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Background: Broodstock nutritional programming improves the offspring utilization of plant-based diets in gilthead sea bream through changes in hepatic metabolism. Attention was initially focused on fatty acid desaturases, but it can involve a wide range of processes that remain largely unexplored. How all this can be driven by a different genetic background is hardly underlined, and the present study aimed to assess how broodstock nutrition affects differentially the transcriptome and genome-wide DNA methylome of reference and genetically selected fish within the PROGENSA® selection program., Results: After the stimulus phase with a low fish oil diet, two offspring subsets of each genetic background received a control or a FUTURE-based diet. This highlighted a different hepatic transcriptome (RNA-seq) and genome-wide DNA methylation (MBD-seq) pattern depending on the genetic background. The number of differentially expressed transcripts following the challenge phase varied from 323 in reference fish to 2,009 in genetically selected fish. The number of discriminant transcripts, and associated enriched functions, were also markedly higher in selected fish. Moreover, correlation analysis depicted a hyper-methylated and down-regulated gene expression state in selected fish with the FUTURE diet, whereas the opposite pattern appeared in reference fish. After filtering for highly represented functions in selected fish, 115 epigenetic markers were retrieved in this group. Among them, lipid metabolism genes (23) were the most reactive following ordering by fold-change in expression, rendering a final list of 10 top markers with a key role on hepatic lipogenesis and fatty acid metabolism (cd36, pitpna, cidea, fasn, g6pd, lipt1, scd1a, acsbg2, acsl14, acsbg2)., Conclusions: Gene expression profiles and methylation signatures were dependent on genetic background in our experimental model. Such assumption affected the magnitude, but also the type and direction of change. Thus, the resulting epigenetic clock of reference fish might depict an older phenotype with a lower methylation for the epigenetically responsive genes with a negative methylation-expression pattern. Therefore, epigenetic markers will be specific of each genetic lineage, serving the broodstock programming in our selected fish to prevent and mitigate later in life the risk of hepatic steatosis through changes in hepatic lipogenesis and fatty acid metabolism., (© 2023. The Author(s).)

Genetic selection programmes in gilthead seabream mainly focus on traits related to growth, disease resistance, skeletal anomalies, or fillet quality. However, the effect of selection for growth on the reproductive performance of seabream broodstock has not received much attention. The present study aimed to determine the effect of selection for growth traits, high (HG) or low (LG) growth, and broodstock feeding with fish oil (FO diet) or rapeseed oil (RO diet) as main lipid sources, on reproductive performance of gilthead seabream. For the first part of the spawning season (Phase I) HG and LG broodstock were fed a commercial diet and the HG broodstock produced a higher number of larvae and higher viable eggs, hatching and larval survival rates than LG broodstock, affecting egg fatty acid profiles. For the second part of the study (Phase II) broodstock were fed one of the two diets containing FO or RO. Fecundity in terms of viable eggs, hatchlings, and larvae produced, as well as fertilization rates, were improved in HG broodstock. Some fatty acids such as 18:0, 20:2n-6, 20:3n-3 or EPA/ARA were also affected by the growth selection. According to the two-way ANOVA analysis, feeding the RO diet did not significantly affect fecundity parameters, but slightly reduced fertilization and hatching rates in HG broodstock. Nevertheless, HG broodstock showed better spawning quality parameters than LG broodstock, even when they were fed the RO diet. Egg fatty acid profiles reflected diet composition, although DHA contents were not affected. In conclusion, broodstock selected for high growth had a positive effect on broodstock performance, and FO replacement by RO did not markedly affect reproduction providing that fatty acid contents were sufficient to fulfill the essential fatty acid requirements of gilthead seabream broodstock., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Several amino acids (AA) are known to regulate key metabolic pathways that are crucial for immune responses. In particular, arginine (ARG) appears to have important roles regarding immune modulation since it is required for macrophage responses and lymphocyte development. Moreover, citrulline (CIT) is a precursor of arginine, and it was reported as an alternative to ARG for improving macrophage function in mammals. The present study aimed to explore the effects of dietary ARG and CIT supplementation on the gilthead seabream ( Sparus aurata ) immune status. Triplicate groups of fish (23.1 ± 0.4 g) were either fed a control diet (CTRL) with a balanced AA profile, or the CTRL diet supplemented with graded levels of ARG or CIT (i.e., 0.5 and 1% of feed; ARG1, CIT1, ARG2, and CIT2, respectively). After 2 and 4 weeks of feeding, fish were euthanized and blood was collected for blood smears, plasma for humoral immune parameters and shotgun proteomics, and head-kidney tissue for the measurement of health-related transcripts. A total of 94 proteins were identified in the plasma of all treatments. Among them, components of the complement system, apolipoproteins, as well as some glycoproteins were found to be highly abundant. After performing a PLS of the expressed proteins, differences between the two sampling points were observed. In this regard, component 1 (61%) was correlated with the effect of sampling time, whereas component 2 (18%) seemed associated to individual variability within diet. Gilthead seabream fed ARG2 and CIT2 at 4 weeks were more distant than fish fed all dietary treatments at 2 weeks and fish fed the CTRL diet at 4 weeks. Therefore, data suggest that the modulatory effects of AA supplementation at the proteome level were more effective after 4 weeks of feeding and at the higher inclusion level (i.e., 1% of feed). The bactericidal activity increased in fish fed the highest supplementation level of both AAs after 4 weeks. Peripheral monocyte numbers correlated positively with nitric oxide, which showed an increasing trend in a dose-dependent manner. The colony-stimulating factor 1 receptor tended to be up-regulated at the final sampling point regardless of dietary treatments. Data from this study point to an immunostimulatory effect of dietary ARG or CIT supplementation after 4 weeks of feeding in the gilthead seabream, particularly when supplemented at a 1% inclusion level., (Copyright © 2020 Ramos-Pinto, Azeredo, Silva, Conceição, Dias, Montero, Torrecillas, Silva and Costas.)

