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ORIGINAL RESEARCH article
Front. Vet. Sci.
Sec. Animal Reproduction - Theriogenology
Volume 12 - 2025 | doi: 10.3389/fvets.2025.1526474
This article is part of the Research Topic Innovative Strategies for Improving Sperm Lifespan during Storage View all articles
Inhibiting Ferroptosis Mitigates Sheep Sperm Freezing Damage
Provisionally accepted- Inner Mongolia Agricultural University, Hohhot, China
Objectives: 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. Methods: 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 postcryopreservation 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 μM. Postthaw 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. Results: Optimal concentrations (2 μM for DFO, Fer-1, and Lip-1; 5 μM 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 2+ 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. Conclusion: 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.
Keywords: Sheep sperm, Cryopreservation, ferroptosis, Apoptosis, Ferroptosis Inhibitors 1. Introduction
Received: 11 Nov 2024; Accepted: 24 Feb 2025.
Copyright: © 2025 Hai, Li, Song, Zhang and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Jiaxin Zhang, Inner Mongolia Agricultural University, Hohhot, China
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