Laura Benedito-Palos

High levels of vegetable oils in plant protein-rich diets fed to gilthead sea bream ( Sparus aurata L.): growth performance, muscle fatty acid profiles and histological alterations of target tissues

The feasibility of fish oil (FO) replacement by vegetable oils (VO) was investigated in gilthead sea bream (Sparus aurata L.) in a growth trial conducted for the duration of 8 months. Four isolipidic and isoproteic diets rich in plant proteins were supplemented with L-lysine (0.55 %) and soya lecithin (1 %). Added oil was either FO (control) or a blend of VO, replacing 33 % (33VO diet), 66 % (66VO diet) and 100 % (VO diet) of FO. No detrimental effects on growth performance were found with the partial FO replacement, but feed intake and growth rates were reduced by about 10 % in fish fed the VO diet. The replacement strategy did not damage the intestinal epithelium, and massive accumulation of lipid droplets was not found within enterocytes. All fish showed fatty livers, but signs of lipoid liver disease were only found in fish fed the VO diet. Muscle fatty acid profiles of total lipids reflected the diet composition with a selective incorporation of unsaturated fatty acids in polar lipids. The robustness of the phospholipid fatty acid profile when essential fatty acid requirements were theoretically covered by the diet was evidenced by multivariate principal components analysis in fish fed control, 33VO and 66VO diets.

Deep sequencing for de novo construction of a marine fish (Sparus aurata)transcriptome database with a large coverage of protein-coding transcripts

BackgroundThe gilthead sea bream (Sparus aurata) is the main fish species cultured in the Mediterranean area and constitutes an interesting model of research. Nevertheless, transcriptomic and genomic data are still scarce for this highly valuable species. A transcriptome database was constructed by de novo assembly of gilthead sea bream sequences derived from public repositories of mRNA and collections of expressed sequence tags together with new high-quality reads from five cDNA 454 normalized libraries of skeletal muscle (1), intestine (1), head kidney (2) and blood (1).ResultsSequencing of the new 454 normalized libraries produced 2,945,914 high-quality reads and the de novo global assembly yielded 125,263 unique sequences with an average length of 727 nt. Blast analysis directed to protein and nucleotide databases annotated 63,880 sequences encoding for 21,384 gene descriptions, that were curated for redundancies and frameshifting at the homopolymer regions of open reading frames, and hosted at http://www.nutrigroup-iats.org/seabreamdb. Among the annotated gene descriptions, 16,177 were mapped in the Ingenuity Pathway Analysis (IPA) database, and 10,899 were eligible for functional analysis with a representation in 341 out of 372 IPA canonical pathways. The high representation of randomly selected stickleback transcripts by Blast search in the nucleotide gilthead sea bream database evidenced its high coverage of protein-coding transcripts.ConclusionsThe newly assembled gilthead sea bream transcriptome represents a progress in genomic resources for this species, as it probably contains more than 75% of actively transcribed genes, constituting a valuable tool to assist studies on functional genomics and future genome projects.

Tissue-specific robustness of fatty acid signatures in cultured gilthead sea bream (Sparus aurata L.) fed practical diets with a combined high replacement of fish meal and fish oil.

The present study aimed to determine the tissue-specific robustness of fatty acid (FA) signatures of gilthead sea bream (Sparus aurata L.) by analyzing the changes in lipid class and FA composition of skeletal muscle, brain, liver, and mesenteric adipose tissue. Triplicate groups of fish were fed to visual satiety over a 14-mo production cycle with 2 practical plant protein-based diets formulated with either fish oil or a blend of vegetable oils (66% of fish oil replacement) to contain 53% CP and 21% crude fat on a DM basis. Growth rates (P = 0.22) and tissue lipid class composition were not altered by the dietary treatment (P = 0.34 and 0.52 for neutral lipids and phospholipids, respectively). The FA signatures of neutral lipids reflected the composition of the diet, although the output of principal components analysis revealed a divergent FA profile for liver compared with skeletal muscle, brain, and mesenteric adipose tissue. Because the theoretical EFA needs were met by the 2 diets, the FA composition of phospholipids remained almost unaltered in all tissues. Interestingly, however, the brain showed the greatest robustness and regulatory capacity to preserve the phenotype of fish fed fish oil-based diets. The FA signatures of total lipids are a combinatory result of neutral and polar lipids, and the most relevant fat storage tissues (mesenteric adipose tissue and skeletal muscle) were more easily influenced by dietary FA composition. The present study provides new insights into fish tissue FA composition and reinforces the use of FA signatures as useful criteria in determining whether EFA requirements for a wide range of physiological processes, including those of neural tissues, can be met with practical fish feeds.

