M.J. Zamorano

Effects of dietary concentrated mannan oligosaccharides supplementation on growth, gut mucosal immune system and liver lipid metabolism of European sea bass (Dicentrarchus labrax) juveniles.

The study assesses the effects of dietary concentrated mannan oligosaccharides (cMOS) on fish performance, biochemical composition, tissue fatty acid profiles, liver and posterior gut morphology and gen expression of selected parameters involved on the intestinal immune response and liver lipid metabolism of European sea bass (Dicentrarchus labrax). For that purpose, specimens of 20 g were fed during 8 weeks at 0 and 1.6 g kg(-1) dietary cMOS of inclusion in a commercial sea bass diet. Dietary cMOS enhanced fish length, specific and relative growth without affecting tissue proximate composition. However, cMOS supplementation altered especially liver and muscle fatty acid profiles by reducing levels of those fatty acids that are preferential substrates for β-oxidation in spite of a preferential retention of long chain polyunsaturated fatty acids (LC-PUFA), such as 20:4n-6 or 22:5n-6, in relation to the down-regulation of delta 6/5 desaturase gene expression found in liver. Besides, dietary cMOS supplementation reduced posterior gut intestinal folds width and induced changes on the gene expression level of certain immune-related genes mainly by down regulating transforming growth factor β (TGFβ) and up-regulating immunoglobulin (Ig), major histocompatibility complex class II (MHCII), T cell receptor β (TCRβ) and Caspase 3 (Casp-3). Thus, dietary cMOS inclusion at 0.16% promoted European sea bass specific growth rate and length, stimulated selected cellular GALT-associated parameters and affected lipid metabolism in muscle and liver pointing to a higher LC-PUFA accumulation and promoted β-oxidation.

Development of the first standardised panel of two new microsatellite multiplex PCRs for gilthead seabream (Sparus aurata L.).

The high number of multiplex PCRs developed for gilthead seabream (Sparus aurata L.) from many different microsatellite markers does not allow comparison among populations. This highlights the need for developing a reproducible panel of markers, which can be used with safety and reliability by all users. In this study, the first standardised panel of two new microsatellite multiplex PCRs was developed for this species. Primers of 138 specific microsatellites from the genetic linkage map were redesigned and evaluated according to their genetic variability, allele size range and genotyping reliability. A protocol to identify and classify genotyping errors or potential errors was proposed to assess the reliability of each marker. Two new multiplex PCRs from the best assessed markers were designed with 11 markers in each, named SMsa1 and SMsa2 (SuperMultiplex Sparus aurata). Three broodstocks (59, 47 and 98 breeders) from different Spanish companies, and a sample of 80 offspring from each one, were analysed to validate the usefulness of these multiplexes in the parental assignation. It was possible to assign each offspring to a single parent pair (100% success) using the exclusion method with SMsa1 and/or SMsa2. In each genotyped a reference sample (Ref-sa) was used, and its DNA is available on request similar to the kits of bin set to genotype by genemapper (v.3.7) software (kit-SMsa1 and kit-SMsa2). This will be a robust and effective tool for pedigree analysis or characterisation of populations and will be proposed as an international panel for this species.

Inheritance of skeletal deformities in gilthead seabream (Sparus aurata) - lack of operculum, lordosis, vertebral fusion and LSK complex.

