Jessica Sales Lobato¹, Denise Damasceno Guerreiro², Sheheryar Sheheryar², João Eudes Farias Cavalcante Filho¹, Yara Silvino Sales¹, Wallisson Bruno de Morais Pacheco¹, Felipe Domingos de Sousa³, Ana Cristina Monteiro Moreira³, Carminda Sandra Brito Salmito-Vanderley¹, Arlindo de Alencar Araripe Noronha Moura^1,2

Tambaqui (Colossoma macropomum) is a rheophilic fish species of high interest for commercial aquaculture. Therefore, biotechnologies focused on its reproduction in captivity are employed to enhance production. In this regard, it is crucial to maintain the quality of semen from through the analysis of motility parameters and its composition, including the study of proteins present in the seminal plasma. This research aimed to evaluate seminal plasma proteins in tambaqui and their potention associations with sperm motility. For this purpose, animals (n=14; 6.5 ± 0.7 kg) were induced to spermiation by applying a synthetic GnRH analog (0.3 pellet/kg). After 14 h, semen was collected by abdominal massage and stored in sterile tubes. Aliquots of semen from each animal were evaluated for sperm motility (AndroScope, Minitube®, Germany), establishing that animals with < 85% of motile sperm was selected to the low motility group, and animals with >85% motile sperm were included in high motility group. Sperm motility in HM and LM animals were 91.5 ± 4.7% (n=7) and 77 ± 6.3% (n=7), respectively (p < 0.05). Sperm motility was different in these two groups (p < 0.05). The remaining samples were treated with protease inhibitor (Sigma-Aldrich®, USA), centrifuged for seminal plasma separation, and processed according to the Filter-Aided Sample Preparation (FASP) protocol for analysis by label-free data-dependent acquisition mass spectrometry. Progenesis (Waters Inc., USA) was used for protein identification, based on Cyprinus carpio Uniprot database. Differentially abundant proteins (DAPs; p < 0.05) in LM and HM animals were determined by Analysis of Variance, followed by Fisher\'s Least Significant Difference post-hoc test. Seminal plasma proteins in greater abundance in HM animals were identified as Nuclear mitotic apparatus protein 1 (NuMA 1), GRAM domain containing 1A (GRAMD1A), 6-phosphogluconolactonase, and Exportin-2. In contrast, LM animals had more Peptidyl-prolyl cis-trans isomerase in the seminal plasma. While NuMA 1 is primarily known for its role in mitotic spindle formation and nuclear organization, its interaction with microtubules suggests it helps to maintain sperm structural integrity and regulate processes affecting motility. The abundance of GRAMD1A in the seminal plasma of HM animals may be associated with its involvement in cholesterol binding and transport, regulation of lipid metabolism and sperm membrane integrity, thereby contributing to superior motility. Furthermore, high levels of 6-phosphogluconolactonase in HM animals may indicate increased antioxidant capacity in the seminal plasma, protecting spermatozoa and, consequently, preserving high motility. Exportin-2 is involved in cell proliferation and apoptosis, has been implicated in tumor cell survival and in mitotic processes. However, Exportin-2 function in sperm physiology and kinetics remains unknown. Peptidyl-prolyl cis-trans isomerase is involved in protein folding, signal transduction, cell cycle regulation, and apoptosis. Thus, these mechanism are certainly dysregulated in LM animals. Therefore, these findings suggest that the identified seminal plasma proteins in C. macropomum play crucial roles in regulating sperm quality and motility, offering potential biomarkers for evaluating reproductive success and guiding breeding strategies in aquaculture.

Agradecimentos: The authors acknowledge the support provided by the National Council for Scientific and Technological Development (CNPq), the Cearense Foundation for Scientific and Technological Development Support (FUNCAP), and the Coordination for the Improvement of Higher Education Personnel (CAPES) for granting scholarships to graduate and post-doctoral students.