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Ex vivo exposure to titanium dioxide and silver nanoparticles mildly affect sperm of gilthead seabream (Sparus aurata) - A multiparameter spermiotoxicity approach

Publication . 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.

2022Journal article

Open Access

Exposure to silver and titanium dioxide nanoparticles at supra-environmental concentrations decreased sperm motility and affected spermatozoa subpopulations in gilthead seabream, Sparus aurata

Publication . Oliveira, Catarina; Ferrão, Leonor; Gallego, Victor; Mieiro, Cláudia; Oliveira, Isabel B.; Carvalhais, Ana; Pachedo, Mário; Cabrita, Elsa

Marine 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.

2023-07Journal article

Open Access

Optimizing rearing and welfare in Senegalese sole (Solea senegalesensis) broodstock: effect of ambient light intensity and handling time on stress response

Publication . Figueiredo, Filipe; Aragão, Cláudia; Pinto, Wilson; Dinis, Maria Teresa; Oliveira, Catarina

Broodstock rearing conditions and handling procedures should be optimized in aquaculture species in order to benefit fish welfare and guarantee optimal conditions for spawning. In teleosts, basal cortisol levels display daily rhythms, oscillating along the 24 h of the day. In this sense, handling fish at different moments of the day may lead to different stress responses. The present study aimed at investigating the optimal rearing conditions for Senegalese sole broodstock, considering ambient light intensity and handling time. The optimal light intensity (50, 100 or 200 lx) was investigated by measuring fish cortisol levels and monitoring locomotor activity rhythms under each intensity tested. Results showed a significant increase in cortisol levels of fish exposed to 200 lx, when compared to values obtained under 100 lx, accompanied by changes in locomotor activity rhythms in both tanks under study. These results suggested that 200 lx may be too high as light intensity for this species, whereas 100 lx seems to be more adequate. Also, daily rhythms of stress response were investigated in breeders from different origins (Wild and first generation, G1). Basal cortisol levels and cortisol stress response after an acute stressor (air exposure) were monitored at two distinct moments of the day (Mid-Light and Mid-Dark). Basal levels were higher during the day in the wild group, while G1 fish seemed to have lost the daily fluctuations in basal cortisol plasma levels, as well as their daily rhythms of locomotor activity. Both groups showed lower stress responses during night-time, an indication that this is an adequate period of the day to handle this species. Senegalese sole breeders born in captivity presented more pronounced stress responses when compared to wild fish, reflecting their different life history in terms of stress challenges.

2020-01Journal article

Open Access

Novel approaches on melatonin role: presence of clock-hormone in fish seminal plasma

Publication . Félix, Francisca; Gallego, Victor; Mendes, Ana; Soares, Florbela; Vera, Luisa M.; Cabrita, Elsa; Oliveira, Catarina

The 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.

2023Journal article

Open Access

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.

2023Journal article

Open Access