The osteogenic and mineralogenic potential of the microalgae Skeletonema costatum and Tetraselmis striata CTP4 in fish models.

Skeletal disorders are problematic aspects for the aquaculture industry as skeletal deformities, which affect most species of farmed fish, increase production costs and affect fish welfare. Following recent findings that show the presence of osteoactive compounds in marine organisms, we evaluated the osteogenic and mineralogenic potential of commercially available microalgae strains Skeletonema costatum and Tetraselmis striata CTP4 in several fish systems. Ethanolic extracts increased extracellular matrix mineralization in gilthead seabream (Sparus aurata) bone-derived cell cultures and promoted osteoblastic differentiation in zebrafish (Danio rerio) larvae. Long-term dietary exposure to both extracts increased bone mineralization in zebrafish and upregulated the expression of genes involved in bone formation (sp7, col1a1a, oc1, and oc2), bone remodeling (acp5a), and antioxidant defenses (cat, sod1). Extracts also improved the skeletal status of zebrafish juveniles by reducing the incidence of skeletal anomalies. Our results indicate that both strains of microalgae contain osteogenic and mineralogenic compounds, and that ethanolic extracts have the potential for an application in the aquaculture sector as dietary supplements to support fish bone health. Future studies should also identify osteoactive compounds and establish whether they can be used in human health to broaden the therapeutic options for bone erosive disorders such as osteoporosis.

Dietary Betaine Mitigates Hepatic Steatosis and Inflammation Induced by a High-Fat-Diet by Modulating the Sirt1/Srebp-1/Pparɑ Pathway in Juvenile Black Seabream (Acanthopagrus schlegelii).

The present study aimed to elucidate the mechanism of dietary betaine, as a lipid-lowering substance, on the regulation of lipid metabolism and inflammation in juvenile black seabream (Acanthopagrus schlegelii) fed a high fat diet. An 8-week feeding trial was conducted in black seabream with an initial weight of 8.39 ± 0.01g fed four isonitrogenous diets including Control, medium-fat diet (11%); HFD, high-fat diet (17%); and HFD supplemented with two levels (10 and 20 g/kg) of betaine, HFD+B1 and HFD+B2, respectively. SGR and FE in fish fed HFD+B2 were significantly higher than in fish fed HFD. Liver histology revealed that vacuolar fat droplets were smaller and fewer in bream fed HFD supplemented with betaine compared to fish fed HFD. Betaine promoted the mRNA and protein expression levels of silent information regulator 1 (Sirt1), up-regulated mRNA expression and protein content of lipid peroxisome proliferator-activated receptor alpha (pparα), and down-regulated mRNA expression and protein content of sterol regulatory element-binding protein-1(srebp-1). Furthermore, the mRNA expression levels of anti-inflammatory cytokines in liver and intestine were up-regulated, while nuclear factor kB (nf-kb) and pro-inflammatory cytokines were down-regulated by dietary betaine supplementation. Likewise, in fish that received lipopolysaccharide (LPS) to stimulate inflammatory responses, the expression levels of mRNAs of anti-inflammatory cytokines in liver, intestine and kidney were up-regulated in fish fed HFD supplemented with betaine compared with fish fed HFD, while nf-kb and pro-inflammatory cytokines were down-regulated. This is the first report to suggest that dietary betaine could be an effective feed additive to alleviate hepatic steatosis and attenuate inflammatory responses in black seabream fed a high fat diet by modulating the Sirt1/Srebp-1/Pparɑ pathway.

Medicinal Plant Leaf Extract From Sage and Lemon Verbena Promotes Intestinal Immunity and Barrier Function in Gilthead Seabream (Sparus aurata).

