Integrated Antioxidants, Nanoparticle, and Antifreeze Protein Strategies Synergistically Enhance Cryotop Vitrification Outcomes of Porcine Parthenogenetic Embryos

Porcine embryo cryopreservation remains challenging due to high lipid content, oxidative stress, and ice recrystallization that compromise post-thaw survival and developmental competence. We evaluated an integrated vitrification approach combining antioxidants (berberine, melatonin), iron oxide (Fe₃O₄) nanoparticles, and antifreeze protein I (AFP I) with post-thaw interventions (glutathione and zona pellucida digestion) to synergistically improve cryosurvival and developmental competence of porcine parthenogenetic embryos. In vitro-matured parthenogenetic embryos were vitrified on Cryotop using a protocol that included berberine and melatonin in embryo culture, Fe₃O₄ nanoparticles and AFP I in cryoprotectant solutions, and post-warming treatment with glutathione and a brief zona pellucida digestion. Survival, hatching, adenosine triphosphate (ATP) content, reactive oxygen species (ROS) levels, cytoskeletal integrity, and the expression of <i>BAX</i>, <i>BCL2</i>, <i>OCT4</i>, and <i>SOX2</i> genes were measured. Both the dual antioxidant (berberine + melatonin) and nanoparticle + AFP interventions produced greater improvements than individual additives. Fully integrating all components yielded the highest post-thaw viability, with ~94% survival and ~90% hatching, values statistically equivalent to those of fresh embryos. Treated embryos also showed significantly higher ATP levels, lower ROS accumulation (approaching levels in fresh embryos), and preserved microtubule structure (~91% normal). Vitrification alone upregulated <i>BAX</i> and downregulated <i>BCL2</i>, <i>OCT4</i>, and <i>SOX2</i>, whereas the integrated protocol restored their expression levels to near control levels. This multi-component antioxidant, nanoparticle, antifreeze strategy synergistically enhances the cryotolerance and developmental competence of vitrified porcine embryos by mitigating oxidative stress and cryoinjury. Post-thaw viability and molecular markers were restored to near-fresh conditions, demonstrating a promising approach to improve embryo cryopreservation outcomes in swine and potentially other species.