AQUAculture infrastructures for EXCELlence in European fish research towards 2020

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The influence of 17β-oestradiol on lymphopoiesis and immune system ontogenesis in juvenile sea bass, Dicentrarchus labrax

Publication . Moreira, Catarina; Paiola, Matthieu; Duflot, Aurélie; Varó, Inma; Sitjà-Bobadilla, Ariadna; Knigge, Thomas; Pinto, P; Monsinjon, Tiphaine

The female sex steroid 17β-oestradiol (E2) is involved in the regulation of numerous physiological functions, including the immune system development and performance. The role of oestrogens during ontogenesis is, however, not well studied. In rodents and fish, thymus maturation appears to be oestrogen-dependent. Never theless, little is known about the function of oestrogen in immune system development. To further the under standing of the role of oestrogens in fish immune system ontogenesis, fingerlings of European sea bass (Dicentrarchus labrax) were exposed for 30 days to 20 ng E2⋅L− 1 , at two ages tightly related to thymic maturation, i.e., 60 or 90 days post hatch (dph). The expression of nuclear and membrane oestrogen receptors was measured in the thymus and spleen, and the expression of several T cell-related gene markers was studied in both immune organs, as well as in the liver. Waterborne E2-exposure at 20.2 ± 2.1 (S.E.) ng⋅L− 1 was confirmed by radioimmunoassay, leading to significantly higher E2-contents in the liver of exposed fish. The majority of gene markers presented age-dependent dynamics in at least one of the organs, confirming thymus maturation, but also suggesting a critical ontogenetic window for the implementation of liver resident γδ and αβ T cells. The oestrogen receptors, however, remained unchanged over the age and treatment comparisons with the exception of esr2b, which was modulated by E2 in the younger cohort and increased its expression with age in the thymus of the older cohort, as did the membrane oestrogen receptor gpera. These results confirm that oestrogen-signalling is involved in thymus maturation in European sea bass, as it is in mammals. This suggests that esr2b and gpera play key roles during thymus ontogenesis, particularly during medulla maturation. In contrast, the spleen expressed low or non-detectable levels of oestrogen receptors. The E2-exposure decreased the expression of tcrγ in the liver in the cohort exposed from 93 to 122 dph, but not the expression of any other immune-related gene analysed. These results indicate that the proliferation/migration of these innate-like T cell populations is estrogensensitive. In regard to the apparent prominent role of oestrogen-signalling in the late thymus maturation stage, the thymic differentiation of the corresponding subpopulations of T cells might be regulated by oestrogen. To the best of our knowledge, this is the first study investigating the dynamics of both nuclear and membrane oestrogen receptors in specific immune organs in a teleost fish at very early stages of immune system development as well as to examine thymic function in sea bass after an exposure to E2 during ontogenesis.

2021-05Journal article

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Disruption of gut integrity and permeability contributes to enteritis in a fish-parasite model: a story told from serum metabolomics

Publication . Sitjà-Bobadilla, Ariadna; Gil-Solsona, Rubén; Estensoro, Itziar; Piazzon, M. C; Martos-Sitcha, Juan A; Picard-Sánchez, Amparo; Fuentes, Juan; Sancho, Juan V; Calduch-Giner, Josep A; Hernández, Félix; Pérez-Sánchez, Jaume

Background In the animal production sector, enteritis is responsible for serious economic losses, and intestinal parasitism is a major stress factor leading to malnutrition and lowered performance and animal production efficiency. The effect of enteric parasites on the gut function of teleost fish, which represent the most ancient bony vertebrates, is far from being understood. The intestinal myxozoan parasite Enteromyxum leei dwells between gut epithelial cells and causes severe enteritis in gilthead sea bream (Sparus aurata), anorexia, cachexia, growth impairment, reduced marketability and increased mortality. Methods This study aimed to outline the gut failure in this fish-parasite model using a multifaceted approach and to find and validate non-lethal serum markers of gut barrier dysfunction. Intestinal integrity was studied in parasitized and non-parasitized fish by immunohistochemistry with specific markers for cellular adhesion (E-cadherin) and tight junctions (Tjp1 and Cldn3) and by functional studies of permeability (oral administration of FITC-dextran) and electrophysiology (Ussing chambers). Serum samples from parasitized and non-parasitized fish were analyzed using non-targeted metabolomics and some significantly altered metabolites were selected to be validated using commercial kits. Results The immunodetection of Tjp1 and Cldn3 was significantly lower in the intestine of parasitized fish, while no strong differences were found in E-cadherin. Parasitized fish showed a significant increase in paracellular uptake measured by FITC-dextran detection in serum. Electrophysiology showed a decrease in transepithelial resistance in infected animals, which showed a diarrheic profile. Serum metabolomics revealed 3702 ions, from which the differential expression of 20 identified compounds significantly separated control from infected groups in multivariate analyses. Of these compounds, serum inosine (decreased) and creatine (increased) were identified as relevant and validated with commercial kits. Conclusions The results demonstrate the disruption of tight junctions and the loss of gut barrier function, a metabolomic profile of absorption dysfunction and anorexia, which further outline the pathophysiological effects of E. leei.

