Sulforaphane acts through the NFE2L2/AMPK signaling pathway to protect boar spermatozoa from cryoinjury by activating antioxidant defenses.

During cryopreservation, a substantial portion of spermatozoa undergoes apoptosis due to cryoinjury, resulting in decreased fertility. Boar spermatozoa are highly sensitive to temperature, with low temperature triggering reactive oxygen species (ROS) generation, leading to oxidative stress and apoptosis. Sulforaphane (SFN), a potent natural compound found in cruciferous vegetables, is efficacious in mitigating oxidative stress. We here supplemented different SFN concentrations (0, 1.25, 2.5, 5, 10, and 20 μM) into the freezing extender to explore its effect on boar sperm during cryopreservation and determine the optimal SFN concentration. Supplementation of 5 μM SFN exhibited the highest sperm motility, motion performance, plasma membrane integrity, acrosome integrity, and antioxidant properties (total antioxidant capacity (T-AOC) and antioxidant enzyme activity) after freezing and thawing. Then, RT group, C group and C + SFN group were established to explore the effect of SFN on the cryopreservation-induced sperm apoptosis level and fertilizing capacity of post-thawed sperms. SFN effectively rescued the apoptosis and fertilizing capacity of post-thawed sperms. Mechanistically, SFN activated the redox-sensitive nuclear factor erythroid 2-related factor 2 (NRF2/NFE2L2) by promoting adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. This activation improved antioxidant defenses, ultimately improving cryoinjury in boar spermatozoa. In summary, SFN suppressed cryopreservation-induced apoptosis of spermatozoa by activating antioxidant defenses and the AMPK/NFE2L2 signaling pathway. These findings suggest a novel approach for augmenting the cryoprotective efficiency and spermatozoa fertility after cryopreservation.