Integration of Proteomics and Phenomics to Reveal Molecular Differences in High and Low Semen Quality in Donkeys

This study investigated the phenotypic and molecular basis of semen-quality variation in donkeys by integrating semen-quality assessment, reproductive ultrasonography and proteomic analysis. High-quality semen (H) and low-quality semen (L) groups were classified according to semen-quality assessment with the parameters of total sperm count (TSC), total motility (TM), progressive motility (PM), acrosome integrity rate (AIR), mitochondrial membrane potential (MMP), sperm abnormality percentage (PSA) and reactive oxygen species (ROS). Our results showed that the H group had greater testicular circumference (TC; 30.20 ± 0.30 cm vs. 28.10 ± 0.40 cm, p < 0.01), long axis of the spermatic cord (LASC; 39.20 ± 4.00 mm vs. 33.50 ± 3.20 mm, p < 0.05), cross-sectional area of the cauda epididymis (CSACE; 352.80 ± 21.60 vs. 281.30 ± 41.80 mm², p < 0.05). Proteomic analysis identified 6 differentially abundant proteins in seminal plasma (SPINKL, HSP-1, GBA2) and sperm (PAFAH1B1, SSMEM1, SPINK2), whose abundances showed potential associations with semen-quality traits and reproductive-organ biometry. The Spearman rank correlation analysis showed that GBA2 abundance was positively correlated with PM (ρ = 0.886), MMP (ρ = 0.943), TC (ρ = 0.986), CSACE (ρ = 1.000) and LASC (ρ = 0.829), and negatively correlated with PSA (ρ = -0.829), CA (ρ = -0.941) and ROS (ρ = -0.886). The other five proteins were positively correlated with PM (ρ > 0.829), while HSP-1, SPINKL, SSMEM1 and SPINK2 were also positively correlated with TC (ρ > 0.812). These findings suggest that semen-quality variation in donkeys is associated with differences in testicular and accessory sex gland biometry, and the 6 identified proteins are potentially associated with semen-quality variation and require further biological validation using targeted proteomics and antibody-based assays in independent populations.