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- Effects of dietary supplementation with macroalgae on sperm quality and antioxidant system in Senegalese solePublication . Félix de Azeredo Pinto e Melo, Francisca; Silva, Nádia; Oliveira, Catarina; Cabrita, Elsa; Gavaia, PauloWithin the efforts in the search for novel feed ingredients in aquaculture, macroalgae are being widely explored due to their diversity of bioactive compounds with considerable prophylactic and therapeutic potential. Plocamium cartilagineum and Sargassum vulgare, have the potential to positively impact fish health, mainly due to their antioxidant properties. The production of Senegalese sole (Solea senegalensis) still faces some constraints due to the low sperm quantity and quality of males from captivity, which is highly affected by oxidative stress. We hypothesized that the scavenger ability of P. cartilagineum and S. vulgare could ameliorate the reproductive performance of Senegalese sole males, once incorporated in the roodstock feeds. In this experiment, broodstock groups were fed with three different formulated experimental diets for 60 days: Control (Ctrl), a diet supplemented with 5% P. cartilagineum (Pc), and a diet supplemented with 5% S. vulgare (Sv). Fish were sampled twice to evaluate sperm quality using different techniques: sperm motility by Computer Assisted Sperm Analysis (CASA) software, and cell viability and Reactive Oxygen Species (ROS) by flow cytometer. At the final sampling, 6 fish per treatment were sacrificed and gonads were extracted to determine the relative expression of anti- and pro-apoptotic genes (bcl2b, bcl-x, mcl1b, badb, bax, boka), and genes involved in fish antioxidant system (sod3, cat, gpx, hsp70). Sex steroids were measured on blood plasma by ELISA. Specific growth rate (SGR) and feed efficiency were also evaluated at the end of the experiment. Senegalese sole breeders fed with Pc diet for 60 days showed enhanced sperm total motility and cell viability at the end of the experiment, when compared with the Ctrl diet. On the other hand, fish fed with Sv diet had lower spermatozoa intracellular ROS and upregulated gpx in the gonads. No differences were obtained in levels of sex steroids and gonadal apoptotic system after 60 days of feeding, however, the macroalgae species and the percentages of incorporation used proved to positively modulate different spermatozoa traits.
- Melatonin production improves Senegalese sole sperm motility at night, but fails as a supplement during cryopreservationPublication . Félix de Azeredo Pinto e Melo, Francisca; Ferrão, Leonor; Gallego, Victor; Oliveira, Catarina; Cabrita, ElsaMelatonin is a powerful antioxidant present in fish seminal plasma. This study aimed to understand melatonin's endogenous and exogenous effects on first-generation Senegalese sole sperm quality for sperm management applications. In the first experiment, samples were collected at mid-light (ML) and mid-dark (MD) daytimes, to evaluate the effects on sperm motility. In a second experiment, using confocal microscopy and melatonin-FITC, spermatozoa permeability to melatonin was evaluated and, after showing that it enters the nucleus and mitochondria by passive diffusion, exogenous melatonin toxicity and antioxidant potential during a cryopreservation assay were performed. The toxicity assay tested different melatonin concentrations (0.01, 0.1, 1, and 10 mM) and exposure times (3, 5, 15 and 30 min), and sperm motility parameters were measured (TM, PM, VCL, VSL, LIN) using CASA system. The best conditions (0.1 and 10 mM) were selected for the cryopreservation assay, and a set of post-thaw sperm quality analyses were performed (motility, viability, reactive oxygen species, lipid peroxidation, and DNA fragmentation). The motility analyzed at ML and MD showed significant differences in all parameters, mainly on velocities (VCL, VSL, VAP), that were significantly higher at MD. Supplemented melatonin did not influence spermatozoa motility, MDA content or DNA fragmentation, although a lower percentage of viable cells was obtained on the 10 mM treatment. Altogether, Senegalese sole spermatozoa motility was enhanced at night, putatively by endogenous melatonin through direct or indirect mechanisms, whereas supplemented melatonin did not confer extra protection during cryopreservation.
- Testis transcriptomic analyses reveal the effects of an algae feed on sperm quality in senegalese sole during the breeding seasonPublication . 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, ElsaThe 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.