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

Artificial insemination underpins both commercial swine production and the conservation of critically endangered breeds. The Wenchang pig, which is classified as a critically endangered breed, faces a genetic bottleneck due to its reduced breeding population. Current estimates suggest that the breeding population has become quite limited, highlighting the need for effective low-temperature semen preservation strategies. Here, we report that inclusion of 1 μM pinobanksin-a propolis-derived dihydroflavonol-in standard extender sustains the functionality and viability of Wenchang pig spermatozoa during 15 days of storage at 4 °C. Compared to control, pinobanksin maintained total and progressive motility and path velocities (VAP, VCL, VSL), preserved membrane and acrosomal integrity, stabilized mitochondrial membrane potential, and prevented ATP and Ca²⁺ depletion. Biochemically, treated sperm exhibited reduced reactive oxygen species and malondialdehyde levels, restored glutathione homeostasis, elevated activities of SOD and GSH-PX, and upregulated expression of NQO1, HO-1 and GPX4. Mechanistic interrogation revealed that pinobanksin elevates intracellular Ca²⁺, promotes phosphorylation of CaMKKβ and AMPK, and increases cellular Nrf2 abundance. Pharmacological blockade of CaMKKβ (STO-609) or AMPK (Compound C) ablated pinobanksin's antioxidant, anti-apoptotic and motility-preserving effects, whereas direct activation of Nrf2 (via TBHQ) reinstated sperm function under AMPK inhibition. These data delineate a coordinated signaling module involving CaMKKβ, AMPK and Nrf2 through which pinobanksin synchronizes redox and energy-metabolic defenses, establishing it as a dual-action cryoprotectant additive with significant promise for AI-based conservation of endangered porcine germplasm.