The present study investigated the effects of nutritional programming through parental feeding on offspring performance and expression of selected genes related to stress resistance in a marine teleost. Gilthead seabream broodstock were fed diets containing various fish oil (FO)/vegetable oil ratios to determine their effects on offspring performance along embryogenesis, larval development and juvenile on-growing periods. Increased substitution of dietary FO by linseed oil (LO) up to 80 % LO significantly reduced the total number of eggs produced by kg per female per spawn. Moreover, at 30 d after hatching, parental feeding with increasing LO up to 80 % led to up-regulation of the fatty acyl desaturase 2 gene (fads2) that was correlated with the increase in conversion rates of related PUFA. Besides, cyclo-oxygenase 2 (cox2) and TNF-α (tnf-α) gene expression was also up-regulated by the increase in LO in broodstock diets up to 60 or 80 %, respectively. When 4-month-old offspring were challenged with diets having different levels of FO, the lowest growth was found in juveniles from broodstock fed 100 % FO. An increase in LO levels in the broodstock diet up to 60LO raised LC-PUFA levels in the juveniles, regardless of the juvenile's diet. The results showed that it is possible to nutritionally programme gilthead seabream offspring through the modification of the fatty acid profiles of parental diets to improve the growth performance of juveniles fed low FO diets, inducing long-term changes in PUFA metabolism with up-regulation of fads2 expression. The present study provided the first pieces of evidence of the up-regulation of immune system-related genes in the offspring of parents fed increased FO replacement by LO.

Nutrition during periconception and early development can modulate metabolic routes to prepare the offspring for adverse conditions through a process known as nutritional programming. In gilthead sea bream, replacement of fish oil (FO) with linseed oil (LO) in broodstock diets improves growth in the 4-month-old offspring challenged with low-FO and low-fishmeal (FM) diets for 1 month. The present study further investigated the effects of broodstock feeding on the same offspring when they were 16 months old and were challenged for a second time with the low-FM and low-FO diet for 2 months. The results showed that replacement of parental moderate-FO feeding with LO, combined with juvenile feeding at 4 months old with low-FM and low-FO diets, significantly (P<0·05) improved offspring growth and feed utilisation of low-FM/FO diets even when they were 16 months old: that is, when they were on the verge of their first reproductive season. Liver fatty acid composition was significantly affected by broodstock or reminder diets as well as by their interaction. Moreover, the reduction of long-chain PUFA and increase in α-linolenic acid and linoleic acid in broodstock diets lead to a significant down-regulation of hepatic lipoprotein lipase (P<0·001) and elongation of very long-chain fatty acids protein 6 (P<0·01). Besides, fatty acid desaturase 2 values were positively correlated to hepatic levels of 18 : 4n-3, 18 : 3n-6, 20 : 5n-3, 22 : 6n-3 and 22 : 5n-6. Thus, this study demonstrated the long-term nutritional programming of gilthead sea bream through broodstock feeding, the effect of feeding a 'reminder' diet during juvenile stages to improve utilisation of low-FM/FO diets and fish growth as well as the regulation of gene expression along the fish's life-cycle.

Omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA) are essential components of the diet of all vertebrates. The major dietary source of n-3 LC-PUFA for humans has been fish and seafood but, paradoxically, farmed fish are also reliant on marine fisheries for fish meal and fish oil (FO), traditionally major ingredients of aquafeeds. Currently, the only sustainable alternatives to FO are vegetable oils, which are rich in C18 PUFA, but devoid of the eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) abundant in FO. Two new n-3 LC-PUFA sources obtained from genetically modified (GM) Camelina sativa containing either EPA alone (ECO) or EPA and DHA (DCO) were compared to FO and wild-type camelina oil (WCO) in juvenile sea bream. Neither ECO nor DCO had any detrimental effects on fish performance, although final weight of ECO-fed fish (117 g) was slightly lower than that of FO- and DCO-fed fish (130 and 127 g, respectively). Inclusion of the GM-derived oils enhanced the n-3 LC-PUFA content in fish tissues compared to WCO, although limited biosynthesis was observed indicating accumulation of dietary fatty acids. The expression of genes involved in several lipid metabolic processes, as well as fish health and immune response, in both liver and anterior intestine were altered in fish fed the GM-derived oils. This showed a similar pattern to that observed in WCO-fed fish reflecting the hybrid fatty acid profile of the new oils. Overall the data indicated that the GM-derived oils could be suitable alternatives to dietary FO in sea bream.

The mode of action of highly unsaturated fatty acids (HUFA) in regulating gilthead sea bream (Sparus aurata) head kidney (HK) cortisol production was studied through in vitro trials using a dynamic superfusion system. Fish were previously fed with different diets containing several inclusion levels of linseed oil (LO) or soyabean oil (SO) for 26 weeks. Five diets were tested; anchovy oil was the only lipid source for the control diet (fish oil, FO) and two different substitution levels (70 and 100 %) were tested using either LO or SO (70LO, 70SO, 100LO and 100SO). Fatty acid compositions of the HK reflected the dietary input, thus EPA, DHA, arachidonic acid and n-3 HUFA were significantly (P < 0·05) reduced in fish fed vegetable oils compared with fish fed the FO diet. Feeding 70 or 100 % LO increased significantly (P < 0·05) cortisol release in HK after stimulation with adrenocorticotrophic hormone (ACTH), while feeding SO had no effect on this response. Cortisol stimulation factor (SF) was increased in fish fed the 70LO and 100LO diets compared with fish fed the control diet. Moreover, eicosanoid inhibition by incubating the HK tissue with indomethacin (INDO) as a cyclo-oxygenase (COX) inhibitor, or nordihydroguaiaretic acid (NDGA) as a lipoxygenase (LOX) inhibitor, significantly reduced (P < 0·05) the cortisol release after ACTH stimulation in the 70LO and 100LO diets. Cortisol SF was reduced in the FO, 70LO and 100LO diets when incubating the HK with INDO or NDGA, while it was increased in the 70SO diet. The present results indicate that changing the fatty acid profile of gilthead sea bream HK by including LO and/or SO in the fish diet affected the in vitro cortisol release, and this effect is partly mediated by COX and/or LOX metabolites.

