Percorrer por autor "Gallego, Victor"
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- Another tool for chondrichthyan ex situ conservation: first-time chimaera monstrosa sperm cryopreservationPublication . García-Salinas, Pablo; Gallego, Victor; Asturiano, Juan F.Chondrichthyans, which comprises elasmobranchs (sharks and rays) and holocephalans (chimaeras), are one of the most endangered group of vertebrates on the planet. Ex situ conservation programs, such as captive breeding, are tools that can be used to improve the status of some of the most sensitive species belonging to these groups. However, the use of reproductive techniques is necessary when planning sustainable breeding programs in controlled environments. In recent years, our group has described the protocols necessary to achieve viable sperm extraction and its cryopreservation in 13 species. However, the use of these techniques in the holocephalan group has not yet been explored. Here, the process of obtaining viable sperm in a holocephalan species, Chimaera monstrosa, is presented for the first time. The sperm was obtained from animals recovered from bottom trawling bycatch. It was possible to recover sperm from both males (n = 3), using cannulation and abdominal massage, and females (n = 2), by directly accessing their oviductal glands. Sufficient sperm was obtained from the males to apply cryopreservation protocols developed for elasmobranchs. For this purpose, the sperm was diluted in an extender for elasmobranchs (1 sperm:9 extender) previously developed by our group. The cryopreservation of sperm was achieved through the addition of different cryoprotectants to the extender: methanol, dimethyl sulfoxide (DMSO) and fresh egg yolk. Samples were frozen inside a Styrofoam box using vapor of liquid nitrogen and preserved in liquid nitrogen. Sperm quality was assessed by studying motility and membrane integrity post-thawing. The initial motility and membrane integrity values were close to 54%. The best post-thawing motility values were obtained with a combination of 5% DMSO, 5% methanol and 10% egg yolk, which induced motility values close to 25% and membrane integrity values close to 24%. This is the first time that sperm from this group of animals has been cryopreserved, expanding our knowledge on their reproductive biology and the tools available for their conservation.
- 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.
- Assessment of the reproductive status of captive populations of endangered leuciscid species from the iberian peninsula: a. hispanica, I. lusitanicum and a. occidentalePublication . Hernandez, Ana; Gil, Fátima; Sousa-Santos, Carla; Cabrita, Elsa; Guerreiro, Pedro Miguel; Gallego, VictorPopulations of freshwater fish species endemic to the Iberian Peninsula have been declining since the mid-20th century, and the captive breeding of highly endangered species is considered to be a useful tool to restock declining populations. A pioneer project of supportive breeding of critically endangered fish started in 2007 at the Aquário Vasco da Gama (AVG), and this work aims to show the reproductive status of the breeders which make up the current captive broodstoks. Populations of different endangered leuciscid species (Anaecypris hispanica, Iberochondrostoma lusitanicum and Achondrostoma occidentale) were sampled at AVG during the spring of 2022. Breeders were counted and sexed, and males were stripped to check for the presence of spermatozoa. The sperm volume was assessed visually, and spermatozoa motility was assessed by a CASA system. Sperm samples were classified into four classes based on the percentage of motile cells: C-I ≤ 25%, C-II = 25–50%, C-III = 50–75%; and C-IV > 75%. The captive population of A. hispanica consisted of 63 individuals and showed a 40% of spermiating males, with an average volume of 5–10 µL. The histogram of sperm quality reported that 15% males had sperm motility of C-II, 50% of males had sperm motility of C-III and, finally, 35% of males had sperm with the high-quality class (C-IV). The population of I. lusitanicum consisted of 599 individuals and showed 93% of spermiating males, with an average volume of 15–20 µL. The histogram of sperm quality reported that most part of the males had good sperm quality belonging to C-III and C-IV class (26% and 71%, respectively), while just 1 male showed bad quality sperm (C-II). The captive population of A. occidentale consisted of 193 individuals, showing a 62% of spermiating males with an average volume of 20–25 µL. The histogram showed that 6% males had sperm motility of C-I, 26% of males had sperm motility of C-II, the most part of the males (45%) showed a sperm quality of C-III and, finally, 23% of males had sperm with the high-quality class (C-IV). Since the project began in 2007, more than 12,000 fish of these three critically endangered species have been released to restock the populations from which the respective wild breeders were caught. All captive fish were released after a maximum of three consecutive generations in captivity, and new stocks were established with wild adults from the target populations, to avoid the negative effects of inbreeding and genetic drift on the original genetic pool.
- 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.
- 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.
- 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.
- 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.