Morphological abnormalities in farmed gilthead seabream (Sparus aurata) are a major problem as it entails significant economic losses. In this study, 3 large scale experiments under different conditions of spawning, offspring handling and breeders phenotype were performed to analyze the inheritance of 4 types of deformities in this species: lack of operculum, lordosis, vertebral fusion, which are 3 of the most important skeletal deformities, and LSK, which is a consecutive repetition of lordosis/scoliosis/kyphosis. In Exp. [1] (mass spawning and fingerling sorting), 900 fish were analyzed at 509 d post-hatching: 846 fish that had been on-grown in a farm and 54 LSK-deformed fish that had been reared separately after being selected during the fingerling sorting process. A total of 89 families were represented. A statistically significant association between 5 of these families (from 6 breeders) and LSK-deformed fish was found. In Exp. [2] (mass spawning and no fingerling sorting), 810 fish were analyzed at 2 ages: 179 and 689 d post-hatching. Significant relationships between 2 of the breeders and 2 of the families with the lack of operculum prevalence of their descendants were found at 689 d but not at 179 d. Heritabilities: 0.09 ± 0.09 at 179 d and 0.17 ± 0.08 at 689 d. Column deformities prevalence was low and no association with family was observed. Family relationships were determined by microsatellites multiplex PCR in both experiments. In Exp. [3] (designed mating), sires suffering from lordosis or lack of operculum or vertebral fusion deformities were mated with non-deformed dams and a mass-spawning mating was considered as a control. After analyzing 11,503 offspring at 159 d post-hatching, a significant relationship between each deformity prevalence and the mating of breeders suffering from the same deformity was observed. In addition, a significant prevalence of lack of operculum in offspring from lordotic matings was observed. Heritabilities ranged from 0.34 to 0.46 for the 3 deformities. The results of the present study suggest that these deformities have a genetic origin. They also suggest that the sorting process is not recommended and that producers should consider these deformities in genetic breeding programs to significantly improve their fish morphological quality and to minimize farmed fish deformities incidence.

Genetic parameters and genotype–environment interactions for skeleton deformities and growth traits at different ages on gilthead seabream (Sparus aurata L.) in four Spanish regions

One of the most important problems of fish aquaculture is the high incidence of fish deformities, which are mainly skeletal. In this study, genetic parameters on gilthead seabream (Sparus aurata L.) for skeleton deformities at different ages (179, 269, 389, 539 and 689 days) and their correlations with growth traits were estimated, as were as their genotype × environment interactions (G × E) at harvesting age. A total of 4093 offspring from the mass spawning of three industrial broodstocks belonging to the PROGENSA(®) breeding programme were mixed and on-grown by different production systems in four Spanish regions: Canary Islands (tanks and cage), Andalusia (estuary), Catalonia (cage) and Murcia (cage). Parental assignment was inferred using the standardized SMsa1 microsatellite multiplex PCR. From three broodstocks, 139 breeders contributed to the spawn and a total of 297 full-sibling families (52 paternal and 53 maternal half-sibling families) were represented. Heritabilities at different ages were medium for growth traits (0.16-0.48) and vertebral deformities (0.16-0.41), and low for any type of deformity (0.07-0.26), head deformities (0.00-0.05) and lack of operculum (0.06-0.11). The genetic correlations between growth and deformity traits were medium and positive, suggesting that to avoid increasing deformities they should be taken into account in breeding programmes when growth is selected. The G × E interactions among the different facilities were weak for length and deformity and strong for growth rate during this period. These results highlight the potential for the gilthead seabream industry to reduce the prevalence of deformities by genetic improvement tools.

Disease resistance and response against Vibrio anguillarum intestinal infection in European seabass (Dicentrarchus labrax) fed low fish meal and fish oil diets

Abstract The aim of this study was to assess the effects of low levels of dietary fish meal (FM) and fish oil (FO) on disease resistance and gut associated lymphoid tissue (GALT) response after an experimental intestinal infection with V. anguillarum in European sea bass (Dicentrarchus labrax) For that purpose, sea bass juveniles were fed one of four diets containing combined levels of FO and FM as follows: 20%FM/6%FO, 20%FM/3%FO, 5%FM/6%FO and 5%FM/3%FO during 153 days. At the end of the feeding trial, fish were subjected to either an in vivo exposure to a sub‐lethal dose of V. anguillarum via anal inoculation or to an ex vivo exposure to V. anguillarum. Additionally, inducible nitric oxide synthase (iNOS) and tumor necrosis factor &agr; (TNF&agr;) gut patterns of immunopositivity were studied. Growth performance was affected by dietary FM level, however ex vivo gut bacterial translocation rates and survival after the in vivo challenge test were affected by dietary FO level. After 5 months of feeding, low dietary FM levels led to a posterior gut up‐regulation of interleukin‐1&bgr; (IL‐1&bgr;) and TNF&agr;, major histocompatibility complex‐II (MHCII) and cyclooxygenase‐2 (COX2), which in turn reduced the gut associated lymphoid tissue (GALT) capacity of response after 24 h post infection and conditioned European sea bass capacity to recover gut homeostasis 7 days post infection. Immunoreactivity to anti‐iNOS and anti‐TNF&agr; presented a gradient of increased immunopositivity towards the anus, regardless of the dietary FM/FO fed. Strong positive anti‐TNF&agr; isolated enterocytes were observed in the anterior gut in relation to low levels of dietary FM/FO. Submucosa and lamina propria immunoreactivity grade was related to the amount of leucocyte populations infiltrated and goblet cells presented immunopositivity to anti‐iNOS but not to anti‐TNF&agr;. Thus, reducing FO content from 6% to a 3% by VO in European sea bass diets increases ex vivo and in vivo gut bacterial translocation rates, whereas reducing FM content from 20% down to 5% up‐regulates the expression of several posterior gut inflammation‐related genes conditioning fish growth and GALT capacity of response after bacterial infection. HighlightsReducing dietary FO levels from 6% to a 3% increases ex vivo and in vivo gut bacterial translocation rates.Diminishing FM dietary content from 20% to 5% up‐regulates several posterior gut inflammation‐related genes.Decreasing FM dietary content of FM from 20% to 5% limits European sea bass growth performance.FM or FO dietary content did not influence gut iNOS and TNF&agr; immunolabelling along the gut.Strong positive anti‐TNF&agr; isolated epithelial cells similar to antigen‐sampling cells were observed particularly in anterior gut.

