This study aimed to evaluate the proteomic profile of seminal plasma from young Nellore bulls. We used 20 bulls aged between 19.8 and 22.7 months, divided into two groups according to the results of the Breeding Soundness Evaluation (BSE): approved (FIT n = 10) and not approved (UNFIT n = 10). The scrotal perimeter was measured and a semen collection was performed through electroejaculation. The percentage of sperm motility, mass motility, and sperm vigor were calculated using conventional microscopy, and the percentage of sperm abnormalities was calculated using phase-contrast microscopy of all ejaculates. Seminal plasma was separated from spermatozoa using centrifugation and processed for proteomic analysis by LC-MS/MS. Seminal plasma proteins were identified using MASCOT Daemon software v.2.4.0 and label-free quantification analysis was carried out by SCAFFOLD Q+ software v.4.0 using the Exponentially Modified Protein Abundance Index (emPAI) method. Functional classification of proteins was performed based on their genetic ontology terms using KOG. Functional cluster analysis was performed on DAVID. There were no differences in scrotal perimeter and physical semen characteristics between FIT and UNFIT groups of bulls. The percentage of sperm abnormalities was higher (p < 0.05) in the UNFIT group of bulls. A total of 297 proteins were identified for the two groups. There were a total of 11 differentially abundant proteins (p < 0.05), two of them more abundant 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 regions, Protein FAM3C, Hemoglobin beta, Seminal ribonuclease, Spermadhesin 2, Seminal plasma protein BSP-30kDa, and Spermadhesin Z13). Spermadhesin-1 was the protein with the highest relative abundance (36.7%) in the seminal plasma among all bulls, corresponding to 47.7% for the FIT bulls and 25,7% for the UNFIT bulls. Posttranslational modification, protein turnover, and chaperones were the functional categories with the highest number of classified proteins. Protein functional annotation clusters were related to Phospholipid efflux, ATP binding, and chaperonin-containing T-complex. The differentially abundant proteins in the group of FIT bulls were related to sperm capacitation and protection against reactive species of oxygen. In contrast, differentially expressed proteins in the group of UNFIT bulls were related to motility inhibition, intramembrane cholesterol removal and oxidative stress. In conclusion, the proteomic profile of the seminal plasma of FIT bulls presents proteins with participation in several biological processes favorable to fertilization, while the proteins of the seminal plasma of UNFIT bulls indicate a series of alterations that can compromise the fertilizing capacity of the spermatozoa. In addition, the relative abundance of spermadhesin-1 found in the seminal plasma of young Nellore bulls could be studied as a reproductive parameter for selection.

Sexual rest (SR) in bulls leads to the accumulation of senescent spermatozoa in the extragonadal reserves, potentially affecting semen quality and reproductive efficiency. Therefore, this study aimed to investigate the impact of SR on the seminal plasma proteome and oxidative status of Nellore bulls. Six adult bulls were subjected to 195 days of SR and sequential semen collections using the electroejaculation method. The ejaculates were analyzed to assess sperm quality. Seminal plasma from the first and last ejaculates was evaluated for oxidative status and proteomic profile using LC-MS. The results revealed significant improvements in sperm motility, vigor, and antioxidant enzyme activity (superoxide dismutase and catalase) in the last ejaculate compared to the first. Conversely, higher levels of oxidative markers, such as malondialdehyde and carbonyl proteins, were observed in the first ejaculate. Proteomic analysis identified 156 proteins, with 28 differentially abundant between ejaculates. The first ejaculate showed a higher abundance of proteins linked to acrosomal exocytosis and energy metabolism, while proteins associated with sperm motility and immune modulation were elevated in the last ejaculate. These findings suggest that SR induces oxidative stress and proteomic alterations in seminal plasma, negatively affecting sperm quality, emphasizing the need for strategic reproductive management in bulls.