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- Post-thaw quality assessment of testicular fragments as a source of spermatogonial cells for surrogate production in the flatfish Solea senegalensisPublication . Cabrita, Elsa; Pacchiarini, Tiziana; Fatsini, Elvira; Sarasquete, Carmen; Herráez, María PazCryopreservation of germ cells would facilitate the availability of cells at any time allow ing the selection of donors and maintaining quality control for further applications such as transplan tation and germline recovery. In the present study, we analyzed the efciency of four cryopreservation protocols applied either to isolated cell suspensions or to testes fragments from Senegalese sole. In tes tes fragments, the quality of cryopreserved germ cells was analyzed in vitro in terms of cell recovery, integrity and viability, DNA integrity (fragmentation and apoptosis), and lipid peroxidation (malondialde hyde levels). Transplantation of cryopreserved germ cells was performed to check the capacity of cells to in vivo incorporate into the gonadal primordium of Senegalese sole early larval stages (6 days after hatch ing (dah), pelagic live), during metamorphosis (10 dah) and at post-metamorphic stages (16 dah and 20 dah, benthonic life). Protocols incorporating dimethyl sulfoxide (DMSO) as a cryoprotectant showed higher number of recovered spermatogonia, especially in samples cryopreserved with L-15 + DMSO (0.39 ± 0.18 × 106 cells). Lipid peroxidation and DNA frag mentation were also signifcantly lower in this treat ment compared with other treatments. An important increase in oxidation (MDA levels) was detected in samples containing glycerol as a cryoprotectant, refected also in terms of DNA damage. Transplan tation of L-15 + DMSO cryopreserved germ cells into larvae during early metamorphosis (10 dah, 5.2 mm) showed higher incorporation of cells (27.30 ± 5.27%) than other larval stages (lower than 11%). Cryopreservation of germ cells using testes fragments frozen with L-15 + DMSO was demonstrated to be a useful technique to store Senegalese sole germline.
- The use of sand substrate modulates dominance behaviour and brain gene expression in a Flatfish SpeciesPublication . Almeida, Maria Mafalda; Cabrita, Elsa; Fatsini, ElviraPhysical complexity adds physical enrichment to rearing conditions. This enrichment promotes fish welfare and reduces detrimental characteristics that fish develop in captivity. Senegalese sole (Solea senegalensis) is an important species for European aquaculture, where it is reared in intensive conditions using fibreglass tanks. However, reproductive dysfunctions present in this species do not allow it to complete its life cycle in captivity. Recently, dominance behaviour has been studied to try to solve this problem. The present study aimed to assess the effect of sand as environmental enrichment in the dominance behaviour and brain mRNA abundance of Senegalese sole juveniles. Four tanks of sole (n = 48 fish in total) were established in two different environments (with and without sand). Juveniles were subjected to dominance tests of feeding and territoriality. Behaviours analysed by video recordings related to the distance from the food delivered and harassment behaviour towards other individuals (e.g., resting of the head on another individual). In both environments, dominant sole were the first to feed, displayed more head-resting behaviour and dominated the area close to the feeding point, where the events were reduced in fish maintained in the sand. mRNA expression related to differentiation of dopamine neurons (nr4a2) and regulation of maturation (fshra) were significantly upregulated in dominant fish in the sand environment compared to dominants maintained without sand. The use of an enriched environment may affect Senegalese sole dominance, enhance welfare and possibly advance future maturation.