Commercial gilthead sea bream feeds are highly energetic, fish oil traditionally being the main lipid source. But the decreased fish oil production together with the increased prices of this oil encourages its substitution by vegetable oils, imposing new nutritional habits to aquaculture species. Partial replacement of fish oil by vegetable oils in diets for marine species allows good feed utilization and growth but may affect fish health, since imbalances in dietary fatty acids may alter fish immunological status. The effect of dietary oils on different aspects of fish immune system has been reported for some species, but very little is known about the effect of dietary oils on immune-related genes expression in fish. Thus, the objective of this study was to elucidate the role of dietary oils on the expression of two pro-inflammatory cytokines, Tumor Necrosis Factor-α (TNF-α) and Interleukine 1β (IL-1β) on intestine and head kidney after exposure to the bacterial pathogen Photobacterium damselae sp. piscicida. For that purpose, 5 iso-nitrogenous and iso-lipidic diets (45% crude protein, 22% crude lipid content) were formulated. Anchovy oil was the only lipid source used in the control diet (FO), but in the other diets, fish oil was totally (100%) or partially (70%) substituted by linseed (rich in n-3 fatty acids) or soybean (rich in n-6 fatty acids) (100L, 100S, 70L, 70S). Fish were fed experimental diets during 80 days and after this period were exposed to an experimental intestinal infection with the pathogen. Serum and tissue samples were obtained at pre-infection and after 1, 3 and 7 days of infection. RNA was extracted and cDNA was synthesized by reverse transcription from intestine and head kidney and the level expression of TNF-α and IL-1β were assayed by using quantitative real time PCR. The expression level of genes analysed was represented as relative value, using the comparative Ct method (2(-ΔΔCt)). Serum anti-bacterial activity was measured as serum bactericidal capacity and lysozyme activity. Reduction of FO tends to reduce basal (pre-infection) genetic expression of both cytokines. However, complete FO replacement caused an over expression of both pro-inflammatory cytokines, particularly after 3 days of induced infection in fish fed soybean oil based diets. On the other hand, fish fed diets with low content of n-6 fatty acids showed better serum bactericidal capacity after infection, suggesting that the substitution of fish oil by vegetable oils containing high levels of n-6 fatty acids may induce imbalances on fish immune response, leading to a lower potential response against infections., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The aim of the present study was to determine the influence of the dietary fatty acid profile on the lipoprotein composition in sea bream fed different vegetable oils. Six experimental diets were formulated combining fish oil with three vegetable oils (soybean, rapeseed, linseed) in order to obtain 60-80 % (w/w) fish-oil replacement. VLDL, LDL and HDL in plasma samples were obtained by sequential centrifugal flotation. The lipid class, protein content and fatty acid composition of each lipoprotein fraction were analysed. HDL was the predominant lipoprotein in sea bream plasma containing the highest proportion of protein (34 %) and phosphatidylcholine. LDL presented a high content of cholesterol, whereas triacylglycerol comprised a larger proportion of VLDL. The lipid class of the lipoprotein fractions was affected by the dietary vegetable oils. Thus, a high dietary inclusion of soyabean and linseed oil (80 %) increased the cholesterol in HDL and LDL in comparison to fish oil. Similarly, the triacylglycerol concentration of VLDL was increased in fish fed 80 % soyabean and linseed oils owing to the low n-3 highly unsaturated fatty acid content of these diets. Lipoprotein fatty acid composition easily responded to dietary fatty acid composition. VLDL was the fraction more affected by dietary fatty acid, followed by LDL and HDL. The n-3 highly unsaturated fatty acid content increased in the order VLDL less than LDL and less than HDL, regardless of dietary vegetable oils.

Despite the good growth performance of several fish species when dietary fish oil is partly replaced by vegetable oils, recent studies have reported several types of intestinal morphological alterations in cultured fish fed high contents of vegetable lipid sources. However, the physiological process implied in these morphological changes have not been clarified yet, since alterations in the physiological mechanisms involved in the different processes of lipid absorption could be responsible for such gut morphological features. The objective of the present study was to investigate the activities of reacylation pathways in fish, the glycerol-3-phosphate and the monoacylglycerol pathways, in order to clarify the intestinal triacylglycerol (TAG) and phospholipid biosynthesis to better understand the morphological alterations observed in the intestine of fish fed vegetable oils. Intestinal microsomes of sea bream fed different lipid sources (fish, soyabean and rapeseed oils) at three different inclusion levels were isolated and incubated with L-[(14)C(U)]glycerol-3-phosphate and [1-(14)C]palmitoyl CoA. The results showed that in this fish species the glycerol-3-phosphate pathway is mainly involved in phospholipid synthesis, whereas TAG synthesis is mainly mediated by the monoacylglycerol pathway. Feeding with rapeseed oil reduced the reacylation activity in both pathways, explaining the high accumulation of lipid droplets in the supranuclear portion of the intestinal epithelium, whereas soyabean oil enhanced phosphatidylcholine synthesis, being associated with the increase in VLDL found in previous studies.

The aim of this study was to investigate the effects of different levels of substitution of fish oil by vegetable oils rich in oleic, linoleic and linolenic acids on gilthead seabream plasma and leukocyte fatty acid compositions and prostaglandin (PG) and leptin production. Juvenile seabream of 24 g initial body mass were fed four iso-energetic and iso-proteic experimental diets for 281 days. Fatty acid composition of plasma lipids was markedly affected by the inclusion of vegetable oils (VO). ARA (arachidonate), EPA (eicosapentaenoate) and DHA (docosahexaenoate) were preferentially incorporated into polar lipids of plasma, and DHGLA (di-homogammalinoleate) accumulated with increased vegetable oil inclusion. Dietary treatments resulted in alterations of DHGLA/ARA ratios, but not ARA/EPA. ARA-derived PGE(2) production in plasma was not affected by vegetable oils, in agreement with similar eicosanoid precursor ratio (ARA/EPA) in leukocytes total lipids and plasma phospholipids among fish fed with the different dietary treatments. Feeding vegetable oils leads to a decrease in plasma EPA which in turn reduced plasma PGE(3) concentration. Moreover, PGE(3) was the major prostaglandin produced in plasma of fish fed fish oil based diet. Such findings point out the importance of EPA as a precursor of prostaglandins in marine fish, at least for the correct function of the blood cells, and correlates well with the predominant role of this fatty acid in immune regulation in this species. A negative correlation was found between plasma PGE(2) and leptin plasma concentration, suggesting that circulating levels of leptin may act as a metabolic signal modulating PGE(2) release. The present study has shown that increased inclusion of vegetable oils in diet for gilthead seabream may profoundly affect the fatty acid composition of plasma and leukocytes, specially HUFA (highly unsaturated fatty acids), and consequently the production of PGE(3), which can be a major PG in plasma. Alteration in the amount and type of PG produced can be at least partially responsible for the changes in the immune system and health parameters of fish fed diets with high inclusion of VO.