Effect of ration size on fillet fatty acid composition, phospholipid allostasis and mRNA expression patterns of lipid regulatory genes in gilthead sea bream (Sparus aurata).

The effect of ration size on muscle fatty acid (FA) composition and mRNA expression levels of key regulatory enzymes of lipid and lipoprotein metabolism have been addressed in juveniles of gilthead sea bream fed a practical diet over the course of an 11-week trial. The experimental setup included three feeding levels: (i) full ration until visual satiety, (ii) 70 % of satiation and (iii) 70 % of satiation with the last 2 weeks at the maintenance ration. Feed restriction reduced lipid content of whole body by 30 % and that of fillet by 50 %. In this scenario, the FA composition of fillet TAG was not altered by ration size, whereas that of phospholipids was largely modified with a higher retention of arachidonic acid and DHA. The mRNA transcript levels of lysophosphatidylcholine acyltransferases, phosphatidylethanolamine N-methyltransferase and FA desaturase 2 were not regulated by ration size in the present experimental model. In contrast, mRNA levels of stearoyl-CoA desaturases were markedly down-regulated by feed restriction. An opposite trend was found for a muscle-specific lipoprotein lipase, which is exclusive of fish lineage. Several upstream regulatory transcriptions were also assessed, although nutritionally mediated changes in mRNA transcripts were almost reduced to PPARα and β, which might act in a counter-regulatory way on lipolysis and lipogenic pathways. This gene expression pattern contributes to the construction of a panel of biomarkers to direct marine fish production towards muscle lean phenotypes with increased retentions of long-chain PUFA.

The nutritional background of the host alters the disease course in a fish-myxosporean system.

The aim of the present work was to determine if a practical plant protein-based diet containing vegetable oils (VO) as the major lipid source could alter the disease course when challenged with the myxosporean Enteromyxum leei, a wide-spread parasite in the Mediterranean basin causing heavy economic losses. Gilthead sea bream (Sparus aurata) fed for 9 months either a fish oil (FO) diet or a blend of VOs at 66% of replacement (66VO diet) were challenged by exposure to parasite-contaminated water effluent. All fish were periodically and non-lethally sampled to obtain biometrical data and to know their infection status. After 102 days of exposure, fish were euthanized and haematological, biometrical, histological, immunological, glutathione and anti-oxidant data were obtained from tissue, blood and serum samples. Anorexia appeared in both exposed groups, but feed intake reduction was higher in 66VO fish. The signs of disease (lower growth, condition factor, specific growth rate, haematocrit) as well as the disease course were worse in fish from 66VO group, with a higher prevalence and intensity of infection, a higher percentage of fish harbouring the parasite in the entire intestinal tract, and a faster establishment of the parasite. Parasite intensity of infection was negatively correlated with growth parameters and haematocrit in both groups, and with complement, lysozyme and hepatic total glutathione in 66VO fish.

Metabolic and transcriptional responses of gilthead sea bream (Sparus aurata L.) to environmental stress: New insights in fish mitochondrial phenotyping

