at 10 μg/mL dilution for two hours and double stained with Hoechst (Sigma, USA) for five min. The slides were evaluated using the Eclipse E600 fluorescent microscope (Nikon, Japan).
Flow Cytometry: Thawed samples were washed with 800 µL of PBS, centrifuged at 170 g for 10 min, and fixed with 2% paraformaldehyde for 30 min at 4 °C.
Thereafter, the aliquots were centrifuged, and the pellets were resuspended in PBS. The aliquots containing 1×105 cells were exposed to FITC-conjugated lectins
at a dilution of 10 μg/mL for two hours at 37 °C. The samples were assessed using the FL1 channel (wavelengths ≈495 nm) and FL3 channel (wavelength >575 nm)
of the FACSCaliburTM flow cytometer (BD Biosciences, USA). The data were analyzed using FlowJo software (BD Biosciences, USA).
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