The objective of this work was to investigate the protein composition of seminal plasma in young Nellore bulls. Twenty animals, aged between 19.8 and 22.7 months, were selected and separated into two categories based on their Breeding Soundness Evaluation (BSE) outcomes: approved (FIT; n = 10) and not approved (UNFIT; n = 10). Scrotal circumference was recorded, and semen samples were collected via electroejaculation. Sperm movement, collective motility, and vigor were assessed by optical microscopy, while sperm defects were evaluated through phase-contrast microscopy. Seminal plasma was separated from sperm cells by centrifugation and analyzed using LC-MS/MS proteomics. Protein identification utilized MASCOT Daemon (v2.4.0), and relative quantification followed the Exponentially Modified Protein Abundance Index (emPAI) via SCAFFOLD Q+ (v4.0). Protein functions were annotated using the KOG ontology, and clustering analyses were carried out in DAVID. No significant differences were found in scrotal size or basic semen traits between groups, but the UNFIT bulls exhibited a higher rate of sperm defects (p < 0.05). Across all samples, 297 proteins were detected, with 11 showing distinct abundances (p < 0.05): two elevated in FIT bulls (Spermadhesin-1 and Ig gamma-1 chain C region) and nine in UNFIT bulls (Vasoactive intestinal peptide, Metalloproteinase inhibitor 2, Ig lambda-1 chain C region, Protein FAM3C, Hemoglobin beta, Seminal ribonuclease, Spermadhesin 2, BSP-30kDa, and Spermadhesin Z13). Spermadhesin-1 represented the most abundant protein overall (36.7%), corresponding to 47.7% in FIT and 25.7% in UNFIT animals. The most represented biological functions included post-translational modification, protein degradation, and chaperone activity. Functionally enriched clusters were linked to phospholipid transport, ATP-binding mechanisms, and chaperonin complexes. Proteins increased in FIT bulls were related to sperm capacitation and antioxidant defense, while those overrepresented in UNFIT bulls were linked to impaired motility, membrane cholesterol loss, and oxidative damage. In summary, seminal plasma from FIT bulls contained proteins that support fertilization processes, whereas the UNFIT group showed patterns associated with reduced fertility potential. The relative concentration of Spermadhesin-1 could serve as a biomarker for reproductive assessment in young Nellore bulls.
