Sperm intracellular pH measurements

After sperm sample liquefaction (30 min at 37 °C), highly motile sperm were recovered by swim-up separation for 1 h in HTF (see above), at 37 °C in an atmosphere of 5% CO₂-95% air. Non-capacitated sperm were diluted to 10⁷ sperm/mL with modified HTF (mHTF: 4 mM NaHCO₃ and 21 mM HEPES were used to replace 25 mM NaHCO₃ in HTF) and loaded with BCECF (2 µM, cell-permeant, dual-excitation ratiometric fluorescent pH probe) in the dark at 37 °C for 30 min. Fluorescence intensity was measured with an Aminco Bowman II spectrofluorometer (λ_Ex1 = 508, λ_Ex2 = 450, λ_Em = 535). Non-capacitated sperm were allowed to stabilize until there was no change, or a small continuous change, in the fluorescent signal. Then Alb or Fab were added and changes in fluorescence were recorded for 400 s at a frequency of 0.5 Hz. High concentrations of Alb likely to cause quenching of fluorescent dyes, so 75 μM was the highest concentration tested. For the C6 group, non-capacitated sperm were incubated with 20 μM C6 for 1 h before the experiment. Conversion of the BCECF Ratio (508/450 nm) to pH value was performed using a calibration curve adjusted with a conventional pH electrode as previous described²⁸. Statistical analyses were performed using the Dunnett Test.