The usage of dietary algae antioxidants to improve fish reproduction is under-explored, especially in terms of the male reproductive system. In this experiment, 6 % of a blended meal of Phaeodactylum tricornutum and Gracilaria gracilis was incorporated in Senegalese sole broodstock feed, to evaluate the effects on sperm quality of F1 males throughout the breeding season. For that, two groups of breeders were fed during 6 months with the control and algae diets (6 % of control wheat meal replaced with 6 % algae blend). Every 2 weeks, fish were sampled for sperm quality evaluation, which included spermatozoa motility (CASA system), lipid peroxidation (MDA quantification), cell viability, reactive oxygen species (ROS), apoptotic status (flow cytometer), and DNA fragmentation (Comet assay). On a final sampling, 6 fish per group were sacrificed to dissect gonadal tissue, extract RNA and perform an RNA sequencing (RNA-seq) for each treatment. Sperm quality variability was high during the breeding season, including within the same month, irrespective of the diet. Cell viability was approximately 80 % during the whole experiment. Nonetheless, in specific sampling points, algae-fed fish showed higher spermatozoa protection against oxidative processes: in the 1st sampling live cells without ROS (%) were 3 times higher than in control group; on the last two samplings, spermatozoa showed half of MDA content; and on the 3rd sampling had less DNA fragmentation. No differences were found regarding apoptotic status. At the end of the reproductive season, gonadal transcriptomic analysis revealed that algae-fed fish were lacking stimuli for sperm production, both in terms of quantity and quality. This fish group seemed to have lipid metabolism and antioxidant capacity enhanced by the diet but, at the same time, were facing a compensatory mechanism due to an unknown algae compound that might be disrupting DNA replication and spermatogenesis. Altogether, this study suggests that algae blends can be used in broodstock feeds for Senegalese sole, however further research is needed to understand how to use only the desirable bioactive compounds and thus obtain higher and consistent sperm quality throughout the breeding season.
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