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- Ex vivo exposure to titanium dioxide and silver nanoparticles mildly affect sperm of gilthead seabream (Sparus aurata) - A multiparameter spermiotoxicity approachPublication . Carvalhais, A.; Oliveira, I. B.; Oliveira, H.; Oliveira, Catarina; Ferrão, L.; Cabrita, Elsa; Asturiano, J. F.; Guilherme, S.; Pacheco, M.; Mieiro, C. L.Nanoparticles (NP) are potentially repmtoxic, which may compromise the success of populations. However, the reprotoxicity of NP is still scarcely addressed in marine fish. Therefore, we evaluated the impacts of environmentally relevant and supra environmental concentrations of titanium dioxide (TiO2: 10 to 10,000 mu g.L-1) and silver NP (Ag: 0.25 to 250 mu g.L-1) on the sperm of gilthead seabream (Sparus aurata). We performed short-term direct exposures (ex vivo) and evaluated sperm motility, head morphometry, mitochondrial function, antioxidant responses and DNA integrity. No alteration in sperm motility (except for supra environmental Ag NP concentration), head morphometry, mitochondrial function, and DNA integrity occurred. However, depletion of all antioxidants occurred after exposure to TiO2 NP, whereas SOD decreased after exposure to Ag NP (lowest and intermediate concentration). Considering our results, the decrease in antioxidants did not indicate vulnerability towards oxidative stress. TiO2 NP and Ag NP induced low spermiotoxicity, without proven relevant ecological impacts.
- Circulating small non-coding RNAs provide new insights into vitamin K nutrition and reproductive physiology in teleost fishPublication . I, Fernández; Fernandes, Jorge M. O.; Roberto, Vânia; Kopp, Martina; Oliveira, Catarina; Riesco, Marta F.; Dias, Jorge; Cox, Cymon J.; Leonor Cancela, M.; Cabrita, Elsa; Gavaia, PauloBackground: Vitamin K (VK) is a fat-soluble vitamin known for its essential role in blood coagulation, but also on other biological processes (e.g. reproduction, brain and bone development) have been recently suggested. Nevertheless, the molecular mechanisms behind its particular function on reproduction are not yet fully understood. Methods: The potential role of VK on reproduction through nutritional supplementation in Senegalese sole (Solea senegalensis) was assessed by gonadal maturation and 11-ketosterone, testosterone and estriol plasma levels when fed with control or VK supplemented (1250 mg kg(-1) of VK,) diets along a six month trial. At the end, sperm production and quality (viability and DNA fragmentation) were evaluated. Circulating small non-coding RNAs (sncRNAs) in blood plasma from males were also studied through RNA-Seq. Results: Fish fed with dietary VK supplementation had increased testosterone levels and lower sperm DNA fragmentation. SncRNAs from blood plasma were found differentially expressed when nutritional and sperm quality conditions were compared. PiR-675//676//4794//5462 and piR-74614 were found up-regulated in males fed with dietary VK supplementation. Let-7g, let-7e(18nt), let-7a-1, let-7a-3//7a-2//7a-1, let-7e(23nt) and piR-675//676//4794//5462 were found to be up-regulated and miR-146a and miR-146a-1//146a-2//146a-3 down-regulated when fish with low and high sperm DNA fragmentation were compared. Bioinformatic analyses of predicted mRNAs targeted by sncRNAs revealed the potential underlying pathways. Conclusions: VK supplementation improves fish gonad maturation and sperm quality, suggesting an unexpected and complex regulation of the nutritional status and reproductive performance through circulating sncRNAs. General significance: The use of circulating sncRNAs as reliable and less-invasive physiological biomarkers in fish nutrition and reproduction has been unveiled.
- 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.
