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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.
- Cognitive appraisal in fish: stressor predictability modulates the physiological and neurobehavioural stress response in sea bassPublication . Cerqueira, Marco; Millot, S.; Felix, A.; Silva, T.; Oliveira, G. A.; Oliveira, Catarina; Rey, S.; MacKenzie, S.; Oliveira, R.The role of cognitive factors in triggering the stress response is well established in humans and mammals (aka cognitive appraisal theory) but very seldom studied in other vertebrate taxa. Predictability is a key factor of the cognitive evaluation of stimuli. In this study, we tested the effects of stressor predictability on behavioral, physiological and neuromolecular responses in the European sea bass (Dicentrarchus labrax). Groups of four fish were exposed to a predictable (signalled) or unpredictable (unsignalled) stressor. Stressor predictability elicited a lower behavioural response and reduced cortisol levels. Using the expression of immediate early genes (c-fos, egr-1, bdnf and npas4) as markers of neuronal activity, we monitored the activity of three sea bass brain regions known to be implicated in stressor appraisal: the dorsomedian telencephalon, Dm (putative homologue of the pallial amygdala); and the dorsal (Dld) and ventral (Dlv) subareas of the dorsolateral telencephalon (putative homologue of the hippocampus). The activity of both the Dm and Dlv significantly responded to stressor predictability, suggesting an evolutionarily conserved role of these two brain regions in information processing related to stressor appraisal. These results indicate that stressor predictability plays a key role in the activation of the stress response in a teleost fish, hence highlighting the role of cognitive processes in fish stress.
- 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.
- Characterization of spawning rhythms of a sparidae aquaculture species, the white seabream (Diplodus sargus), in the south of PortugalPublication . Santos, Cátia; Soares, Florbela; Candeias-Mendes, Ana; Pousão‐Ferreira, Pedro; Dinis, Maria Teresa; Oliveira, CatarinaOne of the most visible features of fish reproduction is seasonality; however, daily rhythmicity is present in most species and should not be disregarded. This study investigated the seasonal and daily spawning rhythms of the white seabream (Diplodus sargus) in captivity, and a possible endogenous control of the daily rhythm. Spawns were monitored daily, during an entire reproductive season by using an automatic and programmable egg collector. The spawning season lasted 5 months, from January until June, when temperatures oscillated between 11.5 and 24.4 degrees C. Female fecundity was higher between 17.5 and 20.4 degrees C, suggesting a temperature influence in spawning. This species showed a very clear daily spawning rhythm (Cosinor analysis, p < 0.001), strongly related with photoperiod. Eggs started to be released at sunset and peaked around 21:00 h. Egg quality parameters also changed along the spawning period, with viability and hatching rates, increasing towards the end of the night period. The spawning rhythm persisted (Cosinor analysis, p < 0.001) for 5 days under constant light conditions, suggesting the presence of an endogenous clock mechanism which controlled this circadian pattern. The present results represent an advantage for the optimization of egg production protocols in this species, and an added value to the current knowledge of marine fish physiology.
- 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.
- Stressor controllability modulates the stress response in fishPublication . Cerqueira, Marco; Millot, Sandie; Silva, Tomé; Félix, Ana S.; Castanheira, Maria Filipa; Rey, Sonia; MacKenzie, Simon; Oliveira, Gonçalo A.; Oliveira, Catarina; Oliveira, Rui F.Background In humans the stress response is known to be modulated to a great extent by psychological factors, particularly by the predictability and the perceived control that the subject has of the stressor. This psychological dimension of the stress response has also been demonstrated in animals phylogenetically closer to humans (i.e. mammals). However, its occurrence in fish, which represent a divergent vertebrate evolutionary lineage from that of mammals, has not been established yet, and, if present, would indicate a deep evolutionary origin of these mechanisms across vertebrates. Moreover, the fact that psychological modulation of stress is implemented in mammals by a brain cortical top-down inhibitory control over subcortical stress-responsive structures, and the absence of a brain cortex in fish, has been used as an argument against the possibility of psychological stress in fish, with implications for the assessment of fish sentience and welfare. Here, we have investigated the occurrence of psychological stress in fish by assessing how stressor controllability modulates the stress response in European seabass (Dicentrarchus labrax). Results Fish were exposed to either a controllable or an uncontrollable stressor (i.e. possibility or impossibility to escape a signaled stressor). The effect of loss of control (possibility to escape followed by impossibility to escape) was also assessed. Both behavioral and circulating cortisol data indicates that the perception of control reduces the response to the stressor, when compared to the uncontrollable situation. Losing control had the most detrimental effect. The brain activity of the teleost homologues to the sensory cortex (Dld) and hippocampus (Dlv) parallels the uncontrolled and loss of control stressors, respectively, whereas the activity of the lateral septum (Vv) homologue responds in different ways depending on the gene marker of brain activity used. Conclusions These results suggest the psychological modulation of the stress response to be evolutionary conserved across vertebrates, despite being implemented by different brain circuits in mammals (pre-frontal cortex) and fish (Dld-Dlv).
- 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.