Effects of thermal stress on the expression of glucocorticoid receptor complex linked genes in Senegalese sole (Solea senegalensis): Acute and adaptive stress responses

The present study examined the short and mid-term effects of a rise in temperature from 18°C to 24°C on the expression of genes related to the stress response regulation in juveniles of Senegalese sole, Solea senegalensis. The animals were exposed to a temperature increase of 6°C, after 1month of acclimation at 18°C. After this process, samples of different tissues were collected from a total of 96 fish at four sampling points: 1h, 24h, 3days and 1week. The transcript levels of a set of genes involved in the stress response such as glucocorticoid receptors 1 and 2, corticotrophin-releasing factor, corticotrophin-releasing factor binding proteins, proopiomelanocortin A and B, and cellular stress defense (heat shock protein 70, 90AA and 90AB) were quantified at these sampling points. Additionally, blood samples were also taken to measure the circulating plasma cortisol concentration. Thermal stress induced by increasing temperature prompted an elevation of plasma cortisol levels in juvenile Senegalese sole after 1h as a short-term response, and a consecutive increase after one week, as a mid-term response. Senegalese sole seemed to respond positively in terms of adaptive mechanisms, with a rapid over-expression of grs and hsps in liver and brain, significantly higher after one hour post stress, denoting the fast and acute response of those tissues to a rapid change on temperature. The ratio hsp90/gr also increased 24h after thermal shock, ratio proposed to be an adaptive mechanism to prevent proteosomal degradation of GR. As a mid-term response, the elevation of brain crfbp gene expression one week after thermal shock could be an adaptive mechanism of negative feedback on HPI axis. Taken together, these data suggested an initial up-regulation of the glucocorticoid receptor complex linked genes in response to a temperature increase in Senegalese sole, with heat shock protein 90 potentially being a regulatory factor for the glucocorticoid receptor in the presence of cortisol.

Quantitative trait loci for a neurocranium deformity, lack of operculum, in gilthead seabream (Sparus aurata L.).

Lack of operculum, a neurocranial deformity, is the most common external abnormality to be found among industrially produced gilthead seabream (Sparus aurata L.), and this entails significant financial losses. This study conducts, for the first time in this species, a quantitative trait loci (QTL) analysis of the lack of operculum. A total of 142 individuals from a paternal half-sibling family (six full-sibling families) were selected for QTL mapping. They had previously shown a highly significant association with the prevalence of lack of operculum in a segregation analysis. All the fish were genotyped for 106 microsatellite markers using a set of multiplex PCRs (ReMsa1-ReMsa13). A linear regression methodology was used for the QTL analysis. Four QTL were detected for this deformity, two of which (QTLOP1 and QTLOP2) were significant. They were located at LG (linkage group) nine and LG10 respectively. Both QTL showed a large effect (about 27%), and furthermore, the association between lack of operculum and sire allelic segregation observed was statistically significant in the QTLOP1 analysis. These results represent a significant step towards including marker-assisted selection for this deformity in genetic breeding programmes to reduce the incidence of the deformity in the species.