Ferroptosis-driven cryoinjury in porcine testicular tissue: Mechanisms, antioxidant-based cryoprotection, and translational strategies for fertility preservation.

Porcine testicular tissue cryopreservation underpins genetic resource banking and fertility preservation. However, conventional dimethyl-sulfoxide (DMSO) protocols recover only a fraction of viable spermatogonial stem cells (SSCs) and often compromise downstream function. This review synthesizes mechanistic evidence showing that cryoinjury is not solely a problem of ice formation and osmotic stress; it is amplified by a surge of reactive oxygen species that peroxidize the exceptionally polyunsaturated, low-cholesterol membranes of porcine germ cells. Together with high testicular iron flux, these features create conditions in which ferroptosis dominates freeze-thaw lethality. In contrast to previous reviews that chiefly attribute cryodamage to generic oxidative stress and advocate broad antioxidant supplementation, this review defines ferroptosis as the dominant mechanism of regulated cell death and proposes targeted antioxidant strategies accordingly. We summarize how glutathione peroxidase-4 (GPX4) insufficiency, labile Fe²⁺, and lipid radical propagation converge during cryostress, and we compile emerging evidence from large animals that targeting this pathway improves outcomes. Lipid-directed radical-trapping agents (ferrostatin-1, liproxstatin-1), vitamin-E analogs, selenium (to support GPX4 activity), and iron chelators each reduce post-thaw lipid peroxidation, preserve membrane and mitochondrial integrity, and enhance sperm/SSC performance. We discuss synergistic combinations and practical delivery considerations, including nano- and liposomal carriers for poorly soluble antioxidants. Translational sections integrate data from xenografting/autografting models showing that cryopreserved testicular tissue can reinitiate spermatogenesis and yield fertile gametes, underscoring the value of mechanism-informed media for both livestock and human fertility biobanking. Finally, we outline priorities for porcine-specific ferroptosis assays, standardized antioxidant-enriched freezing protocols, dose optimization, and long-term reproductive endpoints. Collectively, the evidence supports a paradigm shift: ferroptosis is a central driver of cryodamage in porcine testicular tissue, and ferroptosis-targeted, antioxidant-based cryoprotection offers a rational path to higher SSC survival, improved graft architecture, and better translational fertility outcomes.