- Linking stress coping styles with brain mRNA abundance of selected transcripts for Senegalese sole (Solea senegalensis) juvenilesPublication . Fatsini, Elvira; Rey, Sonia; Ibarra-Zatarain, Zohar; Boltaña, Sebastián; Mackenzie, Simon; Duncan, Neil J.In fish, proactive and reactive individual stress copying styles (SCS) have been used to resolve variation in molecular expression data. Stress coping styles have been previously described in several stages of Solea senegalensis by validating for the species the use of standard behavioural screening tests. The present study aimed to link behavioural SCS tests with brain transcript abundance in early Senegalese sole juveniles in order to observe the natural variation in a molecular pathway in this species. A total of 50 juveniles were subjected to three individual behavioural (Restraining, New environment and Confinement) and one group (Risk-taking) screening tests. The fish were classified in SCS categories by applying a hierarchical cluster to the variable "Total activity" (the total activity time that the fish was moving in each individual test). Three categories were defined, proactive, intermediate and reactive sole. Six transcripts were chosen and tested, one related to basic metabolism (gapdh-2), three to feeding behaviour (per1, igf-Ia, pparß) and two to the stress response (crh-BP and hsp90aa) in 30 juveniles (10 individuals per SCS category) using rt-qPCR to observe differences in the abundance of those transcripts among SCS. Four transcripts were differentially expressed (DETs) among them. The transcript gapdh-2 showed up-regulation for proactive and intermediate SCS sole while reactive individuals showed down-regulation. Target mRNAs per1, igf-Ia and pparß, showed different levels of up-regulation for proactive and reactive fish while intermediates were highly down-regulated. Surprisingly no differences in stress related transcripts were observed. Correlations were found between variation in coping styles and variation in the abundance of mRNAs involved in important biological functions in Senegalese sole. These results are the first evidence of the relationship between the behavioural individual variation and the fluctuation in brain transcripts abundance in Senegalese sole.
- Spawning induction for latin american fishesPublication . Mechaly, Alejandro S.; Batlouni, Sergio R.; Elisio, Mariano; Sanches, Eduardo A.; Guzmán, Jonathan Chacon; García, Minerva Maldonado; Rodríguez‐Forero, Adriana; Vissio, Paula; Fatsini, Elvira; Núñez, Jesús; Duncan, NeilAquaculture offers solutions to meet the growing global demand for fish, and reports from the UN-FAO indicate that aquaculture production in Latin America (LA) has grown at rates above the world average in recent years. One of the major constraints in the diversification of LA aquaculture is the control of reproduction in several popular native fish species for which difficulties in captive propagation have not yet been sufficiently overcome. This article reviews the use of hormone treatments to promote reproduction in females of these native fish species. LA has played a key role in the history of development of hormone administration, including the first hormonally induced spawning. That contribution is included in a historical overview of the discovery of the major hormones used in fish culture. The review provides a summary of difficulties to propagate females of various native fishes and the effects of administering hormones to enhance reproduction. Induced spawning of certain freshwater species was mainly achieved with pituitary extracts or human chorionic gonadotropin (hCG), although gonadotropin-releasing hormone analogues (GnRHa) treatments are being researched, and successful studies suggest that low doses may be more effective. Research on new and emerging aquaculture species has applied both gonadotropins (Gths) and GnRHa-based treatments, and GnRHa treatments have shown potential for marine species. However, native marine species new to aquaculture have also been conditioned to spawn spontaneously without hormones. Finally, we proposed future lines of research to examine reproductive strategies and GnRHa-based hormone treatments to improve reproductive control for economically important fish species of LA.
- 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.