- Solea senegalensis sperm cryopreservation: new insights on sperm qualityPublication . F RIESCO, MARTA; Oliveira, Catarina; Soares, Florbela; Gavaia, Paulo J.; Dinis, Maria Teresa; Cabrita, ElsaCryopreservation of Senegalese sole sperm can represent an alternative to overcome some reproductive problems of this species. However, it is important to guarantee the safe use of cryopreserved sperm by selecting an appropriate protocol according to a high demand quality need to be ensured. It has been demonstrated that traditional assays such as motility and viability do not provide enough information to identify specific damage caused by cryopreservation process (freezing and thawing). Specific tests, including lipid peroxidation and DNA damage, should be performed. In the present study, motility and lipid peroxidation were performed as specific tests allowing us to discard cryopreservation conditions such as methanol as internal cryoprotectant and bovine serum albumin as external cryoprotectant. In addition, a caspase 3/7 detection by flow cytometry was performed to analyze apoptosis activity in the best selected conditions. Moreover, new highly sensitive tests based on transcript number detection have recently been described in fish sperm cryopreservation. For this reason, a transcript level detection assay was performed on certain oxidative and chaperone genes related to fertilization ability and embryo development (hsp70, hsp90BB, hsp90AA, gpx) to select the best cryopreservation conditions. DMSO+ egg yolk proved to be the best cryoprotectant combination in terms of transcript level. This study describes an optimized cryopreservation protocol for Solea senegalensis sperm demonstrating for the first time that transcript degradation is the most sensitive predictor of cell status in this species after cryopreservation.
- Exposure to silver and titanium dioxide nanoparticles at supra-environmental concentrations decreased sperm motility and affected spermatozoa subpopulations in gilthead seabream, Sparus aurataPublication . Oliveira, Catarina; Ferrão, Leonor; Gallego, Victor; Mieiro, Cláudia; Oliveira, Isabel B.; Carvalhais, Ana; Pachedo, Mário; Cabrita, ElsaMarine pollution by nanoparticles (NPs) can be reprotoxic for fish and disturb successful reproduction of wild populations. In gilthead seabream (Sparus aurata), a mild effect on sperm motility was observed after exposure to high concentrations of silver NPs. Considering the great heterogeneity traits within a sperm sample, it is possible that NPs affect spermatozoa accordingly, modulating subpopulation profile. Thus, this work aimed to analyse NP effects in sperm motility in general and considering spermatozoa population structure, using a subpopulation approach. Seabream sperm samples from mature males were exposed for 1 h to increasing concentrations of titanium dioxide (1, 10, 100, 1000 and 10,000 & mu;g L-1) and silver (0.25, 25 and 250 & mu;g L-1) NPs, including Ag NP and Ag+, dissolved in a non-activating medium (0.9 % NaCl). Concentrations chosen include realistic (10-100 and 0.25 & mu;g L-1, respectively, for TiO2 and Ag) and supra-environmental values. The mean particle diameter was determined as 19.34 & PLUSMN; 6.72 and 21.50 & PLUSMN; 8.27 nm in the stock suspension, respectively, for titanium dioxide and silver. After the ex vivo exposure, sperm motility parameters were determined using computer-assisted sperm analysis, and sperm subpopulations were later identified using a two-step cluster analysis. Results revealed a significant reduction in total motility after exposure to the 2 highest concentrations of titanium dioxide NPs, while curvilinear and straight-line velocities were not altered. Exposure to silver NPs (Ag NP and Ag+) lowered significantly total and progressive motilities at all concentrations, while curvilinear and straight-line velocities were significantly lower only at the highest concentration. Sperm subpopulations were also affected by the exposure to both titanium dioxide and silver NPs. In both cases, the highest levels of NPs triggered a decrease in the percentage of fast sperm subpopulations (38.2% in TiO2 1000 & mu;g L-1, 34.8.% in Ag NP 250 & mu;g L-1, and 45.0% in Ag+ 250 & mu;g L-1 vs 53.4% in the control), while an increase on slow sperm subpopulations. A reprotoxic effect was proven for both NPs, but only at supra-environmental concentrations.
