Piceatannol Protects Sperm from Cryopreservation Damage by Modulating the Keap1-Nrf2/ARE Signaling Pathway

The purpose of this study was to explore the mechanism of action of Piceatannol (PIC) in attenuating oxidative damage to sperm during cryopreservation. Semen samples were collected and homogenized into six equal parts for freeze-thawing experiments. Four different concentrations of PIC were utilized as cryoprotectants during the freeze-thawing process, maintaing a semen to PIC ratio of 1:1, while sperm motility after freezing and thawing was analyzed using computer-assisted sperm analysis (CASA). Sperm plasma membrane integrity was assessed via the hypo-osmotic swelling (HOS) test. The levels of reactive oxygen species (ROS), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) activities, long with the ability to scavenge sperm malondialdehyde (MDA), were examined in sperm following the addition of PIC. Quantitative real-time PCR (qRT-PCR) was performed to detect the expression levels of Keap1, Nrf2, GCLC, GCLM, and HMOX1 in sperm. The mechanism by which PIC protects sperm during cryopreservation from oxidative stress damage was further verified. Treatment with PIC at a dose of 5.0 μmol/L significantly improved both sperm motility and viability while effectively reducing ROS levels in frozen sperm. Additionally, the integrity of the sperm plasma membrane was significantly enhanced. Furthermore, the expression level of Keap1 was significantly reduced, whereas the expression levels of GCLC, GCLM, HMOX1, and Nrf2 were significantly increased (p < 0.05) after the addition of PIC. Notably, a significant attenuation of sperm motility and viability was observed in this treatment group when PIC treatment was accompanied by the addition of an Nrf2 inhibitor, resulting in a significant elevation of ROS levels. The finding that PIC modulates ROS in frozen sperm via the Keap1-Nrf2/ARE pathway thereby enhancing sperm viability levels after freezing and thawing provides a novel approach to optimize semen cryopreservation.