Accumulating evidence has demonstrated a decreasing trend of semen quality among males worldwide [[1], [2], [3]]. Pigs are commonly employed as a research model for studying the basal metabolic activity of human sperm, due to similarities in reproductive structure between pigs and humans [4]. Mitochondrial dysfunction induced by adverse factors such as drug residues, heavy metal contamination, and heat stress results in a decline in semen quality in both humans and animals [5]. Sperm quality determines the success of artificial insemination, which in turn impacts major reproductive metrics such as conception rate and litter size [6,7]. Mitochondria play a crucial role in regulating various aspects of reproductive processes, from spermatogenesis to fertilization [5]. Specifically, optimal mitochondrial function and an intact mitochondrial membrane potential are essential for ensuring sperm viability, hyperactivation, acrosome reaction, and the maintenance of DNA integrity [8]. Farms are currently focusing on improving the semen quality of boars by improving nutritional standards, strengthening exercise management and implementing rational breeding [9], however, these approaches require increased economic costs and labor inputs. Therefore, how to effectively improve boar semen quality by regulating spermatozoa mitochondrial function is an urgent challenge that needs to be tackled.

Mitophagy can regulate both mitochondrial quantity and quality, maintaining mitochondrial function by removing damaged or dysfunctional mitochondria [10]. Mitochondrial elimination mediated by mitophagy plays an important role in many processes, including the early embryonic development, cell differentiation, inflammation and apoptosis [11]. It has been found that mitophagy under stress conditions promote cell survival and maintain functional activity [12,13], whereas inhibition of mitophagy causes mitochondrial dysfunction, which further leads to apoptosis or sperm death [14,15]. However, identifying an effective approach that targets mitophagy to mitigate mitochondrial damage and enhance sperm quality remains challenging. Forkhead box O3 (FOXO3) influences the survival of mammalian spermatogonial stem cells and spermatogenesis [16], and plays an important regulatory role in PTEN induced kinase 1 (PINK1)/Parkin-mediated mitophagy [17]. PINK1/Parkin-mediated mitophagy represents the classical mammalian cellular mitophagy pathway, selectively removing redundant or damaged mitochondria [18]. PINK1 functions as a key modulator of mitophagy and is constitutively degraded under homeostatic conditions. During mitophagy, PINK1 stabilizes on the mitochondrial outer membrane, activating Parkin and mitochondrial ubiquitin ligase that ubiquitinates mitochondrial proteins, thereby inducing ubiquitin-mediated mitophagy [19,20].

Non-thermal dielectric barrier discharge (DBD) plasma technology is currently used in sterilization, trauma repair, tissue regeneration, oral cavity, and cancer treatment [21], as well as being utilized in animal husbandry for treatment of wastewater, vaccine production, and animal food preservation [22]. DBD plasma can regulate cell function and sperm viability in an intensity- and time-dependent manner. DBD plasma promotes the proliferation of porcine vascular endothelial cells after low-intensity and short-term treatment [23], while inducing apoptosis in mammalian mammary epithelial cells after high-intensity and prolonged treatment [24]. Our previous research demonstrated that DBD plasma under optimized conditions reduced the number of mitochondria in boar spermatozoa while increasing the linear forward-style motility of spermatozoa [25]. It has been reported that high-viability spermatozoa have significantly fewer mitochondria than low-viability spermatozoa, but their mitochondrial activity is higher [26]. However, whether the reduced mitochondria in DBD plasma-treated spermatozoa can provide sufficient energy and support sperm activity by mitophagy is unclear. Therefore, this study aimed to investigate the effects of non-thermal DBD plasma on boar sperm mitophagy and explored the potential mechanism of mitophagy on optimal DBD plasma-mediated sperm quality improvement.