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  • The protective effect of endogenous melatonin on gilthead seabream sperm during cryopreservation
    Publication . Félix, Francisca; Antunes, Raquel; Vera, L. M.; Oliveira, Catarina; Cabrita, Elsa
    Cryopreservation is widely used for artificial reproduction techniques (ART) but requires good gamete quality to succeed. Considering the endogenously produced melatonin by the fish and its protective role in the organism, the objective of this experiment was to search for the best moment of the day to collect gilthead seabream (Sparus aurata) sperm with better quality and, at the same time, to test the potential protective effect of the exogenous melatonin during sperm freezing and thawing processes. Fish were first sampled every 6 h, corresponding to the beginning of the light period (6 h), mid-light (12h), beginning of the dark period (18 h), and mid-dark (24 h) to determine melatonin concentration in blood plasma by radioimmunoassay (RIA). The analysis showed higher values at 24 h (244 pg/mL) compared with 12 h (99 pg/mL), which allowed the selection of those moments for the next experiment. After, fish were sampled for sperm collection at mid-light (ML, 12 h) and mid-dark (MD, 24 h), and fresh sperm was used to assess gamete quality and for the cryopreservation assay. Sperm samples were cryopreserved to test supplementation of different melatonin concentrations (0.001 mM, 0.01 mM and 0.1 mM) together with a control group without added melatonin. Gamete quality was assessed through spermatozoa concentration and motility (CASA system), cell viability (PI/SYBR-green) and DNA fragmentation (Comet assay). Despite cell viability that was higher at ML, most of the fresh sperm motility parameters did not differ between ML and MD, only linearity (LIN) was enhanced at MD. Nevertheless, in cryopreserved samples, total motility (TM) was significantly higher at MD in all melatonin treatments, control, and fresh samples, revealing an endogenous night-effect. Moreover, spermatozoa concentration was also higher at MD (28.9 x 109/mL) than at ML (20.7 x 109/mL). Supplemented melatonin did not confer extra protection to gilthead seabream sperm during cryopreservation since the tested concentrations did not differ between the control in any sperm quality test. It is here suggested that endogenously produced melatonin may contribute to the improvement of some gamete quality parameters at mid-dark, allowing the aquaculture sector to select better sperm quality in a noninvasive way by choosing it as the best moment of the day for sperm collection.
  • Testis transcriptomic analyses reveal the effects of an algae feed on sperm quality in senegalese sole during the breeding season
    Publication . Félix de Azeredo Pinto e Melo, Francisca; Raposo de Magalhães, Cláudia; Marrero Alemán, Carlos; Duarte, Daniel Filipe Correia; Parente, Pedro; Fatsini Fernández, Elvira; Oliveira, Catarina; Cabrita, Elsa
    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.