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- The protective effect of endogenous melatonin on gilthead seabream sperm during cryopreservationPublication . Félix, Francisca; Antunes, Raquel; Vera, L. M.; Oliveira, Catarina; Cabrita, ElsaCryopreservation 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.
- Novel approaches on melatonin role: presence of clock-hormone in fish seminal plasmaPublication . Félix, Francisca; Gallego, Victor; Mendes, Ana; Soares, Florbela; Vera, Luisa M.; Cabrita, Elsa; Oliveira, CatarinaThe study of melatonin is of great importance for the fundamental knowledge of any living system since it displays many different physiological roles, including being a potent natural antioxidant. To the best of our knowledge, there is no information regarding melatonin in fish seminal plasma. This study aimed to determine this clock-hormone levels in the seminal plasma of three aquaculture fish species: European seabass (Dicentrarchus labrax), gilthead seabream (Sparus aurata), and Senegalese sole (Solea senegalensis) (both wild and F1 breeders), and to explore melatonin potential role in fish reproduction and spermatozoa antioxidant status. Blood and seminal plasma were collected from fish during their reproductive season, at two different times of the day [mid-light (ML) and mid-dark (MD)], and melatonin concentration was determined by radioimmunoassay (RIA). Testosterone (T), 11-ketotestosterone (11KT), and total antioxidant status (TAS) were also determined, to investigate the putative role of seminal melatonin in fish reproduction, both at endocrine and antioxidant levels. For each species, Pearson's correlation analysis was performed between all possible factors. Blood plasma melatonin showed higher average values at night in the three species: gilthead seabream (808 +/- 139 pg/mL), European seabass (364 +/- 85 pg/mL), and Senegalese sole (248 +/- 40 and 88 +/- 11 pg/mL in F1 and wild males, respectively). However, melatonin levels in seminal plasma were species-specific: in European seabass, melatonin levels were not detectable at any time-point, whereas in gilthead seabream it was only found at MD (average of 21 pg/mL), and in Senegalese sole, different melatonin patterns were observed between F1 and wild males, but both had higher melatonin at MD (6.84 and 14.26 pg/mL, respectively). In gilthead seabream, at MD seminal melatonin levels correlated with the antioxidant status of seminal plasma. A relationship between blood melatonin and seminal TAS levels was observed in European seabass at ML: in this species, seminal melatonin could not be detected and the lowest seminal TAS levels were found. Regarding steroid analysis, opposite patterns in the seminal plasma of F1 and wild Senegalese sole were observed: at MD, wild Senegalese sole had substantially greater 11KT levels (2.53 ng/mL), whereas F1 males had higher T levels (1.92 ng/mL). In gilthead seabream, a positive correlation between T and ML blood melatonin and seminal TAS was observed. This study unraveled the species-specificity and daily changes of melatonin in fish seminal plasma.
- First study in cryopreserved Crassostrea angulata spermPublication . F RIESCO, MARTA; Félix, Francisca; Matias, Domitilia; Joaquim, Sandra; Suquet, Marc; Cabrita, ElsaSperm cryopreservation is a widely employed technique that promotes alternative techniques to contribute to broodstock management or restoration programs for species of commercial interest, endangered species or species with an interesting genotype. The preservation of genetic material from improved stocks or from the original population is extremely important for the oyster aquaculture industry to prevent the potential impacts of epidemic diseases and natural disasters. The Portuguese oyster, Crassostrea angulata, was the most important species commercialized by the shellfish industry. However, inadequate management of this industry and pathology occurrences resulted in a significant decrease in natural populations. For this reason, in this work a successful sperm cryopreservation protocol for this important species has been developed for the first time. Different internal cryoprotectants (DMSO, ethylene glycol, polyethylene glycol and methanol) at several concentrations (5, 10, 20%), containers (straws vs cryovials) and freezing rates (slow and fast rates) were tested. Cryoprotectant toxicity tests corroborated that this assay did not take into account the following steps of cryopreservation protocol as sperm agglutination. A fast freezing rate of cells diluted in 10% DMSO and the use of straws as containers were the best cryopreservation conditions for Portuguese oyster sperm. Finally, fertilization assays confirmed the efficiency of the cryopreservation protocol in oyster sperm. These results demonstrated that different susceptibilities have been detected concerning sperm cryopreservation depending on oyster species or genetic material composition. (C) 2016 Elsevier Inc. All rights reserved.
