María J. Zamorano

Starvation before slaughtering as a tool to keep freshness attributes in gilthead sea bream (Sparus aurata)

Instrumental quality parameters related to freshness were evaluated for gilthead sea bream (Sparus aurata) after different storage periods on ice (2, 4, 7 and 11 days of storage) for one, four and eight days of pre-slaughter starvation. The study was carried out on 360 gilthead sea bream from five different groups reared on a commercial fish farm. Starvation period had a significant effect on fish texture: the force necessary to compress 4 mm of flesh after two days ice storage in 1 day starved fish (16.50 ± 0.59 Newtons) was less than that for four day (18.98 ± 0.63 Newtons) and eight day starved fish (20.43 ± 1.12 Newtons). Significant differences in flesh puncture and flesh compression were not detected, although these values did increase with increasing starvation period. A significant increase in pH and myofibrilar protein solubility was also detected for fish starved for one day.

Total substitution of fish oil by vegetable oils in Senegalese sole (Solea senegalensis) diets: effects on fish performance, biochemical composition, and expression of some glucocorticoid receptor-related genes

To study the substitution of fish oil by vegetable oils in fish diets, juveniles Senegalese sole (Solea senegalensis) were fed diets (56xa0% crude protein, 12xa0% crude lipid) containing either linseed (100LO) or soybean (100SO) oils in comparison with a 100xa0% fish oil-based diet (100FO) for 90xa0days. Samples of muscle, liver, and intestine were collected for biochemical analysis and for glucocorticoid receptor-related genes, including GR1 and GR2, and the associated heat shock proteins HSP70, HSP90AA, and HSP90AB. Besides, basal levels of plasma cortisol were also determined. After the feeding period, a stress test, consisting on 5xa0min of net chasing, was applied to a selected population of each dietary group. Total replacement of fish oil by vegetable oils did not induced changes in fish growth and performance, but affected fatty acid profile of muscle, liver, and intestine, reflecting those tissues the characteristic fatty acids of each type of dietary oil. A tendency to conserve the ARA/EPA ratio could be observed in the different tissues, despite of the level of these fatty acids in diet. Chasing stress induced an increase of muscle GR1 and a reduction in intestinal GR2 relative expressions at any of the experimental diets assayed. In liver, chasing stress induced an increase in both GR1 and GR2 gene expression in fish fed fish oil diets. Similarly, chasing stress induced an increase of muscle HSP70 and decrease of HSP90AB in liver at any of the experimental diet assayed. Besides, vegetable oils decreased the expression of HSP70 in intestine, being the relative expression of liver HSP90AA increased by the inclusion of linseed oil in the diet, at any of the experimental conditions assayed.

Dietary vegetable oils: effects on the expression of immune-related genes in Senegalese sole (Solea senegalensis) intestine.

The decreased availability of fish oil, traditionally used as oil source in marine aquafeeds, has lead to the search for alternatives oils. Vegetable oils (VO) are being extensively used as lipid sources in marine fish diets, inducing an imbalance on certain dietary fatty acids. Alteration on the dietary ratio of w-6/w-3 has been described to have detrimental effects on fish immunity. Senegalese sole has high susceptibility to stress and diseases, and little is known on the effects of dietary VO on its immunity. In this study, Senegalese sole juveniles were fed diets (56% crude protein, 12% crude lipid) containing linseed (100LO), soybean (100SO) or fish (100FO) oils as unique oil source. Growth, cortisol and intestinal fatty acid composition were determined after 90 days. Moreover, at the final of the experiment a stress test (5 min of net chasing) was carried out. To evaluate the effect of diets and stress on intestine immunology, expression profiles of a set of 53 immune-related genes using RT-qPCR was also performed. The use of VO did not induced changes in fish growth, but affected fatty acid profile of intestine and expression of immune-related genes. The use of SO (rich in n-6 fatty acids) induced an over-expression of those genes related to complement pathway, recognizing pathogen associated to molecular patterns, defensive response against bacteria, defensive response against viruses, antigen differentiation, cytokines and their receptors. This general over-expression could indicate an activation of inflammatory processes in fish gut. When a stress was applied, a decrease of mRNA levels of different immune-related genes with respect to the unstressed control could be observed in fish fed 100FO. However, fish fed 100LO, with a higher ALA/LA ratio, seemed to ameliorate the effects of combined effects of FO substitution plus stressful situation whereas fish fed 100SO did not show this type of response.

