Proteomic analysis of cryopreserved Prochilodus lineatus seminal plasma: effects of melatonin on sperm quality preservation

Abstract

This study evaluated the effects of melatonin supplementation on sperm cryopreservation and seminal plasma proteomics in the neotropical fish Prochilodus lineatus. Sperm Cryopreservation is a key technique for aquaculture and species conservation but often causes oxidative stress, structural damage, and metabolic impairment in sperm cells. In this study, sperm samples (n = 9 per group) were cryopreserved with or without 2 mM melatonin, followed by post-thaw analyses of motility, vigor, membrane integrity, morphology, and proteomic profile. Melatonin significantly improved sperm quality after thawing, increasing motility from 43.15 ± 16.95% to 65.72 ± 16.32% (p = 0.042), as well as straight-line velocity (VSL; 10.53 ± 4.19 to 16.57 ± 5.64 μm/s; p = 0.033) and average path velocity (VAP; 15.73 ± 5.48 to 22.90 ± 6.75 μm/s; p = 0.046), while curvilinear velocity (VCL; p = 0.075) and beat-cross frequency (BCF; p = 0.061) showed positive but non-significant trends. Morphological evaluation revealed a reduction in strongly coiled tails, whereas membrane integrity (42–84%) and total abnormality rates (49–51%) were unaffected. Proteomic analysis identified 1,238 proteins, including eight exclusively expressed under melatonin treatment, mainly associated with energy metabolism, antioxidant defense, and proteostasis. Melatonin upregulated enzymes related to ATP production, redox balance, and protein degradation, while downregulating proteins linked to cryo-induced damage. These findings demonstrate that melatonin exerts a multifaceted protective effect during sperm cryopreservation by preserving energy metabolism, reinforcing antioxidant capacity, maintaining structural and genomic integrity, and improving post-thaw motility. Overall, this study provides the most comprehensive proteomic dataset of P. lineatus seminal plasma to date and highlights melatonin as a promising additive for enhancing sperm cryopreservation protocols in aquaculture and conservation.