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- Assessment of male reproductive traits in endangered leuciscids from the Iberian Peninsula: first attempts to store gametes both at short- and long-termPublication . Hernandez, Ana; Sousa-Santos, Carla; Gil, Fátima; Cabrita, Elsa; Guerreiro, Pedro; Gallego, VictorDuring the spring of 2022, several endangered leuciscid species (Anaecypris hispanica, Squalius aradensis, Anachondrostoma Occidentale, and Iberochondrostoma lusitanicum) were sampled both at the Vasco da Gama aquarium facilities and in some rivers of the Algarve region, Portugal. Sperm samples were extracted by gentle abdominal pressure and sperm motion parameters were assessed for the first time in four species, using a computerized analysis system. The results obtained showed that spermatozoa kinetic patterns were similar for all 4 species, with high motility and velocity values after the sperm activation time and with a marked decrease after 20. On the other hand, sperm longevity was highly variable between species, with short longevities (around 40 s) for A. hispanica and S. aradensis, and longer longevities (100-120 s) for A. occidentale and I. lusitanicum, which could indicate a latitudinal pattern in terms of sperm longevity. At the same time, morphometric analysis was carried out for the four target species, revealing that spermatozoa showed similar sizes and shapes to other external fertilizers belonging to Leuscididae, with small spherical heads, uniflagellate, and without acrosomes. In addition, a short-term gamete storage trail was performed by diluting sperm in 1:9 (sperm:extender) and storing them at 4oC. Although the results obtained were uneven among the species studied, the dilution and extender used generated motilities above 40% up to day 4 of storage in S. aradensis and I. lusitanicum, and up to days 1-2 in A. hispanica and A. occidentale, respectively. Finally, gamete cryopreservation trials were also carried out on these threatened species. Although cryopreserved samples showed significantly lower motility than fresh samples, some protocols generate acceptable percentages of viability, DNA integrity, and sperm motility in some species such as I. lusitanicum and A. occidentale. The data revealed that the protocol based on 10% DMSO plus 7.5% egg yolk generated the best results.This study is the first to assess the reproductive traits of wild and captive populations of endangered leuciscids endemic from the Iberian Peninsula, describing the spermatozoa kinetics and developing protocols for managing male gametes both in short- and long-term storage. Outcomes will provide new and useful tools to complement the management and conservation of ex situ breeding programs that are being developed for these four endangered species.
- The importance of developing and Standardizing Gamete, Embryo and Larvae Handling in aquatic animalsPublication . Paredes, Estefanía; Gallego, VictorArtificial reproduction in aquatic animals usually involves the collection and handling of gametes both from males and females in a way that secures their quality and optimizes the fertilization event. There are innumerable specie- and family-specific protocols among the various aquatic species that inhabit bodies of water, from freshwater lagoons to seawater oceans. In that sense, this Special Issue aims to gather high-quality papers addressing different areas, focusing on gamete collection and handling, gamete storage (both shortand long-term storage), in vitro fertilization techniques, embryo development studies and the larvae management of different aquatic animals.
- 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.
- 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.
- 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.
- 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.