- 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.
- Kisspeptin influences the reproductive axis and circulating levels of microRNAs in Senegalese solePublication . Oliveira, Catarina; Fatsini, Elvira; Fernández, Ignacio; Anjos, Catarina; Chauvigné, François; Cerdà, Joan; Mjelle, Robin; Fernandes, Jorge M. O.; Cabrita, ElsaKisspeptin regulates puberty and reproduction onset, acting upstream of the brain-pituitary-gonad (HPG) axis. This study aimed to test a kisspeptin-based hormonal therapy on cultured Senegalese sole (G1) breeders, known to have reproductive dysfunctions. A single intramuscular injection of KISS2-10 decapeptide (250 µg/kg) was tested in females and males during the reproductive season, and gonad maturation, sperm motility, plasma levels of gonadotropins (Fsh and Lh) and sex steroids (11-ketotestosterone, testosterone and estradiol), as well as changes in small non-coding RNAs (sncRNAs) in plasma, were investigated. Fsh, Lh, and testosterone levels increased after kisspeptin injection in both sexes, while sperm analysis did not show differences between groups. Let7e, miR-199a-3p and miR-100-5p were differentially expressed in females, while miR-1-3p miRNA was up-regulated in kisspeptin-treated males. In silico prediction of mRNAs targeted by miRNAs revealed that kisspeptin treatment might affect paracellular transporters, regulate structural and functional polarity of cells, neural networks and intracellular trafficking in Senegalese sole females; also, DNA methylation and sphingolipid metabolism might be altered in kisspeptin-treated males. Results demonstrated that kisspeptin stimulated gonadotropin and testosterone secretion in both sexes and induced an unanticipated alteration of plasma miRNAs, opening new research venues to understand how this neuropeptide impacts in fish HPG axis.
- 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.
- Cutting-Edge methods in Teleost and Chondrichthyan reproductive biologyPublication . Mechaly, Alejandro S.; Awruch, Cynthia; Cabrita, Elsa; Costas, Benjamín; Fernandes, Jorge M.O.; Gallego, Victor; Hirt-Chabbert, Jorge; Konstantinidis, Ioannis ; Oliveira, Catarina; Ramos Júdez, Sandra; Ramos-Pinto, Lourenço ; Fatsini Fernández, ElviraReproduction is a fundamental biological process for producing offspring and ensuring the continuation of the species. With nearly 35,000 fish species worldwide exhibiting a very diverse range of reproductive systems and strategies, the study of fish reproduction is a significant challenge. Research into various fish reproductive models is driven by their use in human biomedical applications, improving the diversification and sustainability of aquaculture species, and developing strategies for biodiversity conservation. These models must be approached from an animal ethics perspective, considering the welfare of the species under study and applying the 3Rs principle (Replacement, Reduction and Refinement). This principle focusses on replacing animals with alternative methods wherever possible, reducing the number of animals used and refining procedures to minimize suffering. While destructive sampling of wild aquatic species should be minimized, the need remains to provide information on reproduction as an important parameter for delineating management and conservation programs. One of the practices that helps reduce and replace the use of animal testing is the scientific study of individuals obtained through bycatch. This is the case for some chondrichthyans, and endangered fish species included in conservation programs. Regarding fish species that are kept under human care (commonly called captivity), studies on reproduction are essential to improve well-being and increase production of farmed species. Research on fish reproduction requires expanding basic knowledge of broodstock management tools, such as gamete quality assessment, reproductive behavior studies, the use of hormone therapies to stimulate reproduction, and the development of non-lethal or less invasive methods using body fluids (e.g., mucus, seminal plasma, blood plasma, or urine) to study reproductive status, as well as novel markers from state-of-the-art omics research. This review describes a multidisciplinary approach that includes the aforementioned reproductive management tools, indicators of welfare, and next-generation sequencing techniques using samples collected by minimally invasive methods.