Evaluation of VIE (Visible Implant Elastomer) and PIT (Passive Integrated Transponder) physical tagging systems for the identification of red porgy fingerlings (Pagrus pagrus)

The effect of two physical tagging systems, Visible Implant Elastomer (VIE) and Passive Integrated Transponder (PIT), was evaluated in red porgy (Pagrus pagrus) fingerlings, at different sizes. For VIE tagging, the weight classes were <1 and 1–5xa0g and no tag effect was detected on fish growth. There was a significant difference in mortality between tagged and untagged <1xa0g fish, but not for the 1–5xa0g fish. The tag loss rate was null for all sizes, however, tags showed fragmentation. This partial tag loss was evaluated and quantified. Seven VIE colours were compared and arranged from best to worst visibility: green, red, pink, orange, yellow, white and blue. For PIT tagging, 2–6xa0g, 5–10xa0g and 10–20xa0g weight classes were analysed. There was no significant tag effect on fish growth, for any size, nor on mortality from 10xa0g. Tag loss rate ranged from 2.9 to 5.9%. These results demonstrate that, in red porgy, VIE is a successful tagging system from 2xa0g onwards whereas PIT system is successful from 10xa0g onwards. The combination of both systems allows traceability of fish from a very small size on, which is necessary for the implementation of genetic breeding programmes.

Modulation of the expression of components of the stress response by dietary arachidonic acid in European sea bass (Dicentrarchus labrax) larvae

This study reports for the first time on European sea bass, Dicentrarchus labrax (L.), larvae, the effect of different levels of dietary arachidonic acid (ARA; 20:4n-6) on the expression of genes related to the fish stress response. Copies of mRNA from genes related to steroidogenesis [StAR (steroidogenic acute regulatory protein), c-Fos, and CYP11β (11β-hydroxylase gene)], glucocorticoid receptor complex [GR (glucocorticoid receptor) and HSP (heat shock proteins) 70 and 90) and antioxidative stress (catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase] were quantified. Eighteen day-old larvae were fed for 14xa0days with three experimental diets with increasing levels of ARA (0.3, 0.6 and 1.2xa0% d.w.) and similar levels of docosahexaenoic (22:6n-3) and eicosapentaenoic (20:5n-3) acids (5 and 3xa0%, respectively). The quantification of stress-related genes transcripts was conducted by One-Step TaqMan real time RT-PCR with the standard curve method (absolute quantification). Increase dietary levels of ARA induced a significantly (pxa0<xa00.05) down-regulation of genes related to cortisol synthesis, such as StAR and CYP11β and up-regulated genes related to glucocorticoid receptor complex, such as HSP70 and GR. No effects were observed on antioxidant enzymes gene expression. These results revealed the regulatory role of dietary ARA on the expression of stress-related genes in European sea bass larvae.

Modulation of adrenocorticotrophin hormone (ACTH)-induced expression of stress-related genes by PUFA in inter-renal cells from European sea bass (Dicentrarchus labrax).