The aim of the current study was to phenotype fish metabolism and the transcriptionally-mediated response of hepatic mitochondria of gilthead sea bream to intermittent and repetitive environmental stressors: (i) changes in water temperature (T-ST), (ii) changes in water level and chasing (C-ST) and (iii) multiple sensory perception stressors (M-ST). Gene expression profiling was done using a quantitative PCR array of 60 mitochondria-related genes, selected as markers of transcriptional regulation, oxidative metabolism, respiration uncoupling, antioxidant defense, protein import/folding/assembly, and mitochondrial dynamics and apoptosis. The mitochondrial phenotype mirrored changes in fish performance, haematology and lactate production. T-ST especially up-regulated transcriptional factors (PGC1α, NRF1, NRF2), rate limiting enzymes of fatty acid β-oxidation (CPT1A) and tricarboxylic acid cycle (CS), membrane translocases (Tim/TOM complex) and molecular chaperones (mtHsp10, mtHsp60, mtHsp70) to improve the oxidative capacity in a milieu of a reduced feed intake and impaired haematology. The lack of mitochondrial response, increased production of lactate and negligible effects on growth performance in C-ST fish were mostly considered as a switch from aerobic to anaerobic metabolism. A strong down-regulation of PGC1α, NRF1, NRF2, CPT1A, CS and markers of mitochondrial dynamics and apoptosis (BAX, BCLX, MFN2, MIRO2) occurred in M-ST fish in association with the greatest circulating cortisol concentration and a reduced lactate production and feed efficiency, which represents a metabolic condition with the highest allostatic load score. These findings evidence a high mitochondrial plasticity against stress stimuli, providing new insights to define the threshold level of stress condition in fish.

Effects of fish oil replacement and re-feeding on the bioaccumulation of organochlorine compounds in gilthead sea bream (Sparus aurata L.) of market size.

Organochlorine pesticide residues and polychlorinated biphenyls were determined in raw materials, fish feeds and fillets from fish exposed through the productive cycle (14 months) to experimental diets with different percentages of fish oil replacement with vegetable oils. Detectable amounts of organochlorine compounds were found in raw materials derived from fish sources with none being detected in vegetable ingredients. Fish feeds presented trace concentrations of contaminants at the ng/g level, which varied according to the contribution of the different resources used in their manufacture. Contaminants did not accumulate during the first 11 months of exposure, and low concentrations of organochlorine compounds were found both at the start and at the end of this feeding period. Fillets from fish fed the fish oil diet presented the highest concentrations of organochlorine compounds, with these decreasing in proportion to fish oil replacement. Three months of fish oil re-feeding during the finishing phase only produced significant bioaccumulation over the course of the first month. By optimizing fish meal and fish oil replacement with vegetable oils alternative feeds can contribute to significantly reduce the risk of organochlorine uptake by consumers.

A reliable analytical approach based on gas chromatography coupled to triple quadrupole and time-of-flight mass analyzers for the determination and confirmation of polycyclic aromatic hydrocarbons in complex matrices from aquaculture activities.

The potential of gas chromatography coupled to tandem mass spectrometry (GC/MS/MS) with a triple quadrupole analyzer (QqQ) has been investigated for the quantification and reliable identification of sixteen polycyclic aromatic hydrocarbons (PAHs) from the EPA priority list in animal and vegetable samples from aquaculture activities, whose fat content ranged from 5 to 100%. Matrices analyzed included fish fillet, fish feed, fish oil and linseed oil. Combining optimized saponification and solid-phase extraction led to high efficiency in the elimination of interfering compounds, mainly fat, from the extracts. The developed procedure minimized the presence of these interfering compounds in the extracts and provided satisfactory recoveries of PAHs. The excellent sensitivity and selectivity of GC/(QqQ)MS/MS in selected reaction monitoring (SRM) allowed to reach limits of detection at pg/g levels. Two SRM transitions were acquired for each analyte to ensure reliable identification of compounds detected in samples. Confirmation of positive findings was performed by GC coupled to high-resolution time-of-flight mass spectrometry (GC/TOFMS). The accurate mass information provided by GC/TOFMS in full acquisition mode together with its high mass resolution makes it a powerful analytical tool for the unequivocal confirmation of PAHs in the matrices tested. The method developed was applied to the analysis of real-world samples of each matrix studied with the result of detecting and confirming the majority of analytes at the microg/kg level by both QqQ and TOF mass spectrometers.

Effects of dietary NEXT ENHANCE®150 on growth performance and expression of immune and intestinal integrity related genes in gilthead sea bream (Sparus aurata L.).