- Antioxidants in fish sperm and the potential role of melatoninPublication . Félix, Francisca; Oliveira, Catarina; Cabrita, ElsaIn recent years, the effects of novel antioxidants have played an important role in the research focusing on fish cell protection. As food demand grows, aquaculture production becomes more intensive, and fish are more exposed to oxidative stress conditions, like high densities, temperature shifting, frequent fish handling and samplings, and prophylactic or disease treatments, which expose fish to a different environment. Particularly in reproduction, germ cells lose antioxidant capacity with spermatogenesis, as spermatozoa are more prone to oxidative stress. Antioxidants have been used in a variety of fish physiological problems including in reproduction and in the establishment of cryopreservation protocols. From the most used antioxidants to natural plant food and herbs, and endogenously produced antioxidants, like melatonin, a review of the literature available in terms of their effects on the protection of fish spermatozoa is presented here in a classified structure. Several direct and indirect approaches to improve gamete quality using antioxidants administration are mentioned (through feed supplementation or by adding in cryopreservation media), as well as factors affecting the efficiency of these molecules and their mechanisms of action. Special attention is given to the unclear melatonin pathway and its potential scavenger activity to prevent and counteract oxidative stress damage on fish spermatozoa.
- Comparative study on cellular and molecular responses in oyster sperm revealed different susceptibilities to cryopreservationPublication . F RIESCO, MARTA; Félix, Francisca; Matias, Domitilia; Joaquim, Sandra; Suquet, Marc; Cabrita, ElsaThe Portuguese (Crassostrea angulata) and Pacific oyster (Crassostrea gigas), both from the genus Crassostrea, are two important species for production and conservation. Although they have common characteristics, different susceptibilities to rearing conditions have been described in these species. Overall, in the case of C. angulata, only a few remaining populations are present in the south of Portugal and Spain. The preservation of genetic material from improved stocks or from the original population is crucial in oysters to prevent the potential impacts of epidemic diseases and natural disasters. Sperm cryopreservation in oysters has progressed in recent years. However many issues, such as protocol standardization, are still unsolved for the application of research results. In the present study a sperm cryopreservation protocol, previously published in C. angulata by our group, were analyzed in terms of cellular and molecular damage, in an effort to determine the most sensible parameters to standardize the cryopreservation protocols for both species. Different approaches in the analysis of sperm quality were performed for the first time in this genus to detect different susceptibilities between the two valuable species. Our results revealed that our previously published protocol containing 10% DMSO as cryoprotectant is more suitable comparing to 10% EG in both species. In addition, an integrative analysis was performed in both oyster species comparing all cellular parameters and C. gigas showed a higher susceptibility to cryopreservation using this optimized protocol. Moreover, higher susceptibility to transcript degradation was detected in C. gigas using this optimized cryopreservation protocol. This study highlights the importance of using different techniques and exhaustive analyses for selecting the most suitable cryopreservation protocol and its standardization, thus ensuring the total safety of the technique.
- 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.
- Melatonin rhythms beyond the pineal organ: gene expression of receptors and biosynthesis enzymes in wild and F1 Senegalese solePublication . Félix de Azeredo Pinto e Melo, Francisca; Oliveira, Catarina; Martín, Ignacio; Manchado, Manuel; Sánchez-Vázquez, Francisco J.; Vera, Luisa M.; Cabrita, ElsaFish gonadal melatonin production is still unexplored and could contribute to a better understanding of its role in reproduction control, especially for species with reproductive impairments. This study aimed to comprehend if Senegalese sole testes are an extra-pineal production site of melatonin and if it has seasonal and daily variations. Wild and F1 broodstocks were sampled in the breeding season (BS) and out of the reproductive season (OS), at mid-light (ML) and mid-dark (MD) daytimes. Blood plasma melatonin concentration was determined by radioimmunoassay (RIA). The expression of genes involved in melatonin biosynthesis (tph1a, tph2, hiomt1, aanat1a, aanat1b, and aanat2) and melatonin receptors (mel1, mel1c, and mel2) was evaluated in the brain, eye, and testis by quantitative real-time PCR (qPCR). Plasma melatonin concentration in wild sole displayed day/night differences in both seasons (average ML: 36 +/- 22 pg/mL, MD: 108 +/- 63 pg/mL), whereas differences in the F1 broodstock were only found OS (ML: 100 +/- 54 pg/mL, MD: 187 +/- 88 pg/mL). Gene expression of mel1 and mel2 receptors, and tph1a, aanat1a, aanat2, and hiomt1 enzymes was detected and quantified in the fish testes. Moreover, daily and seasonal fluctuations in the expression of those genes were found in all tissues and broodstock groups. However, the F1 group showed distinct gene expression patterns compared to the wild type, suggesting a disruption in the circadian system. This study revealed that Senegalese sole testes are a melatonin production site and, at the same time, suggested a dysregulation in the hypothalamus-pituitary-gonad (HPG) axis of F1 males.