Proteomic profiling of bull spermatozoa and seminal plasma to inform the rational development of functionally targeted semen extenders for tropical cattle

Background and Aim: Artificial insemination (AI) is a cornerstone technology for genetic improvement in livestock; however, the fertility outcomes of cryopreserved semen often remain inconsistent, particularly in tropical production systems where heat stress and oxidative damage compromise sperm function. Conventional semen extenders are largely developed through empirical approaches and may not adequately reflect the molecular characteristics of locally adapted cattle breeds. Proteomic profiling offers an opportunity to identify endogenous proteins involved in sperm function and resilience, thereby enabling the rational design of functionally targeted semen extenders. This study aimed to characterize the proteomic profiles of bull spermatozoa and seminal plasma and to identify functional proteins associated with semen quality traits to inform the development of biologically informed extender formulations for tropical cattle. Materials and Methods: Semen samples were collected from three sexually mature Bali bulls maintained under standardized management conditions. Three ejaculates were obtained from each bull, resulting in nine ejaculates for evaluation. Semen quality parameters, including motility, viability, abnormality, acrosome integrity, and membrane integrity, were assessed using conventional microscopic techniques and computer-assisted sperm analysis. For proteomic analysis, spermatozoa and seminal plasma fractions were separated by centrifugation and subjected to protein extraction, enzymatic digestion, and high-resolution liquid chromatography–tandem mass spectrometry. Identified proteins were analyzed using bioinformatics tools for functional annotation, Gene Ontology classification, and protein–protein interaction analysis to determine their biological roles and potential relevance to semen preservation. Results: Fresh semen exhibited high motility (86.28% ± 3.26%), membrane integrity (86.35% ± 2.88%), and acrosome integrity (79.65% ± 6.93%), indicating overall favorable semen quality. Proteomic analysis identified 371 proteins, including 101 unique to spermatozoa and 270 shared between spermatozoa and seminal plasma. Functional annotation revealed that sperm proteins were predominantly associated with energy metabolism, cytoskeletal organization, and spermatogenesis, whereas seminal plasma proteins were enriched in antioxidant activity, immune response, and proteolytic processes. Key proteins involved in mitochondrial function, antioxidant defense, acrosomal activity, and structural integrity were identified and associated with semen quality parameters, including motility, viability, and membrane stability. Interaction network analysis further demonstrated coordinated relationships among mitochondrial enzymes, structural proteins, and fertilization-related molecules. Conclusion: The integration of semen quality assessment with proteomic profiling provides molecular insights into the biochemical environment supporting sperm function in Bali bulls. The identified proteins highlight critical pathways associated with energy production, oxidative stress protection, structural stability, and fertilization competence. These findings provide a conceptual framework for translating proteomic information into targeted additives for semen extenders, thereby supporting the development of biology-informed cryopreservation strategies tailored to tropical cattle production systems. Keywords: Bali cattle, cryopreservation, proteomics, seminal plasma, semen extender, sperm motility, spermatozoa, tropical cattle.