- The feasibility of using gas mixture to stun seabream (Sparus aurata) before slaughtering in aquaculture productionPublication . Roque, A.; Gras, N.; Rey-Planellas, S.; Fatsini, Elvira; Pallisera, J.; Duncan, N.; Muñoz, I.; Velarde, A.; Hernandez, M. D.Current European Union regulation explicitly states that farmed fish should be spared any avoidable pain, distress or suffering at the time of slaughter. It has been shown that fish suffer when they are killed in an ice slurry, the most common method of killing farmed fish in the Mediterranean. Thus, it is necessary to find a method of slaughtering Mediterranean fish that is, (1) efficient in inducing unconsciousness with minimal pain and distress, (2) practical to be applied to a large group of animals at the same time, and (3) feasible to be used at sea. The present study assesses the welfare of Gilthead seabream (Sparus aurata) stunned by two different gas mixtures authorised for stunning other farmed species. To achieve this objective, commercial sized seabream were stunned and/or sacrificed under different protocols: a) killed directly in ice slurry, b) exposed to a mixture of 30% CO2 + 70% N-2, and then moved to ice slurry and c) exposed to a mixture of 40% CO2 + 30% N-2 + 30% O-2 and then moved to ice slurry. Electroencephalograms (EEG) were recorded to evaluate the state of consciousness of seabream during stunning, while blood and brains were sampled to obtain acute stress indicators and relative gene expression, respectively. Additionally, dead fish were kept for in situ meat quality evaluation. When exposed to the gas mixtures, fish lost balance at 1 min 23 s +/- 31 s with CO2 + N-2 and 1 min 12 s +/- 32 s, with CO2 + N-2 + O-2, respectively. Cortisol, lactate and glucose levels were significantly lower in all fish exposed to gas prior to ice slurry than in fish slaughtered directly in ice slurry (p < 0.05). Electroencephalogram records indicated that fish started to lose consciousness when they lost balance and sank to the bottom of the tank. No differences were found in the meat quality (pH and rigor mortis) among the three treatments. Altogether, the study concludes that the use of carbon dioxide together with nitrogen prior to immersion in ice slurry is more humane than ice slurry alone.
- The presence of wild Senegalese sole breeders improves courtship and reproductive success in cultured conspecificsPublication . Fatsini, Elvira; Gonzalez, Wendy; Ibarra-Zatarain, Zohar; Napuchi, Juvenal; Duncan, Neil J.The life cycle of Senegalese sole (Solea senegalensis) is not closed in captivity due to a reproductive dysfunction related to the lack of participation of cultured male breeders in the courtship. To discern a possible solution to this social reproductive dysfunction, the main objective of this study was to determine the effect of the presence of spawning wild Senegalese sole breeders on the courtship and reproduction of cultured breeders. Three experimental groups were formed: Control group (n = 10) formed with only cultured sole; groups M1 and M2 constituted of mixed-origin sole (10 cultured and 8 or 9 wild breeders). All cultured breeders were from the same stock, which had never successfully reproduced, whilst the wild broodstock had spawned viable eggs in captivity. All groups were held in the same captive conditions and spawning, and behaviour were recorded for each group over four spawning seasons. All spawns were collected, and the parameters registered were floating and non-floating eggs, fertilization and hatching rates. In addition, parental analysis was made of larvae from viable spawns. Behaviour was studied with video recordings to compare locomotor activity and courtship behaviours including the "Follow" behaviours where sole swim after each other in a procession. Fertilized spawns were obtained from the mixed-origin groups (M1 and M2) including spawns involving a cultured male. The cultured males also participated actively in the "Follow" behaviours with the courting wild sole and this participation of cultured males increased significantly over the four years of the experiment. Male percentage participation in the "Follow" behaviours was positively correlated (R = 0.81) to participation in spawning to indicate the importance of increasing cultured male participation in the "Follow" behaviours. A total of seven spawns were obtained from a cultured male that fertilized eggs from one cultured female and two wild females. The same cultured female also spawned with a wild male. No viable spawns were obtained from the Control group and locomotor activity and courtship behaviour counts were significantly lower than in the experimental mixedorigin groups. This is the first report of cultured male breeders participating in reproductive behaviour and spawning, which could be associated with social learning processes, mate selection and dominance where cultured males copied the behaviour and spawning of wild Senegalese sole breeders.
