Resveratrol and antifreeze protein during slow freezing enhance post-thaw mitochondrial activity but increase oxidative stress in in vivo–produced ovine embryos

This study evaluated whether resveratrol (RESV), alone or in combination with antifreeze protein type I (AFP I), improves slow freezing cryopreservation of in vivo-derived ovine embryos. Good-quality embryos recovered non-surgically from 35 superovulated ewes were assigned to three groups: AFP (0.1 µg/mL AFP I in the freezing solution), RESV (pre-incubation for 6 h in holding medium supplemented with 1 µM RESV before cryopreservation), or RESV + AFP (combined treatment). After thawing, embryos were cultured in vitro for 48 h and assessed for several endpoints. Resveratrol exposure (RESV and RESV + AFP) reduced intracellular levels of glutathione (GSH), reactive oxygen species (ROS), and ROS/GSH ratio after incubation (P < 0.05), without affecting mitochondrial activity. After thawing, embryos in the RESV + AFP group exhibited higher mitochondrial activity than the others (P < 0.05), accompanied by increased ROS/GSH ratios compared to AFP alone (P < 0.05). After culture, redox parameters did not differ across treatments. There was no difference in survival rates or dead cell index among groups (P > 0.05), but RESV + AFP presented the highest content of intracellular lipid (P < 0.05). Finally, expression of AQP3 and PRDX1 genes was downregulated in the AFP group compared with RESV, with no difference between RESV and RESV + AFP (P > 0.05). Altogether, combining RESV with AFP I enhanced post-thaw mitochondrial activity but disrupted redox homeostasis and increased lipid accumulation, without improving embryo survival or hatching. These findings indicate that RESV combined with AFP I does not improve embryo cryotolerance, while RESV pretreatment may help reduce oxidative stress before cryopreservation.