Fig. 3
Effect of HCO3− on boar epididymal sperm intracellular pH (pHi). Samples were loaded with 5 μmol/L of the pH-sensitive dye BCECF-AM for 30 min at 38.5 °C, centrifuged at 700×g for 3 min to remove the excess of dye and resuspended in PBS without Ca2+ and Mg2+ and incubated again for 15 min at 38.5 °C for the de-esterification of the dye. After that, sperm were incubated for 1 and 60 min in capacitating medium (TALP) containing different concentrations of HCO3− (0 mmol/L, 5 mmol/L, 15 mmol/L and 25 mmol/L) and non-capacitating medium (NCAP) in 4 replicates. The fluorescence was monitored using a spectrofluorometer every 2 s for a total time of 300 s. The emitted fluorescence ratio from the excitation at 490/440 nm was calculated and the regression line for pHe vs. the 490/440 nm ratio was obtained (Additional file 1). The pHi of sperm cells was estimated from the regression line. Results are shown as mean ± SEM. Different letters (a, b) in the same time of incubation indicate statistically significant differences (P < 0.05)