Beneficial effects of EGCG on boar sperm quality during liquid storage at 4 °C are mediated by DRD2 receptor

Epigallocatechin gallate (EGCG), a natural antioxidant, plays a vital role in modulating sperm function, yet its protective impact on boar spermatozoa during liquid preservation at 4 °C remains elusive. This study aimed to investigate the beneficial effects of EGCG on boar semen preservation, and elucidate the potential mechanism. Multiple parameters including sperm quality, anti-oxidative status, protein phosphorylation levels, membrane receptor and cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling pathways were analyzed using computer-assisted semen analysis system, Western blot and molecular docking techniques. Results revealed that supplementation with EGCG, particularly with 10 μg/mL, significantly increased sperm motility, acrosome integrity, mitochondrial membrane potential and intracellular ATP content. Moreover, EGCG enhanced the antioxidant defenses of sperm through eliminating excessive reactive oxygen species. Intriguingly, the antioxidant property of EGCG partly prevented protein dephosphorylation, thereby indirectly enhancing protein phosphorylation. Additionally, the dopamine receptor (DRD2) was detected in boar spermatozoa and inhibition of DRD2 greatly prevented EGCG-caused enhancement of protein phosphorylation levels and sperm motility, suggesting the role of DRD2 in regulation of the beneficial effects of EGCG. Molecular docking results indicated that EGCG has favorable binding interactions with the active sites of DRD2, involving crucial hydrogen bonding and hydrophobic interactions, further suggesting that EGCG might directly interact with DRD2, mediate protein phosphorylation via activating the DRD2/cAMP/PKA pathway and thus boost sperm motility. The present study is the first to explore the interacting cell-surface receptor of EGCG on boar sperm and provides comprehensive insights into the protective mechanism of EGCG during hypothermic liquid storage.