2019-10-16Journal article

Open Access

Dietary butyrate helps to restore the Intestinal status of a marine teleost (Sparus aurata) fed extreme diets low in fish meal and fish oil

Publication . Estensoro, Itziar; Ballester-Lozano, Gabriel; Benedito-Palos, Laura; Grammes, Fabian; Martos-Sitcha, Juan Antonio; Mydland, Liv-Torunn; Calduch-Giner, Josep Alvar; Fuentes, Juan; Karalazos, Vasileios; Ortiz, Alvaro; Overland, Margareth; Sitja-Bobadilla, Ariadna; Perez-Sanchez, Jaume

There is a constant need to find feed additives that improve health and nutrition of farmed fish and lessen the intestinal inflammation induced by plant-based ingredients. The objective of this study was to evaluate the effects of adding an organic acid salt to alleviate some of the detrimental effects of extreme plant-ingredient substitution of fish meal (FM) and fish oil (FO) in gilthead sea bream diet. Three experiments were conducted. In a first trial (T1), the best dose (0.4%) of sodium butyrate (BP-70 (R) NOREL) was chosen after a short (9 weeks) feeding period. In a second longer trial (T2) (8 months), four diets were used: a control diet containing 25% FM (T2-D1) and three experimental diets containing 5% FM (T2-D2, T2-D3, T2-D4). FO was the only added oil in D1, while a blend of plant oils replaced 58% and 84% of FO in T2-D2, and T2-D3 and T2-D4, respectively. The latter was supplemented with 0.4% BP-70. In a third trial (T3), two groups of fish were fed for 12 and 38 months with D1, D3 and D4 diets of T2. The effects of dietary changes were studied using histochemical, immunohistochemical, molecular and electrophysiological tools. The extreme diet (T2-D3) modified significantly the transcriptomic profile, especially at the anterior intestine, up-regulating the expression of inflammatory markers, in coincidence with a higher presence of granulocytes and lymphocytes in the submucosa, and changing genes involved in antioxidant defences, epithelial permeability and mucus production. Trans-epithelial electrical resistance (Rt) was also decreased (T3-D3). Most of these modifications were returned to control values with the addition of BP-70. None of the experimental diets modified the staining pattern of PCNA, FABP2 or ALPI. These results further confirm the potential of this additive to improve or reverse the detrimental effects of extreme fish diet formulations.

2016-11Journal article

Open Access

Comparative study on cellular and molecular responses in oyster sperm revealed different susceptibilities to cryopreservation

Publication . F RIESCO, MARTA; Félix, Francisca; Matias, Domitilia; Joaquim, Sandra; Suquet, Marc; Cabrita, Elsa

The Portuguese (Crassostrea angulata) and Pacific oyster (Crassostrea gigas), both from the genus Crassostrea, are two important species for production and conservation. Although they have common characteristics, different susceptibilities to rearing conditions have been described in these species. Overall, in the case of C. angulata, only a few remaining populations are present in the south of Portugal and Spain. The preservation of genetic material from improved stocks or from the original population is crucial in oysters to prevent the potential impacts of epidemic diseases and natural disasters. Sperm cryopreservation in oysters has progressed in recent years. However many issues, such as protocol standardization, are still unsolved for the application of research results. In the present study a sperm cryopreservation protocol, previously published in C. angulata by our group, were analyzed in terms of cellular and molecular damage, in an effort to determine the most sensible parameters to standardize the cryopreservation protocols for both species. Different approaches in the analysis of sperm quality were performed for the first time in this genus to detect different susceptibilities between the two valuable species. Our results revealed that our previously published protocol containing 10% DMSO as cryoprotectant is more suitable comparing to 10% EG in both species. In addition, an integrative analysis was performed in both oyster species comparing all cellular parameters and C. gigas showed a higher susceptibility to cryopreservation using this optimized protocol. Moreover, higher susceptibility to transcript degradation was detected in C. gigas using this optimized cryopreservation protocol. This study highlights the importance of using different techniques and exhaustive analyses for selecting the most suitable cryopreservation protocol and its standardization, thus ensuring the total safety of the technique.

2019-01Journal article

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Optimization of sterlet sperm concentration for cryopreservation

Publication . Nascimento, João Pedro; Horokhovatskyi, Yevhen; Kholodnyy, Vitaliy; Rodina, Marek; Dzyuba, Viktoriya; Stechkina, Taisiya; Boryshpolets, Sergii; Dzyuba, Borys

One of the critical points in low temperature storage is the expense of storage capacities. Moreover, sperm concentration in a sample is not only a parameter for effective use of a cryobanks but also a parameter affecting cryopreservation outcomes. Sturgeon sperm in comparison to other species is significantly less concentrated due to the specificity of sperm maturation process, during which sperm is mixed with urine. In this study, we evaluated whether the artificial modification of the sterlet spermatozoa concentration before freezing can be useful during the routine application of sturgeon sperm cryopreservation. The sperm was first concentrated by centrifugation of native sperm samples. Then, the excess of seminal fluid was collected. The concentrated sperm samples were further diluted with the same seminal fluid to obtain different sperm concentrations in suspension, and they were subjected to freeze-thawing. The percentage of postthaw sperm motility depended on the sperm concentration in the samples. The highest post-thaw sperm motility was found in sperm samples with concentration of 0.2 and 1 × 109 spz mL􀀀 1. The sperm concentration of 3 × 109 spz mL􀀀 1, which is higher than the native sperm concentration in sterlet, was found to be appropriate for use in cryopreservation procedures since the sperm fertilizing ability remained at a high level even though a significant decline in percentage of sperm motility was observed. These findings supported the conclusion that the procedure of artificially increasing the sperm concentration before freezing could be useful for the reduction of the volume kept in cryostorage and to decrease the sample volume required for artificial fertilization.

2021Journal article

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