All procedures involving T. aduncus, including animal handling and semen collection, were reviewed and approved by the Animal Ethics Committee of the School of Veterinary Medicine and Biomedical Sciences, IPB University, Bogor, Indonesia (Approval No. 092/KEH/SKE/VIII/2023). The study was conducted in accordance with the institutional guidelines and ethical principles governing the use, care, and welfare of animals in research. Particular attention was given to minimizing animal discomfort and stress during all procedures.

The study involved two healthy adult male T. aduncus, aged 11 and 18 years and weighing approximately 110 and 105 kg, respectively, maintained under identical environmental, nutritional, and reproductive manage-ment conditions at Wersut Seguni Indonesia Animal Park, Kendal Regency, Central Java, Indonesia. Semen collection was performed only from animals previously trained and conditioned for the procedure. Each dolphin voluntarily positioned its ventral surface above the water level in response to trainer commands, after which manual penile stimulation was performed until ejaculation. The ejaculate was collected directly into a sterile collection bottle and immediately transported to the laboratory. No sedation or invasive procedures were used during semen collection, and all procedures were performed by trained personnel with measures to minimize stress and safeguard animal welfare throughout the study.

The study was conducted on 26 January 2024. Semen samples were collected from captive T. aduncus housed at Wersut Seguni Indonesia Animal Park, Kendal Regency, Central Java, Indonesia. Laboratory analyses were subsequently performed according to the study protocol.

This descriptive proteomic study aimed to characterize the protein composition of seminal plasma and sperm. Equal volumes of semen collected from two adult males were pooled before protein extraction to obtain a representative proteomic profile. Consequently, the analyzed seminal plasma and sperm fractions represented a single biological sample rather than biological or technical replicates.

Two healthy adult male T. aduncus, aged 11 and 18 years and weighing approximately 110 and 105 kg, respectively, were included in the study. Both animals were maintained under identical environmental, nutritional, and reproductive management conditions. The dolphins were housed in outdoor seawater pools maintained at 24°C–32°C.

A single ejaculate was collected from each trained dolphin according to the method described by Fahrudin et al. [17]. During semen collection, each dolphin voluntarily positioned its ventral surface above the water level in response to trainer commands. Manual penile stimulation was then performed until ejaculation, and the semen was collected directly into a sterile collection bottle before being transported immediately to the laboratory. The semen characteristics of both individuals are presented in Table 1.

Equal volumes of semen from the two dolphins were pooled and centrifuged at 4,000 × g for 30 min to separate the seminal plasma from the sperm pellet. Both fractions were stored at −20°C until protein extraction.

Only two adult male T. aduncus trained for semen collection were available in captivity in Indonesia. Because this study aimed to characterize the overall semen proteome rather than evaluate inter-individual variation, semen from both males was pooled before analysis. Therefore, the resulting seminal plasma and sperm samples represented a single biological sample, which precluded statistical comparisons.

Sperm proteins were extracted using PRO-PREP™ Protein Extraction Solution (17081, iNtRON Biotechnology, Seongnam, South Korea). The sperm pellet was resuspended in the extraction solution. Before sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the total soluble protein concentrations of the seminal plasma and sperm fractions were determined using a bicinchoninic acid (BCA) protein assay kit (A65453, Thermo Fisher Scientific, Waltham, MA, USA).

Proteins were separated according to molecular weight using SDS-PAGE, and protein bands were visualized after electrophoresis. Separation was performed on a SurePAGE™ Bis-Tris gel (10 × 8 cm, 12 wells, 4%–20% gradient; M00656, GenScript, Piscataway, NJ, USA) using Tris-MOPS-SDS running buffer (M00138; GenScript). Electrophoresis was performed at 200 V and 100–120 mA for 50 min. A Broad Multi Color Pre-Stained Protein Standard (5-270 kDa; M00624, GenScript) was used as the molecular weight marker.

