{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T10:42:33Z","timestamp":1785321753489,"version":"3.55.0"},"reference-count":43,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,3,7]],"date-time":"2025-03-07T00:00:00Z","timestamp":1741305600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Vet. Sci."],"abstract":"<jats:sec><jats:title>Objectives<\/jats:title><jats:p>To evaluate the roles of apoptosis and ferroptosis in cryopreservation-induced damage to sheep sperm, with a focus on assessing the effectiveness of inhibitors targeting these pathways.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Initial analysis compared the expression of apoptotic marker Cleaved-caspase3 (CL-caspase3) and ferroptotic marker Transferrin receptor (TFRC) between fresh and cryopreserved sheep sperm. Elevated CL-caspase3 expression and sustained high TFRC expression post-cryopreservation suggested concurrent occurrence of apoptosis and ferroptosis. Consequently, the study employed Deferoxamine Mesylate (DFO), ferrostatin-1 (Fer-1), liproxstatin-1 (Lip-1), and the apoptosis inhibitor Z-VAD-FMK (Z-VAD) at concentrations ranging from 0 to 10 \u03bcM. Post-thaw assessments encompassed plasma membrane integrity, acrosome integrity, and ferroptosis biomarkers. Additional experiments were conducted to measure the expression of GPX4, a key regulator of ferroptosis.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Optimal concentrations (2 \u03bcM for DFO, Fer-1, and Lip-1; 5 \u03bcM for Z-VAD) significantly improved sperm motility and membrane integrity. Among these, Fer-1 demonstrated the greatest efficacy, reducing reactive oxygen species (ROS), lipid peroxidation, and Fe<jats:sup>2+<\/jats:sup> levels. Z-VAD primarily decreased ROS but was less potent than ferroptosis inhibitors. Notably, Glutathione Peroxidase 4 (GPX4) expression was reduced post-cryopreservation, while Fer-1 supplementation restored its levels to those comparable with fresh sperm.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>Both apoptosis and ferroptosis play critical roles in sheep sperm cryopreservation. Fer-1 effectively enhanced cryopreservation outcomes by inhibiting ferroptosis, as evidenced by the restoration of GPX4 expression and improvement in sperm quality indicators. These findings highlight ferroptosis inhibition as a promising strategy for preserving genetic material, with implications for animal breeding and biodiversity conservation.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fvets.2025.1526474","type":"journal-article","created":{"date-parts":[[2025,3,7]],"date-time":"2025-03-07T10:47:01Z","timestamp":1741344421000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":10,"title":["Inhibiting ferroptosis mitigates sheep sperm freezing 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