- Reproduction techniques applied to chondrichthyans conservationPublication . García-Salinas, Pablo; Gallego, Victor; Asturiano, Juan F.Chondrichthyan fishes, which comprise sharks, rays, and chimaeras, are one of the most threatened groups of vertebrates. Given this situation, one possible strategy for the protection of these species could be the use of ex situ conservation projects. However, to develop sustainable ex situ conservation programs, captive breeding techniques, such as sperm extraction and its preservation, should be used. Two main obstacles must be overcome to develop these techniques: first, the lack of knowledge and the scarce previous work focused on the conservation of gametes from these animals; secondly, the peculiarities of the reproductive anatomy of each particular species. Through a detailed description of their reproductive anatomy, we have been able to develope the best techniques to obtain viable sperm from 17 species. Extraction has been performed in both live and dead animals, using cannulation, abdominal massage, and dissection. Exceptionally, we have even been able to recover viable sperm from the reproductive tract of females. Moreover, we have formulated artificial seminal plasma that can be used as an extender to maintain sperm motility for 36 days at 4 ◦C. By supplementing this extender with different combinations of cryoprotectants, i.e., methanol, dimethyl sulfoxide (DMSO), and fresh egg yolk, we were able to successfully cryopreserve (for the first time in most of these species) the sperm of 14 chondrichthyan species. Sperm samples were frozen inside a styrofoam box using the vapour of liquid nitrogen and were preserved in liquid nitrogen. The sperm quality was assessed by studying the motility and membrane integrity post thawing, demonstrating its effectiveness in the 14 species tested. In rays, the use of 10% DMSO or 10% methanol rendered post-thawing motility values higher than 40%. In sharks and the chimaera species, the best postthawing motility values were obtained with a combination of 5% DMSO, 5% methanol and 10% egg yolk, which induced mean values close to 35%. All this information broadens our knowledge on the reproductive techniques that can be applied to chondrichthyans, laying the foundations for the first cryobanks for their sperm.
- Sperm characterization of endangered leuciscids endemic from the iberian peninsula: gamete storage as a tool for helping ex-situ breeding programsPublication . Hernandez, Ana; Sousa-Santos, Carla; Gil, Fátima; Guerreiro, Pedro Miguel; Cabrita, Elsa; Gallego, VictorPopulations of freshwater fish species endemic to the Iberian Peninsula have been declining since the mid-20th century, and several types of actions (from in situ to ex situ measurements) have been applied over the past decades for preserving these species. However, limited knowledge about their reproductive biology makes it necessary to investigate different aspects of the reproductive cycle for improving breeding programs. The main objectives of this work were to advance knowledge concerning sperm kinetics and spermatozoa morphology and to develop protocols for the short- and long- term storage of gametes. Populations of different endangered leuciscid species (Anaecypris hispanica, Iberochondrostoma lusitanicum, Achondrostoma occidentale, and Squalius aradensis) were sampled during the spring of 2022 both in captive populations kept at Aquário Vasco da Gama (AVG) and in wild populations from different Portuguese rivers. Sperm samples were collected and sperm motion parameters were assessed, for the first time, for these four species. Sperm kinetics differed between species in motility and velocity traits, also showing a different number of sperm subpopulations. The longevity of sperm (swimming period) was also different among species: the shortest period was obtained for the wild population of S. aradensis (values close to zero at 40 s), and the longest swimming period for the captive population of I. lusitanicum (values close to zero at 120 s). Furthermore, different storage trials were carried out diluting the sperm in a extender solution (75 mM NaC1, 70 mM KC1, 2 mM CaC12, 1 mM, MgSO4, l0 mM Hepes, pH 8) at a ratio 1:20 (sperm:extender). Sperm quality (>40% of motile cells) was kept for a maximum of four days of storage, depending on the species. In addition, new cryopreservation protocols (using DMSO, Methanol and/or egg yolk) were tested for cryobanking the sperm of these threatened species. Cryopreserved samples showed significantly lower motility when compared with fresh samples, and the best results were obtained for I. lusitanicum, reaching 20% of motile cells after thawing using 10% of DMSO supplemented with 10% of egg yolk. This study is the first of its kind to successfully achieve gamete cryopreservation of Iberian endemic and endangered freshwater fish species, developing new and useful tools to complement the management and conservation programs.
- Sperm handling in aquatic animals for artificial reproductionPublication . Beirão, José; Boulais, Myrina; Gallego, Victor; O'Brien, Justine K.; Peixoto, Silvio; Robeck, Todd R.; Cabrita, ElsaArtificial reproduction involves collection and handling of gametes in a way that secures their quality and maximizes the fertilization outcome. In addition to initial sperm quality, numerous steps can affect the final result of fertilization, from the sperm collection process until gamete mixing (or co-incubation) when the spermatozoon enters or fuses with the oocyte. In this review, we summarize the whole process of sperm handling, from collection until fertilization for fish, penaeid shrimp, bivalve mollusks and marine mammals. To obtain sperm from captive animals, techniques vary widely across taxa, and include stripping by abdominal massage or testis surgical removal in fish, spermatophore collection in penaeid shrimps, gonadal scarification or temperature shock in bivalve mollusks, and voluntary collection via positive reinforcement in mammals. In most cases, special care is needed to avoid contamination by mucus, seawater, urine, or feces that can either activate sperm motility and/or decrease its quality. We also review techniques and extender solutions used for refrigerated storage of sperm across the aforementioned taxa. Finally, we give an overview of the different protocols for in vivo and in vitro fertilization including activation of sperm motility and methods for gamete co-incubation. The present study provides valuable information regarding breeder management either for animal production or species conservation.