Protein bands were excised from the gel, destained with 100 mM ammonium bicarbonate (A6141, Merck, Darmstadt, Germany) in 50% acetonitrile (A-0621-PB17, Fisher Chemical, Waltham, MA, USA), and dehydrated with 100% acetonitrile. The samples were dried at 30°C for 15 min using a SpeedVac (Thermo Fisher Scientific). Proteins were reduced with 10 mM dithiothreitol (3483-12-3, Sigma-Aldrich, St. Louis, MO, USA) in 100 mM ammonium bicarbonate for 30 min at 56°C and alkylated with 55 mM iodoacetamide (I1149, Sigma-Aldrich) in 100 mM ammonium bicarbonate for 30 min in the dark. After evaporation for 10 min, proteins were digested overnight at 37°C with sequencing-grade trypsin (10 μg/mL; V5111, Promega, Madison, WI, USA) prepared in 10 mM ammonium bicarbonate (A6141, Merck) containing 10% acetonitrile. Peptides were extracted with 10% formic acid and 70% acetonitrile at 5,000 × g for 30 min. The supernatant was evaporated to dryness in a SpeedVac (Eppendorf, Hamburg, Germany), resuspended in 1 mL MS-grade water (047146.Y0, Fisher Chemical), filtered through a 0.2 μm polyvinylidene fluoride membrane (88518, Thermo Fisher Scientific), and used for LC–MS/MS analysis.

LC–MS/MS data were analyzed using Proteome Discoverer version 2.5 (Thermo Fisher Scientific, Waltham, MA, USA). Common contaminant proteins were excluded before downstream analyses. Trypsin was specified as the digestion enzyme, allowing up to one missed cleavage. Carbamidomethylation of cysteine was set as a fixed modification, whereas oxidation of methionine was specified as a variable modification. The precursor mass tolerance was set to 10 ppm, and protein identifications were accepted at a false discovery rate (FDR) of 1%.

Proteins were identified using the UniProt T. truncatus protein database. Only proteins identified with at least two unique peptides and a High Troughput score >0 were retained for further analysis. GO enrichment and protein-protein interaction analyses were performed using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) version 12.0 (https://string-db.org/) with the Tursiops background database. Statistical significance was defined as an FDR of <0.05 with a confidence score >0.4.

Statistical analyses were not performed because semen samples from the two dolphins were pooled before protein analysis. Consequently, the resulting measurements represented a single biological sample rather than independent biological replicates, making statistical comparisons inappropriate.

A total of 199 proteins were identified in this study. Of these, 84 proteins were detected in seminal plasma and 119 in sperm, with only four proteins, namely aldolase C (ALDOC), basigin (BSG), creatine kinase B-type (CKB), and lactate dehydrogenase A (LDHA), shared between the two fractions. Consequently, 80 proteins were unique to seminal plasma and 115 were unique to sperm (Figure 1). Complete lists of the identified proteins are provided in Supplementary Tables S1 (seminal plasma) and S2 (sperm).

GO analysis classified the identified proteins into the categories of biological process, molecular function, and cellular component (Figure 2).

Within the biological process category, seminal plasma proteins were enriched in carbohydrate metabolic process (GO:0005975), catabolic process (GO:0009056), small molecule metabolic process (GO:0044281), monosaccharide metabolic process (GO:0005996), organic substance catabolic process (GO:1901575), membrane lipid catabolic process (GO:0046466), and regulation of endopeptidase activity (GO:0052548). In contrast, sperm proteins were enriched in cellular respiration (GO:0045333), aerobic respiration (GO:0009060), oxidative phosphorylation (GO:0006119), respiratory electron transport chain (GO:0022904), mitochondrial adenosine triphosphate (ATP) synthesis-coupled electron transport (GO:0042775), aerobic electron transport chain (GO:0019646), tricarboxylic acid cycle (GO:0006099), sexual reproduction (GO:0019953), and spermatogenesis (GO:0007283).

