Proper functioning of mitochondrial complex I is essential for the preservation of porcine semen at 17 °C

Artificial insemination in swine primarily relies on semen preserved at 17 °C; however, semen quality progressively declines during storage. Although liquid storage aims to keep sperm metabolism low, and liquid-stored semen is understood to rely mainly on glycolysis rather than oxidative phosphorylation, whether mitochondrial Complex I (MCI) contributes to sperm preservation has not been investigated. Herein, we blocked MCI with different concentrations of the inhibitor rotenone (0, 10, 25, 50, and 100 μM) and stored the samples at 17 °C for 7 days. Blocking MCI reduced (P < 0.05) the percentage of viable sperm with intact acrosomes and increased membrane lipid disorder in viable sperm. Furthermore, inhibiting MCI with rotenone increased oxidative stress, decreased Ca²⁺ and mitochondrial membrane potential, and impaired total and progressive motility (P < 0.05). Interestingly, as revealed by motile subpopulations analysis, blocking MCI with rotenone led sperm to exhibit slower, less linear movement. These findings suggest that MCI is crucial for maintaining sperm quality and function during the preservation of porcine semen at 17 °C, supporting the conclusion that basal activity of the mitochondrial electron transport chain remains essential, even though liquid-stored sperm catabolism relies more heavily on glycolysis than on oxidative phosphorylation.