Pinobanksin alleviates 4℃ semen preservation-induced oxidative damage and apoptosis in Wenchang Pig spermatozoa via the CaMKKβ/AMPK/Nrf2 pathway.

Pinobanksin preserves Wenchang pig sperm viability via CaMKKβ/AMPK/Nrf2 pathway activation during cold storage.

This study demonstrates that pinobanksin, a propolis-derived dihydroflavonol, significantly improves the preservation of Wenchang pig spermatozoa during 15-day storage at 4°C. At 1 μM concentration added to standard extender, pinobanksin maintained sperm motility, membrane integrity, mitochondrial function, and ATP/Ca²⁺ levels while reducing oxidative stress markers (ROS, MDA) and enhancing antioxidant enzyme activities (SOD, GSH-PX). The mechanism involves activation of the CaMKKβ/AMPK/Nrf2 signaling pathway, with pharmacological studies confirming that blocking CaMKKβ or AMPK eliminated pinobanksin's protective effects, while direct Nrf2 activation partially restored function under AMPK inhibition. These findings are particularly significant for conservation of the critically endangered Wenchang breed, whose reduced breeding population faces genetic bottleneck risks. The dual-action cryoprotectant properties make pinobanksin a promising additive for artificial insemination programs in swine, offering both immediate practical applications and insights into oxidative stress mitigation mechanisms in preserved gametes.

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