Sperm Functional Analysis by Flow Cytometry

Information about flow cytometry analyses is given according to the recommendations of the International Society for Advancement of Cytometry (ISAC) [38] (link). These analyses were conducted to evaluate some functional parameters of spermatozoa, such as plasma membrane integrity and permeability, membrane lipid disorder, intracellular calcium levels, or ROS levels in extended and FT spermatozoa after a HT of 3 h or 24 h. In each case, sperm concentration was adjusted to 1×10⁶ spermatozoa·mL⁻¹ in a final volume of 0.5 mL, and spermatozoa were then stained with the appropriate combinations of fluorochromes. Plasma membrane integrity was assessed through SYBR-14/PI assay according to the protocol described by Garner and Johnson [39] (link), as well as through PNA-FITC/PI co-staining following the procedure described by Nagy et al. [40] (link). In addition, changes in the permeability of sperm plasma membrane were evaluated through co-staining with YO-PRO-1 and PI, following Martin et al. [24] (link), and membrane lipid disorder was assessed using the protocol for Merocyanine 540 (M-540) and YO-PRO-1 described by Harrison et al. [41] (link). Intracellular calcium levels of spermatozoa were determined through Fluo3-AM/PI co-staining [42] (link). Levels of peroxides and superoxides were evaluated through H₂DCFDA/PI and HE/YO-PRO-1, respectively, according the protocol described by Guthrie and Welch [43] (link). Finally, data was corrected following Petrunkina et al. [44] (link) by determining the percentage of non-DNA-containing particles, to avoid an overestimation of sperm particles. All protocols are described in detail in Information S1.
In all cases, samples were evaluated through a Cell Laboratory QuantaSC™ cytometer (Beckman Coulter; Fullerton, CA, USA; Serial Number: AL300087) using single-line visible light (488 nm) from an argon laser. A minimum of 10,000 events per replicate was evaluated, and data was collected in List-mode Data files (.LMD) and analysed using the Cell Lab Quanta SC MPL Analysis Software (version 1.0; Beckman Coulter). In all cases except for the SYBR-14/PI assessment, data obtained from flow cytometry experiments were corrected according to the procedure set by Petrunkina et al. [44] (link). Each assessment for each sample and parameter was repeated three times in independent tubes, prior to calculating the corresponding mean±SEM. Technical details are also given in Information S1.