Ferroptosis contributes to boar sperm deterioration during liquid semen storage

Ferroptosis is an iron-dependent form of regulated cell death characterized by labile iron accumulation, glutathione depletion, mitochondrial dysfunction, and lipid peroxidation. Although ferroptosis has been linked to impaired spermatogenesis and male infertility in vivo, its occurrence and functional relevance in ejaculated spermatozoa during liquid semen storage at 17 °C has not been established. This study evaluated how ferroptosis inducers and inhibitors modulate boar sperm function during liquid semen storage at 17 °C and determined whether ferroptosis contributes to spontaneous sperm deterioration over time. Semen samples from five Duroc boars were treated with ferroptosis inducers: Erastin (system Xc^- antagonist) and RSL-3 (GPX4 inhibitor), inhibitors: Ferrostatin-1 and N-acetylcysteine (NAC), and vehicle/untreated controls. Sperm motility was assessed by computer-aided sperm analysis, and comprehensive ferroptosis-associated biomarkers were quantified using spectral flow cytometry with a 7-fluorophore panel including sperm identification (Hoechst 33342), labile ferrous iron (FerroOrange), glutathione (monochlorobimane), reactive oxygen species (DCFDA), lipid peroxidation (BODIPY 581/591 C11), mitochondrial membrane potential (MitoTracker Deep Red), and plasma membrane integrity (propidium iodide). RSL-3 induced declines in total and progressive motility accompanied by elevated Fe²⁺ accumulation, glutathione depletion, mitochondrial depolarization, and membrane disruption by day 3 (p < 0.05). Erastin produced similar but delayed effects, reaching maximum ferroptotic markers by day 7. Both Ferrostatin-1 and NAC mitigated oxidative injury and preserved motility and cellular integrity relative to inducers. Critically, untreated controls exhibited time-dependent increases in all ferroptosis-associated biomarkers over 7 days of liquid storage, indicating spontaneous ferroptotic activity independent of chemical induction. These findings establish ferroptosis as a mechanistically relevant contributor to sperm.