ABSTRACT

Background and Aim: Proteomic characterization of semen provides molecular insights into reproductive function beyond conventional semen evaluation. Although the seminal plasma and sperm proteomes of the common bottlenose dolphin (Tursiops truncatus) have been reported, corresponding information for the Indo-Pacific bottlenose dolphin (Tursiops aduncus) is lacking. This study aimed to characterize the protein composition of seminal plasma and sperm in T. aduncus to identify proteins and biological pathways associated with reproductive function.

Materials and Methods: Semen samples were collected from two healthy adult captive male T. aduncus exhibiting >70% sperm motility. Equal volumes of semen from both animals were pooled, and seminal plasma and sperm fractions were separated by centrifugation. Proteins were extracted, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and identified using liquid chromatography-tandem mass spectrometry. Protein identification was performed with Proteome Discoverer 2.5 using a 1% false discovery rate. Functional annotation was conducted using Gene Ontology enrichment and Search Tool for the Retrieval of Interacting Genes/Proteins protein-protein interaction analyses.

Results: A total of 199 proteins were identified, including 84 proteins in seminal plasma and 119 in sperm, with only four proteins shared between the two fractions. Seminal plasma proteins were predominantly enriched in carbohydrate and lipid catabolism, proteolysis, and regulation of endopeptidase activity, indicating roles in maintaining the extracellular environment, protecting sperm, and supporting post-ejaculatory survival. In contrast, sperm proteins were significantly enriched in oxidative phosphorylation, aerobic respiration, mitochondrial adenosine triphosphate synthesis, respiratory electron transport, spermatogenesis, sperm flagellum organization, and motility. Protein interaction analysis demonstrated a highly interconnected mitochondrial network in sperm, whereas seminal plasma proteins formed a more dispersed regulatory network. These findings indicate distinct functional specialization of the two semen fractions and suggest that oxidative phosphorylation represents the principal energy-producing pathway supporting sperm function in this marine mammal.

Conclusion: This study provides the first comprehensive proteomic characterization of seminal plasma and sperm in the Indo-Pacific bottlenose dolphin. The identified proteins and enriched biological pathways advance understanding of cetacean reproductive physiology, reveal molecular adaptations associated with mitochondrial energy metabolism, and establish a valuable resource for developing fertility biomarkers and assisted reproductive technologies to support the conservation and reproductive management of this Near Threatened species.

Keywords: assisted reproductive technology, cetacean reproduction, Indo-Pacific bottlenose dolphin, LC–MS/MS, oxidative phosphorylation, proteomics, seminal plasma, sperm proteome.