Figure 5: (a) The FCM results of intracellular calcium ions of spermatozoa: left, sperm cells without either incubation of 1,25(OH)2D or addition of FLUO-4AM; middle, sperm cells without incubation of 1,25(OH)2D but with addition of FLUO-4AM; right, sperm cells with both incubation of 1,25(OH)2D and addition of FLUO-4AM. (b) Effects of 1,25(OH)2D on intracellular calcium ions of spermatozoa (P < 0.05). (c) Effects of 1,25(OH)2D on NADH concentration of spermatozoa (P < 0.05). (d) Effects of MCU inhibitor (Ru360) on the ATP production of spermatozoa (P > 0.05). Data for each group are expressed as mean ± standard deviation. The sperm concentration used was 1 × 107 ml−1. The differences among different groups and between two groups were assessed by one-way ANOVA with Holm–Sidak post hoc test and paired samples t-test, respectively.*P < 0.05 and **P < 0.01, groups treated with 1,25(OH)2D compared with the control group. FCM: flow cytometry; FLUO-4AM: the acetoxymethyl ester form of fluo-4; NADH: nicotinamide adenine dinucleotide; MCU: mitochondrial calcium uniporter; ATP: adenosine triphosphate; ANOVA: analysis of variance; RU360: Ruthenium 360; CV: coefficient of variation; FL1-H: FL1 pulse height; arb.units: arbitrary units.