Dietary fatty acids have been shown to exert a clear effect on the stress response, modulating the release of cortisol. The role of fatty acids on the expression of steroidogenic genes has been described in mammals, but little is known in fish. The effect of different fatty acids on the release of cortisol and expression of stress-related genes of European sea bass (Dicentrarchus labrax) head kidney, induced by a pulse of adenocorticotrophin hormone (ACTH), was studied. Tissue was maintained in superfusion with 60 min of incubation with EPA, DHA, arachidonic acid (ARA), linoleic acid or α-linolenic acid (ALA) during 490 min. Cortisol was measured by RIA. The quantification of stress-related genes transcripts was conducted by One-Step TaqMan real-time RT-PCR. There was an effect of the type of fatty acid on the ACTH-induced release of cortisol, values from ALA treatment being elevated within all of the experimental period. The expression of some steroidogenic genes, such as the steroidogenic acute regulatory protein (StAR) and c-fos, were affected by fatty acids, ALA increasing the expression of StAR after 1 h of ACTH stimulation whereas DHA, ARA and ALA increased the expression of c-fos after 20 min. ARA increased expression of the 11β-hydroxylase gene. Expression of heat shock protein 70 (HSP70) was increased in all the experimental treatments except for ARA. Results corroborate previous studies of the effect of different fatty acids on the release of cortisol in marine fish and demonstrate that those effects are mediated by alteration of the expression of steroidogenic genes.

Identification of Quantitative Trait Loci Associated with the Skeletal Deformity LSK complex in Gilthead Seabream (Sparus aurata L.)

Morphological abnormalities, especially skeletal deformities, are some of the most important problems affecting gilthead seabream (Sparus aurata L.) aquaculture industry. In this study, a QTL analysis for LSK complex deformity in gilthead seabream is reported. LSK complex is a severe deformity consisting of a consecutive repetition of three vertebral deformities: lordosis, scoliosis, and kyphosis. Seventy-eight offspring from six breeders from a mass-spawning were analyzed: five full-sibling families, three maternal, and two paternal half-sibling families. They had shown a significant association with the LSK complex prevalence in a previous segregation analysis. Fish were genotyped using a set of multiplex PCRs (ReMsa1-13), which includes 106 microsatellite markers. Two methods were used to perform the QTL analysis: a linear regression with the GridQTL software and a linear mixed model with the Qxpak software. A total of 18 QTL were identified. Four of them (QTLSK3, 6, 12, and 14), located in LG5, 8, 17, and 20, respectively, were the most solid ones. These QTL were significant at genome level and showed an extremely large effect (>35xa0%) with both methods. Markers close to the identified QTL showed a strong association with phenotype. Two of these molecular markers (DId-03-T and Bt-14-F) were considered as potential linked-to-this-deformity markers. The detection of these QTL supposes a critical step in the implementation of marker-assisted selection in this species, which could decrease the incidence of this deformity and other related deformities. The identification of these QTL also represents a major step towards the study of the etiology of skeletal deformities in this species.

Inorganic, organic, and encapsulated minerals in vegetable meal based diets for Sparus aurata (Linnaeus, 1758)

Substituting fishmeal (FM) with vegetable meal (VM) can markedly affect the mineral composition of feeds, and may require additional mineral supplementation. Their bioavailability and optimal supplementation levels depend also on the form of delivery of minerals. The aim of the study was to determine the effect of different delivery forms of three major trace elements (Zn, Mn and Se) in a marine teleost. Gilthead sea bream juveniles of 22.5 g were fed a VM-based diet for 12 weeks that was either not supplemented with these minerals or supplemented with inorganic, organic, or encapsulated inorganic forms of minerals in triplicate and compared to a FM-based diet. Our results showed that mineral delivery form significantly affected the biochemical composition and morphology of posterior vertebrae. Supplementation of VM-based diets with inorganic forms of the target minerals significantly promoted growth, increased the vertebral weight and content of ash and Zn, enhanced bone mineralization and affected the vertebral shape. Conversely, encapsulation of inorganic minerals reduced fish growth and vertebral mineral content, whereas supplementation of organic minerals, enhanced bone osteogenesis by upregulating bone morphogenetic protein 2 (bmp2) gene and produced vertebrae with a larger length in relation to height. Furthermore, organic mineral forms of delivery downregulated the expression of oxidative stress related genes, such as Cu/Zn superoxide dismutase (Cu/Zn sod) and glutathione peroxidase 1 (gpx-1), suggesting thus that dietary minerals supplemented in the organic form could be reasonably considered more effective than the inorganic and encapsulated forms of supply.