- Comparative transcriptome analysis reveals molecular damage associated with cryopreservation in Crassostrea angulata D-larvae rather than to cryoprotectant exposurePublication . Anjos, Catarina; Duarte, Daniel Filipe Correia; Fatsini Fernández, Elvira; Domitília Matias; Cabrita, ElsaBackground The Portuguese oyster Crassostrea angulata, a bivalve of significant economic and ecological importance, has faced a decline in both production and natural populations due to pathologies, climate change, and anthropogenic factors. To safeguard its genetic diversity and improve reproductive management, cryopreservation emerges as a valuable strategy. However, the cryopreservation methodologies lead to some damage in structures and functions of the cells and tissues that can affect post-thaw quality. Transcriptomics may help to understand the molecular consequences related to cryopreservation steps and therefore to identify different freezability biomarkers. This study investigates the molecular damage induced by cryopreservation in C. angulata D-larvae, focusing on two critical steps: exposure to cryoprotectant solution and the freezing/thawing process. Results Expression analysis revealed 3 differentially expressed genes between larvae exposed to cryoprotectant solution and fresh larvae and 611 differentially expressed genes in cryopreserved larvae against fresh larvae. The most significantly enriched gene ontology terms were "carbohydrate metabolic process", "integral component of membrane" and "chitin binding" for biological processes, cellular components and molecular functions, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified the "neuroactive ligand receptor interaction", "endocytosis" and "spliceosome" as the most enriched pathways. RNA sequencing results were validate by quantitative RT-PCR, once both techniques presented the same gene expression tendency and a group of 11 genes were considered important molecular biomarkers to be used in further studies for the evaluation of cryodamage. Conclusions The current work provided valuable insights into the molecular repercussions of cryopreservation on D-larvae of Crassostrea angulata, revealing that the freezing process had a more pronounced impact on larval quality compared to any potential cryoprotectant-induced toxicity. Additionally, was identify 11 genes serving as biomarkers of freezability for D-larvae quality assessment. This research contributes to the development of more effective cryopreservation protocols and detection methods for cryodamage in this species.
- Comparative transcriptome analysis reveals molecular damage associated with cryopreservation in Crassostrea angulata D-larvae rather than to cryoprotectant exposurePublication . Anjos, Catarina; Duarte, Daniel Filipe Correia; Fatsini Fernández, Elvira; Matias, Domitília; Cabrita, ElsaThe Portuguese oyster Crassostrea angulata, a bivalve of significant economic and ecological importance, has faced a decline in both production and natural populations due to pathologies, climate change, and anthropogenic factors. To safeguard its genetic diversity and improve reproductive management, cryopreservation emerges as a valuable strategy. However, the cryopreservation methodologies lead to some damage in structures and functions of the cells and tissues that can affect post-thaw quality. Transcriptomics may help to understand the molecular consequences related to cryopreservation steps and therefore to identify different freezability biomarkers. This study investigates the molecular damage induced by cryopreservation in C. angulata D-larvae, focusing on two critical steps: exposure to cryoprotectant solution and the freezing/thawing process. Results Expression analysis revealed 3 differentially expressed genes between larvae exposed to cryoprotectant solution and fresh larvae and 611 differentially expressed genes in cryopreserved larvae against fresh larvae. The most significantly enriched gene ontology terms were "carbohydrate metabolic process", "integral component of membrane" and "chitin binding" for biological processes, cellular components and molecular functions, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified the "neuroactive ligand receptor interaction", "endocytosis" and "spliceosome" as the most enriched pathways. RNA sequencing results were validate by quantitative RT-PCR, once both techniques presented the same gene expression tendency and a group of 11 genes were considered important molecular biomarkers to be used in further studies for the evaluation of cryodamage. Conclusions The current work provided valuable insights into the molecular repercussions of cryopreservation on D-larvae of Crassostrea angulata, revealing that the freezing process had a more pronounced impact on larval quality compared to any potential cryoprotectant-induced toxicity. Additionally, was identify 11 genes serving as biomarkers of freezability for D-larvae quality assessment. This research contributes to the development of more effective cryopreservation protocols and detection methods for cryodamage in this species.
- 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.