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Abstract The Wannan black pig is a superior local breed in Anhui province, renowned for its exceptional meat quality and remarkable adaptability to various environmental conditions. Semen, being a crucial indicator of male sexual maturity and fertility, significantly influences the performance of breeding boars. The molecular basis for comprehending the fecundity of boars in practical production lies in understanding the disparities in sperm proteins among boars of varying ages. In this investigation, spermatozoa from three one-year-old and three seven-year-old Wannan black pigs were individually chosen. Employing a tandem mass tag (TMT)-based quantitative proteomics approach, a total of 4050 proteins were identified, out of which 130 proteins exhibited significant differences between the two groups. GO enrichment analysis revealed that these proteins primarily participated in energy metabolism, spermatogenesis, fertilization, and reproduction. KEGG enrichment analysis demonstrated that the differential proteins predominantly resided within the ribosome pathway. A protein-protein interaction (PPI) network was constructed to identify core proteins such as Small ribosomal subunit protein uS7 (RPS5). Ultimately, parallel reaction monitoring (PRM) was conducted on the selected differential proteins to validate result accuracy. The findings of this study establish a foundation for elucidating the molecular mechanism underlying variations in spermatozoa protein levels among Wannan Black Pig with different age.

Abstract VPS28 (vacuolar protein sorting 28) is a subunit of the endosomal sorting complexes required for transport (ESCRTs) and is involved in ubiquitination. Ubiquitination is a critical system for protein degradation in eukaryotes. Considering the recent findings on the role of ubiquitination in the regulation of lipid metabolism, we hypothesized that VPS28 might affect the expression of genes involved in milk fat synthesis. To test this hypothesis, we modulated VPS28 expression in the bovine mammary epithelial cell line (MAC-T) and measured the effects on triglyceride (TG) synthesis using lentivirus-mediated techniques. The results showed that VPS28 knockdown significantly upregulated the levels of the fatty acid transporter CD36 molecule (CD36) and adipose differentiation-related protein (ADFP), leading to increased TG and fatty acid production, along with elevated ubiquitin (UB) levels, while reducing proteasome activity. In contrast, VPS28 overexpression increased CD36 levels while not significantly affecting ADFP or TG levels, with a trend toward reduced lipid droplets and increased UB expression and proteasome activity. In addition, inhibition of the ubiquitin-proteasome system and the endosomal-lysosomal pathway using epoxomicin and chloroquine, respectively, further increased CD36, ADFP, and TG levels, thereby enhancing cell viability. These in vitro findings were validated in vivo in a mouse model, where VPS28 knockdown increased mammary CD36, ADFP, UB expression, TG content, and lipid droplets without pathological changes in mammary tissue or blood TG alterations. These results confirm the pivotal role of VPS28 in regulating TG synthesis via the ubiquitination pathway, offering novel insights into the molecular mechanisms of milk fat production in a bovine cell model.