Dolphin semen metabolome characterization and relationships with sperm quality

Introduction Identification and quantification of metabolites in semen can augment assisted reproductive technologies by indicating reproductive health and fertility. Species-specific semen metabolites associated with improved semen quality remain largely unknown in exotic species due to limited sample availability. Objectives Study objectives were to establish metabolome profiles of common bottlenose dolphin ( Tursiops truncatus ) semen and seminal plasma and identify metabolites associated with semen quality. Methods Semen was collected from sexually mature common bottlenose dolphins (aged 17–46 years) trained to perform voluntary semen collection behavior. One to five semen samples were collected from each of four dolphins in managed care, resulting in 12 ejaculates for metabolomic analyses. Semen quality parameters were measured including volume, pH, and sperm concentration, motility, kinematics, and viability. Metabolites in whole semen and sperm-free seminal plasma were analyzed using ultra-high performance liquid chromatography coupled with Orbitrap Fusion Tribrid mass spectrometry. Metabolome profile variability was assessed using principal component analyses with permutational multivariate analysis of variance and volcano plots with t-tests. Metabolomic pathway analyses were conducted using a species-specific systems biology markup language (SBML) metabolite model. Correlations between semen quality parameters and normalized metabolite peak areas were assessed using repeated-measures correlation. Results An untargeted analysis identified 2,198 metabolites in dolphin semen and seminal plasma. Biological matrix, individual variation, and collection date contributed to differences in metabolome profiles. The relative abundances of 353 metabolites differed significantly between semen and seminal plasma ( p < 0.05). Targeted analysis detected eight hormones in semen and/or seminal plasma. Pathway enrichment analysis indicated inter-individual variation including enriched nucleotide metabolism, protein degradation, and transport reaction pathways. Two individuals exhibited enriched inflammatory, reactive oxygen species detoxification, and antioxidant metabolism pathways, suggesting oxidative stress. A significant correlation ( p < 0.0005) occurred between 98 distinct metabolites and semen quality parameters. Conclusion This study provides the first semen metabolome profile for any marine mammal species and identifies novel metabolomic biomarkers of dolphin semen quality.