Forskolin-Induced Lipid-Droplet Remodeling Is Associated with Improved Redox Homeostasis and Cryotolerance of Vitrified Porcine Oocytes and Parthenogenetic Embryos

Cryopreservation of mammalian oocytes and embryos is essential for the long-term conservation of livestock germplasm resources. Porcine oocytes and embryos accumulate high levels of intracellular lipids. This heavy lipid load aggravates vitrification-related oxidative stress and mitochondrial redox disturbance, greatly reducing cellular cryotolerance and hindering reliable preservation of indigenous pig germplasm. This study aimed to develop a non-invasive metabolic intervention strategy to mitigate lipid-associated oxidative cryodamage and improve the post-thaw quality of porcine oocytes and parthenogenetic embryos. Porcine cumulus–oocyte complexes were treated with forskolin, an adenylate cyclase activator, throughout in vitro maturation. For embryo assays, parthenogenetic embryos received continuous forskolin supplementation during oocyte maturation and were vitrified at Day 2 (cleavage stage). Vitrification disrupted lipid-droplet structural integrity and perturbed multiple metabolic pathways. Enrichment of starch, sucrose and galactose metabolism should be interpreted with caution as computational outputs, alongside changes in glycine, serine and threonine metabolism. Cryoinjury further induced mitochondrial depolarization, excessive reactive oxygen species production, redox imbalance and early apoptosis, ultimately impairing cell viability and embryonic developmental competence. Continuous forskolin exposure during oocyte in vitro maturation (with matching treatment for embryo groups) triggered lipid-droplet remodeling. This remodeling was associated with restored mitochondrial redox homeostasis and suppressed oxidative stress and apoptosis in warmed oocytes and embryos. Forskolin treatment enhanced post-thaw survival and cleavage rates of vitrified oocytes, as well as survival of cryopreserved embryos. By contrast, blastocyst formation rate showed only a numerical, non-significant increase. In conclusion, non-invasive forskolin-driven lipid-droplet remodeling can mitigate vitrification-triggered oxidative damage to lipid droplets and mitochondria. This work provides a practical pre-cryopreservation optimization strategy based on redox protection. It supports improved oocyte experimental protocols and offers useful references for germplasm conservation of rare and endangered pig breeds.