This study investigates the effect of dietary vitamin E on juveniles of gilthead seabream under stressful situations, focusing on the effects on growth, haematology, some immune parameters and plasma cortisol as indicators of stress. Two sardine meal-based experimental diets, one of them supplemented with 150 mg of alpha tocopherol kg(-1) of diet (control) and another one without vitamin E supplementation (diet NE), were assayed under two different stress conditions: overcrowding as a chronic stressor (during 15 weeks) and repetitive chasing as an acute repetitive stressor. Low levels of vitamin E in the diet depleted alternative complement pathway activity [from 167.23 U ml(-1) (control fish) down to 100.99 U ml(-1)] and also nonspecific haemagglutination. Also, fish fed a non-supplemented diet showed an elevation of plasma cortisol basal levels without a stressor influence [from 3.91 ng cortisol ml(-1) plasma (control fish) up to 21.70 ng cortisol ml(-1) plasma]. Low levels of vitamin E in the diet also produced an increase of erythrocyte fragility. Under chronic stress, fish fed the vitamin E-deficient diet showed a reduction in growth and survival, and alterations in haematological parameters, such as an additional haemoconcentration in response to overcrowding when compared with control fish. Under repetitive stress, fish fed the vitamin E deficient diet showed faster elevation of plasma cortisol levels in response to stress and a lower survival rate than control fish. Production of oxygen radicals by blood neutrophils was reduced under repetitive stress in fish fed the non-supplemented diet. These results suggest that fish fed the vitamin E-deficient diet had lower stress resistance.

Abstract Gilthead sea bream Sparus aurata Linnaeus, 1758 were subjected to repeated stresses, namely confinement and handling once a month for 5 consecutive months. Plasma cortisol concentration was determined as a primary stress response, and plasma glucose and osmolality, serum agglutination and growth rate were analysed as indicators of the secondary and tertiary effects of stress. The results of the mean values after the five trials showed that most indicators followed a consistent pattern with time. Cortisol and growth rate were positively correlated, and cortisol and osmolality were negatively correlated after stress. Analysis of the individual responses throughout all 5 months showed that 6–20% of the fish displayed a consistently high or low response depending on the parameter analysed. [ABSTRACT FROM AUTHOR]

Abstract: The post mortem degradation processes that take places in fish muscle gradually modify the initial state of freshness. Tissue degradation is accompanied by drastic myofibrillar proteolysis produced as a consequence of the activation of proteolytic enzymes. In this study, structural changes were correlated with the textural properties of sea bream (Sparus aurata) muscle during 14 days of post mortem cold storage through the immunohistochemical detection of muscle filament proteins (desmin, actin and dystrophin), as well as endoproteases (µ-calpain, m-calpain) and their endogenous inhibitor (calpastatin). Actin and desmin were detected in fish muscle as late as 10 days post mortem (dpm) while dystrophin could not be detected after 4 dpm. In contrast, labelling for both calpains and calpastatin persisted during the entire storage period. Fracturability was clearly reduced after 7 dpm, while greatest changes in hardness, gumminess and chewiness were observed during the first week of storage. Our immunohistochemical observations indicate the disappearance of cytoskeleton proteins at different times post mortem. Thus, actin and desmin persist even when the muscle tissue shows a deteriorated appearance and texture, while dystrophin vanishes soon after the death of the fish. Detachment between myofibres and the myocommata was concomitant with the loss of dystrophin and also corresponded to the time when the reduction in flesh hardness observed was most significant. On the contrary, after 4 days of storage, calpain activity remained practically unaltered in refrigerated muscle specimens. [Copyright &y& Elsevier]