CTC and indirect immunofluorescence assays

The CTC was performed as described previously [13] using the following protocol. After the capacitation process (60, 120, 180, 240 min) sperm suspensions were centrifuged at 200 x g, for 5 min; the capacitation medium was removed and kept at − 20 °C. Sperm were re-suspended in phosphate-buffered saline (PBS) and mixed with equal volume (45 μl/45 μl) of CTC solution (750 mmol/l CTC in 130 mmol/l NaCl, 5 mmol/l cysteine, 20 mmol/l Tris-HCl, pH 7.8) and incubated for 30 min. Cells were then fixed in 8 μl of 12.5% paraformaldehyde in 0.5 mol/l Tris-HCl (pH 7.4). After incubation, sperm suspension was smeared onto a glass slide covered by a cover slip. To avoid evaporation and CTC fading, slides were kept in a dark wet chamber and immediately evaluated.

ACR.2 (Exbio 11–260-C100) immunofluorescent analysis was described previously [20]. After the capacitation process, sperm suspensions from all incubation times (60, 120, 180, 240 min) were centrifuged (200 x g, 5 min); the capacitation medium was removed, and kept at − 20 °C. Sperm were re-suspended in equal volume of phosphate-buffered saline (PBS), smeared onto glass slides, dried and kept at 4 °C. During fluorescent labelling preparation, sperm slides were fixed with acetone for 10 min, rinsed with PBS, treated with ACR.2 monoclonal antibody (50 μg/ml), anti-pY antibody (Sigma-Aldrich P5872; 10 μg/ml) or FITC-phall (Sigma-Aldrich P5282; 50 μg/ml) binding specifically to actin filaments, and incubated in a wet chamber for 60 min at 37 °C. After thorough washing in PBS, the ACR.2 and anti-pY smears were treated with FITC-conjugated anti-mouse IgG antibody (Sigma-Aldrich F0257; 1:500) and incubated in a wet chamber for 60 min at 37 °C. After washing in PBS and water, smears were mounted by the Vectashield mounting medium with DAPI (Vector Lab., Burlingame, CA).

Samples were examined with a Nikon Labothot-2 fluorescent microscope equipped with 40x Nikon Plan 40/0.65 and photographed with a COHU 4910 CCD camera (Inc. Electronics Division, San Diego, USA) using LUCIA imaging software (Laboratory Imaging Ltd., Prague, Czech Republic). Sperm cells were classified according to their cellular (acrosomal) staining patterns into non-capacitated, acrosome intact sperm; capacitated, acrosome-intact sperm; and acrosome-reacted sperm (Table 1; Fig. 1). In each sample, 200 cells were evaluated.

Specific fluorescent patterns of the boar sperm (chilled 17 °C/diluted) as detected by individual fluorescent methods

Fluorescent microscopy pictures of sperm stained with CTC, ACR.2, anti-pY and FITC-phall. Acrosomal and sperm head fluorescent patterns prominent in distinct stages of capacitation process. a1 – a3 sperm treated by CTC: a1 Non-capacitated, acrosome-intact sperm - bright fluorescence over the entire sperm head and positive mid-piece of the tail; a2 Capacitated, acrosome-intact sperm - prominent fluorescent positive equatorial segment and mid-piece, fluorescence-free (dark) band in the post-acrosomal region; a3 Acrosome-reacted sperm - low fluorescent signal throughout the sperm head, with a remaining positive signal in the equatorial segment and mid-piece. B1 – B3 representative pictures of three specific ACR.2 acrosomal fluorescent patterns: b1 Non-capacitated, acrosome-intact sperm - moderate uniform fluorescence in the acrosomal area; b2 Capacitated, acrosome-intact sperm - intensive fluorescence of the acrosome; b3 Acrosome-reacted sperm - low or no fluorescent signal in the sperm head. Anti-pY: C1 – C3 pictures of three specific pY staining patterns: c1 Non-capacitated sperm – moderate signal in the acrosomal area, visible triangular segment; c2 Intensive fluorescence of the sperm head, triangular segment and tail – capacitated, acrosome-intact sperm; c3 Very low/no signal in the acrosomal area, visible triangular segment – acrosome reacted sperm. D1 – D3 representative pictures of three specific FITC-phall staining: d1 Non-capacitated sperm – moderate fluorescence in the acrosomal and sperm head/tail area; d2 Intensive fluorescence of the acrosome and the tail – capacitated, acrosome-intact sperm; d3 Low intensity in the acrosomal and apical sperm head area – sperm after AR. b1 – b3, c1 – c3 nuclei stained with a Blue DAPI dye