Axioskop epifluorescence microscope filter 1

The acrosome damages during vitrification and warming were assessed by Pisum sativum agglutinin lectin (PSA) conjugated to fluorescein isothiocyanate (FITC). Briefly, 20 μl of sperm suspension (about 20 × 10⁶/ml) of the swim-up-prepared fresh sperm and the vitrified sperm of each treatment group was washed with Dulbecco’s phosphate-buffered saline (DPBS; #14190-094, Gibco, Waltham, MA, United States) by centrifugation to remove semen diluents; then, the pellet was resuspended in PBS. The sperm suspension was smeared onto a slide and fixed by cold 4% paraformaldehyde for 30 min at room temperature. The PSA–FITC solution (0.1 mg/ml; #L0770, Sigma-Aldrich, Steinheim, Germany) was placed over the smear and incubated in a humidified chamber at 37°C for 20 min, rinsed with PBS, and dried. The slides were examined under Zeiss Axioskop epifluorescence microscope filter I (excitation filter, 450–490 nm; dichroic mirror, 510 nm; and emission filter, 515–565 nm) with a × 100 oil objective. About 200 cells were counted on each slide. Each determination was carried out in duplicate. The bright fluorescence with a clear-cut acrosomal cap in the acrosomal region was considered an intact acrosome, while no fluorescence or only fluorescence of the equatorial segment or an irregular acrosomal cap was considered a damaged acrosome.

The live and dead sperm were evaluated using a live/dead sperm viability kit (#L7011, Molecular Probes, Eugene, OR, United States) as per the manufacturer’s instructions. Briefly, 20 μl spermatozoa suspension (about 20 × 10⁶/ml) of the swim-up-prepared fresh sperm and the vitrified sperm of each treatment group was incubated with SYBR-14 (1 μM) and propidium iodide (PI, 120 μM) in an Eppendorf tube at 37°C for 10 min. A 10-μl drop of the mix was placed on a slide under coverslips and evaluated under epifluorescence using Zeiss Axioskop epifluorescence microscope filter I (excitation filter, 450–490 nm; dichroic mirror, 510 nm; and emission filter, 515–565 nm) at × 630 magnification. Both live (green) and dead (red) sperm were counted simultaneously. About 200 sperm cells were counted on each slide. Each determination was carried out in duplicate.