Factors affecting staining to discriminate between bull sperm with greater and lesser mitochondrial membrane potential

The sperm mitochondrial membrane potential (MMP) is usually evaluated using the JC-1 dye. This study aimed to verify the effect of incubation temperature (25 °C or 38 °C), incubation time (10, 30, and 45 min), JC-1 stain concentration (0.2 μM, 2 μM, 8 μM, 12 μM), and the presence of glycerol (6.6% compared with 0%), on the capacity of the stain to discriminate between sperm with high mitochondrial membrane potential (hMMP) and low mitochondrial membrane potential (lMMP) in fresh and frozen bull sample by both flow cytometry and epifluorescence microscopy. The temperature (38 °C for 10 min) and the dye concentration (8 μM and 12 μM) resulted in a greater proportion of hMMP (P < .05). The incubation for 45 min at 38 °C resulted in a significant reduction of hMMP in samples stained with JC-1 dye at 8 μM and 12 μM (P < .01). A longer incubation time (45 min) and greater dye concentration (8 μM and 12 μM) resulted in an increased proportion of hMMP sperm in cryopreserved samples. Fresh sperm incubated with glycerol had a hMMP (P < .05). Data for the present study indicate that the optimal incubation temperature was 38 °C, with an incubation time differing between fresh (10-30 min) and cryopreserved sperm (at least 45 min). Furthermore, the JC-1 dye concentration used that could reliably detect the proportion of hMMP sperm was 2 μM in fresh samples, and at least 8 μM in cryopreserved sperm.