For the molecular function category, seminal plasma proteins were enriched in endopeptidase inhibitor activity (GO:0004866), hydrolase activity acting on glycosyl bonds (GO:0016798), peptidase activity (GO:0008233), endopeptidase activity (GO:0004175), oxidoreductase activity (GO:0016614), and L-lactate dehydrogenase activity (GO:0004459). In contrast, sperm proteins were enriched in electron transfer activity (GO:0009055), Nicotinamide adenine dinucleotide (NADH) dehydrogenase activity (GO:0003954), NADH dehydrogenase (ubiquinone) activity (GO:0008137), oxidoreduction-driven active transmembrane transporter activity (GO:0015453), primary active transmembrane transporter activity (GO:0015399), porin activity (GO:0015288), active transmembrane transporter activity (GO:0022804), and succinate dehydrogenase activity (GO:0000104).

Within the cellular component category, seminal plasma proteins were enriched in lysosome (GO:0005764), extracellular region (GO:0005576), extracellular space (GO:0005615), and endoplasmic reticulum lumen (GO:0005788). In contrast, sperm proteins were enriched in mitochondrial inner membrane (GO:0005743), inner mitochondrial membrane protein complex (GO:0098800), mitochondrial protein-containing complex (GO:0098798), mitochondrial respirasome (GO:0005746), motile cilium (GO:0031514), 9+2 motile cilium (GO:0097729), and sperm flagellum (GO:0036126).

Protein-protein interactions were analyzed using STRING (Figure 3). The seminal plasma proteins formed a relatively dispersed interaction network, whereas the sperm proteins exhibited a more interconnected network. In the seminal plasma network, beta-actin (ACTB), serum albumin (ALB), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) had the highest interaction scores (Figure 3A). In contrast, the sperm protein network comprised several interconnected clusters associated with cellular respiration, intracellular structural organization, cell motility, and reproductive processes (Figure 3B).

This study provides the first comprehensive proteomic characterization of seminal plasma and sperm from T. aduncus. The findings establish a species-specific molecular reference and expand the current understanding of cetacean reproductive biology. Although the seminal plasma and sperm proteomes of T. truncatus have previously been characterized [16], corresponding proteomic information for T. aduncus has not been reported.

Dolphin seminal plasma originates primarily from the testes, epididymides, and prostate, the only accessory sex gland described in cetaceans [11]. A total of 199 proteins were identified in the seminal plasma and sperm fractions. Only four proteins were shared between the two fractions: aldolase C, fructose-bisphosphate (ALDOC), basigin (BSG), creatine kinase B-type (CKB), and L-lactate dehydrogenase (LDHA). ALDOC and LDHA have also been detected in both seminal plasma and sperm of T. truncatus [16].

ALDOC is involved in sperm energy metabolism, including phosphocreatine and ATP synthesis, as well as NAD/NADH metabolism [18]. LDHA contributes to sperm function and fertility [19]. Both proteins are localized in the fibrous sheath of spermatozoa, where they anchor metabolic enzymes along the flagellum and provide a localized adenosine triphosphate (ATP) supply required for sperm motility [20].

BSG belongs to the immunoglobulin superfamily and is required for both male and female reproduction. It is initially localized in the principal piece of testicular spermatozoa, relocates to the midpiece during epididymal maturation, and subsequently moves to the sperm head after capacitation, indicating its role in sperm-zona pellucida interaction [21].

CKB rapidly regenerates ATP from adenosine diphosphate (ADP) using phosphocreatine, thereby supporting efficient energy generation, transport, and utilization in spermatozoa [22]. Phosphorylation of CKB by protein kinase C further enhances its enzymatic activity by facilitating ATP regeneration.

GO analysis showed enrichment of the organic substance catabolic process in seminal plasma. Proteins associated with this process included Beta-hexosaminidase (HEXA and HEXB), Alpha-L-fucosidase (FUCA1), Beta-mannosidase (MANBA), Deoxyribonuclease-2-alpha (DNASE2), Cathepsin B (CTSB), Cathepsin S (CTSS), Legumain (LGMN), Lipase (LIPA and LIPF), angiotensin-converting enzyme (ACE), Xaa-Pro aminopeptidase 1 isoform X1 (XPNPEP1), Glyceraldehyde-3-phosphate (GAPDH), Aspartate aminotransferase (GOT1), Calreticulin (CALR), Endoplasmic reticulum chaperone BIP (HSPA5), 5’-nucleotidase (NT5E), Sphingomyelin phosphodiesterase (SMPD1), and ALDOC.