Gilthead sea bream juveniles were fed different doses (0, 50, 100, 200, 300 ppm) of NEXT ENHANCE®150 (NE) for 9 weeks. Feed gain ratio (FGR) was improved by a 10% with all the doses, but feed intake decreased in a dose dependent manner. The optimum inclusion level to achieve maximum growth was set at 100 ppm. The hepatosomatic index did not vary and only at the highest dose, viscerosomatic and splenosomatic indexes were significantly decreased. No significant changes were found in haematological parameters, plasma biochemistry, total antioxidant capacity and respiratory burst. In a second trial, NE was given at 100 ppm alone (D1) or in combination with the prebiotic PREVIDA® (0.5%) (PRE) (D2) for 17 weeks. There were no differences in the growth rates, and FGR was equally improved for D1 and D2. No significant changes in haematology and plasma antioxidant capacity were detected. The histological examination of the liver and the intestine showed no outstanding differences in the liver, but the number of mucosal foldings appeared to be higher in D1 and D2 vs CTRL diet and the density of enterocytes and goblet cells also appeared higher, particularly in the anterior intestine. A 87-gene PCR-array was constructed based on our transcriptomic database (www.nutrigroup-iats.org/seabreamdb) and applied to samples of anterior (AI) and posterior (PI) intestine. It included 54 new gene sequences and other sequences as markers of cell differentiation and proliferation, intestinal architecture and permeability, enterocyte mass and epithelial damage, interleukins and cytokines, pattern recognition receptors (PRR), and mitochondrial function and biogenesis. More than half of the studied genes had significantly different expression between AI and PI segments. The functional significance of this differential tissue expression is discussed. The experimental diets induced significant changes in the expression of 26 genes. The intensity of these changes and the number of genes that were significantly regulated were higher at PI than at AI. At PI, both diets invoked a clear down-regulation of genes involved in cell differentiation and proliferation, some involved in cell to cell communication, cytokines and several PRR. By contrast, up-regulation was mostly found for genes related to enterocyte mass, cell epithelial damage and mitochondrial activity at AI. The changes were of the same order for D1 and D2, except for fatty acid-binding proteins 2 and 6 and the PRR fucolectin, which were higher in D2 and D1 fed fish, respectively. Thus, NE alone or in combination with PRE seems to induce an anti-inflammatory and anti-proliferative transcriptomic profile with probable improvement in the absorptive capacity of the intestine that would explain the improved FGR.

Feed restriction up-regulates uncoupling protein 3 (UCP3) gene expression in heart and red muscle tissues of gilthead sea bream (Sparus aurata L.): New insights in substrate oxidation and energy expenditure

The physiological regulation of the mitochondrial uncoupling protein 3 (UCP3) remains practically unexplored in fish and the aim of this study was to examine the effects of ration size on the regulation of UCP3 in heart, red skeletal muscle and white skeletal muscle of gilthead sea bream (Sparus aurata L.). Juvenile fish were fed at three different levels for 11 weeks: i) full ration until visual satiety (R(100) group), ii) 70% of satiation (R(70) group) and iii) 70% of satiation with two finishing weeks at the maintenance ration (20% of the satiation level) (R(70-20) group). The thirty percent feed restriction improved fish performance, increasing feed conversion efficiency and circulating levels of insulin-like growth factor-I (IGF-I). Fish of the R(70-20) group showed reduced growth and low circulating levels of IGF-I in combination with increased circulating concentrations of growth hormone and free fatty acids. Feed restriction did not alter UCP3 transcript levels in white skeletal muscle, but improved this tissues oxidative capacity as assessed by changes in glycolytic and oxidative mitochondrial enzyme activities. In contrast, in cardiac and red skeletal muscle tissues, this dietary treatment primarily increased UCP3 mRNA expression. The respiratory control ratio of freshly isolated heart mitochondria was slightly lower in R(70-20) fish than in R(100) fish, which suggests that there was an increase in mitochondrial uncoupling concomitant with the enhanced UCP3 mRNA expression. Altogether, these findings highlight the different adaptive mechanism of glycolytic and highly oxidative muscle tissues for their rapid adjustment to varying feed intake.