These proteins participate in the degradation and processing of proteins, lipids, and nucleic acids that may influence the post-ejaculatory environment of spermatozoa. This finding is consistent with the enrichment of GO molecular function terms associated with endopeptidase and peptidase activities. Protease regulation can occur without new protein synthesis, making it suitable for controlling extracellular processes, including interactions between seminal plasma proteins and spermatozoa, as well as between spermatozoa and the female reproductive tract. However, proteolysis must be tightly regulated because it also contributes to remodeling sperm surface proteins during preparation for capacitation while preventing premature activation and tissue damage [23].

Membrane lipid catabolism was also enriched in seminal plasma. Plasma membrane lipids are essential for maintaining the structural integrity and physiological functions of mammalian spermatozoa. Coordinated lipid metabolism contributes to sperm motility, capacitation, the acrosome reaction, and sperm-oocyte fusion. Consequently, sperm fertility depends greatly on maintaining lipid homeostasis [24].

Carbohydrate metabolism was also enriched in seminal plasma and involved HEXA, HEXB, FUCA1, MANBA, beta-galactosidase (GLB1), retinol-binding protein (RBP4), GAPDH, 6-phosphogluconate dehydrogenase (PGD), alpha-mannosidase (MAN2A2), GOT1, and ALDOC. Although proteins associated with carbohydrate metabolism were enriched in seminal plasma, previous studies have shown that dolphin spermatozoa maintain motility primarily through β-oxidation of fatty acids rather than glycolysis [25].

Several seminal plasma proteins may also help protect spermatozoa against oxidative stress. HSPA5 functions as a molecular chaperone that protects cells by enhancing antioxidant synthesis, stabilizing proteins, and activating compensatory energy pathways [26]. GAPDH is an essential glycolytic enzyme that is susceptible to oxidative inactivation, thereby redirecting glucose metabolism toward the pentose phosphate pathway. This metabolic shift reduces NADH production through glycolysis while increasing NADPH production through the pentose phosphate pathway, contributing to short-term adaptation to oxidative stress [27]. RBP4 is the principal transport protein for retinol (vitamin A) [28] and has also been identified previously in the seminal plasma of T. truncatus [16].

Collectively, the identified proteins, enriched GO terms, and protein-protein interaction network suggest that seminal plasma provides a supportive extracellular environment that promotes sperm function and protection. This observation is consistent with previous reports demonstrating that seminal plasma provides nutrients, protects spermatozoa, acts as a buffering medium, and supports sperm motility [29].

GO biological process enrichment analysis of spermatozoa proteins revealed enrichment of terms associated with energy production, including oxidative phosphorylation (GO:0006119), aerobic respiration (GO:0009060), proton motive force-driven mitochondrial ATP synthesis (GO:0042776), proton motive force-driven ATP synthesis (GO:0015986), respiratory electron transport chain (GO:0022904), and mitochondrial ATP synthesis-coupled electron transport (GO:0042775). Collectively, these enriched GO terms indicate that energy production in dolphin sperm is predominantly supported by aerobic respiration through oxidative phosphorylation.

The proteomic findings further support the distinctive metabolic characteristics previously described for dolphin sperm [25, 30]. Unlike humans and rodents, which rely primarily on glycolysis, and ruminants and canines, which use both glycolysis and oxidative phosphorylation for energy production [31], dolphin sperm depend predominantly on endogenous fatty acid oxidation as an adaptation to the carbohydrate-limited marine environment [25]. Although glycolysis-associated proteins such as ALDOC and LDHA were detected in seminal plasma, they may originate from somatic cells of the male reproductive tract or extracellular secretions rather than reflecting active glycolytic metabolism in spermatozoa. In contrast, enrichment of oxidative phosphorylation, respiratory electron transport chain, mitochondrial ATP synthesis, and proton motive force-driven ATP synthesis pathways supports the importance of mitochondrial ATP production in delphinid sperm [25, 30]. The substrates for oxidative phosphorylation are likely derived from the high fatty acid content previously reported in dolphin sperm. Fatty acids are transported into mitochondria through the carnitine shuttle, which depends on Carnitine O-palmitoyltransferase 1, muscle isoform (CPT1A), and subsequently undergo β-oxidation to generate acetyl-CoA. Acetyl-CoA then enters the tricarboxylic acid cycle, producing NADH and FADH₂, which supply electrons to the mitochondrial electron transport chain and drive oxidative phosphorylation [25]. These findings are consistent with fatty acid β-oxidation serving as the principal energy source supporting sperm function in delphinids.

Other enriched GO biological processes were associated with flagellated sperm motility (GO:0030317), spermatogenesis (GO:0007283), spermatid development (GO:0007286), cilium-dependent cell motility (GO:0060285), cilium movement involved in cell motility (GO:0060294), and sperm capacitation (GO:0048240). These processes involved proteins including Ropporin-1-like protein (ROPN1L), Sperm-associated antigen 6 isoform X1 (SPAG6), Calcium-binding tyrosine phosphorylation-regulated protein (CABYR), Fibrous sheath-interacting protein 2 (FSIP2), Parkin coregulated gene protein (PACRG), Tektin (TEKT2, TEKT3, and TEKT5), Tubulin beta chain (TUBB4B), cAMP-dependent protein kinase catalytic subunit alpha (PRKACA), and Glutathione peroxidase (GPX4). Several proteins identified in the present study, including A-kinase anchor protein 3 (AKAP3), outer dense fiber protein 2 (ODF2), TUBB, GSTM3, and ROPN1, have also been reported in the sperm proteome of T. truncatus [16].

AKAP3 is an essential structural component of the fibrous sheath and plays a critical role in sperm flagellar formation, motility, and fertility [32]. The fibrous sheath is a sperm-specific cytoskeletal structure surrounding the outer dense fibers and axoneme that contributes to the regulation of flagellar movement. Loss of AKAP3 causes widespread alterations in the sperm proteome, mislocalization of sperm proteins, and dysregulation of protein kinase A (PKA) activity [32]. ROPN1 and ROPN1L bind to AKAP3 and function cooperatively to maintain its incorporation into the fibrous sheath. Simultaneous deficiency of both proteins reduces cAMP-dependent protein kinase phosphorylation and capacitation-associated tyrosine phosphorylation, resulting in impaired fertility [33]. Reduced abundance of these proteins has also been associated with poor blastocyst development [34].

SPAG6 is a component of the central apparatus of the “9 + 2” axoneme and is essential for ciliary and flagellar motility. In addition to its structural role, SPAG6 functions as a chaperone during spermatogenesis, and its deficiency disrupts meiosis and causes morphological abnormalities of the acrosome, manchette, and flagella [35, 36]. ODF2 is a major component of the outer dense fibers of the sperm flagellum and is initially stored in the granulated body of post-meiotic spermatids before being incorporated into the sperm tail during elongation [37]. ODF2 contributes to flagellar stability and sperm transport, whereas its deficiency causes separation of the neck and midpiece [38]. ODF proteins stabilize the axoneme, and their reduced expression has been associated with asthenozoospermia [39], whereas greater abundance has been associated with improved fertility and semen quality [40].

CABYR is localized to the fibrous sheath, an accessory structure of the sperm flagellar principal piece. Functional studies have demonstrated that CABYR knockout results in severe subfertility characterized by impaired sperm motility, marked disorganization of the fibrous sheath, and abnormal doublet microtubule configuration [41]. In addition, CABYR is required to maintain sperm chirality, a process influenced by intracellular Ca²⁺ homeostasis [42]. CABYR also interacts with AKAP3 and ROPN1 to facilitate the assembly of signaling complexes within the fibrous sheath [43]. These findings highlight the importance of CABYR in maintaining the structural integrity and functional organization of the sperm flagellum.

The tubulin proteins identified in this study, TUBA4A and TUBB4B, are essential cytoskeletal components involved in microtubule organization and the development of cilia and flagella [44]. However, increased TUBA4A expression has also been reported in sperm from men with asthenozoospermia compared with normozoospermic controls [45]. Tektins are a family of microtubule-stabilizing proteins indispensable for the assembly and structural stability of cilia and flagella. Deficiency of tektin family members disrupts microtubule integrity and impairs sperm motility [46]. TEKT3 and TEKT4 are enriched in male germ cells, and TEKT4 is essential for coordinated and progressive sperm motility in mice. Male mice lacking TEKT3 produce sperm with reduced motility, diminished forward progression, and increased flagellar bending defects [46]. TEKT2 has also been reported to be downregulated in teratozoospermia and is essential for ciliary and flagellar development [47]. TEKT5 is expressed exclusively in the testes. Collectively, tektins play indispensable roles in sperm motility and male fertility. Deficiency of TEKT2 or TEKT4 results in infertility or subfertility, whereas loss of TEKT3 reduces sperm motility and forward progression. Furthermore, the organization of TEKT1, TEKT2, and TEKT4 filaments is highly conserved among sea urchin sperm, mammalian sperm, and mammalian respiratory cilia [48].

Several identified proteins are also associated with cellular detoxification and protection against oxidative stress. GSTM3, a member of the glutathione S-transferase family, catalyzes the conjugation of electrophilic compounds to reduced glutathione and contributes to cellular antioxidant defense. GSTM3 has also been proposed as a potential biomarker of male infertility or subfertility and is distributed throughout the midpiece, principal piece, and end piece of the sperm tail [49]. PRDX5 provides additional antioxidant protection and is localized to the acrosomal cap and post-acrosomal region [50]. Together, these proteins may help preserve sperm function by protecting spermatozoa from oxidative damage.

The identification of highly connected proteins, including AKAP3, ODF2, SPAG6, and Glutathione S-transferase Mu 3 (GSTM3), suggests that these molecules may serve as candidate biomarkers for non-invasive assessment of male fertility in captive and wild dolphin populations.

Compared with the T. truncatus seminal proteome reported by Fuentes-Albero et al. [16], the overall number of proteins identified differed substantially. Nevertheless, the five most abundant sperm proteins reported in that study (AKAP3, ODF2, TUBB, GSTM3, and ROPN1) were also identified in the present study, indicating conservation of core structural and motility-associated proteins across Tursiops species. In contrast, the five most abundant seminal plasma proteins reported by Fuentes-Albero et al. [16] (CST11, LTF, ALB, HSP90B1, and PIGR) differed from the proteins with the highest interaction scores identified in the seminal plasma of T. aduncus (ACTB, ALB, and GAPDH), with ALB being the only protein common to both datasets. This difference may reflect species-specific variation in seminal plasma composition, although methodological differences between studies cannot be excluded. Because a species-specific T. aduncus reference proteome is currently unavailable, protein identification in the present study relied on the UniProt and STRING databases for T. truncatus. This database limitation should be considered when interpreting potential species-specific proteins and underscores the need to develop a dedicated T. aduncus reference proteome for future proteomic investigations.

This study has several limitations. First, the proteomic analysis used a single pooled semen sample from two adult T. aduncus, which limited statistical power, precluded assessment of inter-individual biological variation, and prevented formal quantitative comparisons of protein abundance between seminal plasma and sperm. Second, protein identification relied on the T. truncatus reference database because a dedicated T. aduncus proteome is not currently available, which may have introduced bias in identifying species-specific proteins. Third, the candidate proteins identified in this study were not validated using orthogonal techniques, such as Western blotting or immunofluorescence, and their associations with fertility were not evaluated. Future studies should include larger sample sizes with individual-level analyses, quantitative proteomic approaches, functional validation of candidate biomarkers, and correlation of protein expression with reproductive performance.