The inclusion of a medicinal plant leaf extract (MPLE) from sage (Salvia officinalis) and lemon verbena (Lippia citriodora), rich in verbascoside and triterpenic compounds like ursolic acid, was evaluated in gilthead seabream (Sparus aurata) fed a low fishmeal-based diet (48% crude protein, 17% crude fat, 21.7 MJ kg-1, 7% fishmeal, 15% fish oil) for 92 days. In particular, the study focused on the effect of these phytogenic compounds on the gut condition by analyzing the transcriptomic profiling (microarray analysis) and histological structure of the intestinal mucosa, as well as the histochemical properties of mucins stored in goblet cells. A total number of 506 differentially expressed genes (285 up- and 221 down-regulated) were found when comparing the transcriptomic profiling of the intestine from fish fed the control and MPLE diets. The gut transcripteractome revealed an expression profile that favored biological mechanisms associated to the 1) immune system, particularly involving T cell activation and differentiation, 2) gut integrity (i.e., adherens and tight junctions) and cellular proliferation, and 3) cellular proteolytic pathways. The histological analysis showed that the MPLE dietary supplementation promoted an increase in the number of intestinal goblet cells and modified the composition of mucins' glycoproteins stored in goblet cells, with an increase in the staining intensity of neutral mucins, as well as in mucins rich in carboxylated and weakly sulfated glycoconjugates, particularly those rich in sialic acid residues. The integration of transcriptomic and histological results showed that the evaluated MPLE from sage and lemon verbena is responsible for the maintenance of intestinal health, supporting gut homeostasis and increasing the integrity of the intestinal epithelium, which suggests that this phytogenic may be considered as a promising sustainable functional additive for aquafeeds.

Carvacrol, Thymol, and Garlic Essential Oil Promote Skin Innate Immunity in Gilthead Seabream (Sparus aurata) Through the Multifactorial Modulation of the Secretory Pathway and Enhancement of Mucus Protective Capacity.

One of the main targets for the use of phytogenics in aquafeeds is the mucosal tissues as they constitute a physical and biochemical shield against environmental and pathogenic threats, comprising elements from both the innate and acquired immunity. In the present study, the modulation of the skin transcriptional immune response, the bacterial growth capacity in skin mucus, and the overall health condition of gilthead seabream (Sparus aurata) juveniles fed a dietary supplementation of garlic essential oil, carvacrol, and thymol were assessed. The enrichment analysis of the skin transcriptional profile of fish fed the phytogenic-supplemented diet revealed the regulation of genes associated to cellular components involved in the secretory pathway, suggesting the stimulation, and recruitment of phagocytic cells. Genes recognized by their involvement in non-specific immune response were also identified in the analysis. The promotion of the secretion of non-specific immune molecules into the skin mucus was proposed to be involved in the in vitro decreased growth capacity of pathogenic bacteria in the mucus of fish fed the phytogenic-supplemented diet. Although the mucus antioxidant capacity was not affected by the phytogenics supplementation, the regulation of genes coding for oxidative stress enzymes suggested the reduction of the skin oxidative stress. Additionally, the decreased levels of cortisol in mucus indicated a reduction in the fish allostatic load due to the properties of the tested additive. Altogether, the dietary garlic, carvacrol, and thymol appear to promote the gilthead seabream skin innate immunity and the mucus protective capacity, decreasing its susceptibility to be colonized by pathogenic bacteria.

Diet, Immunity, and Microbiota Interactions: An Integrative Analysis of the Intestine Transcriptional Response and Microbiota Modulation in Gilthead Seabream (Sparus aurata) Fed an Essential Oils-Based Functional Diet.

Essential oils (EOs) are promising alternatives to chemotherapeutics in animal production due to their immunostimulant, antimicrobial, and antioxidant properties, without associated environmental or hazardous side effects. In the present study, the modulation of the transcriptional immune response (microarray analysis) and microbiota [16S Ribosomal RNA (rRNA) sequencing] in the intestine of the euryhaline fish gilthead seabream (Sparus aurata) fed a dietary supplementation of garlic, carvacrol, and thymol EOs was evaluated. The transcriptomic functional analysis showed the regulation of genes related to processes of proteolysis and inflammatory modulation, immunity, transport and secretion, response to cyclic compounds, symbiosis, and RNA metabolism in fish fed the EOs-supplemented diet. Particularly, the activation of leukocytes, such as acidophilic granulocytes, was suggested to be the primary actors of the innate immune response promoted by the tested functional feed additive in the gut. Fish growth performance and gut microbiota alpha diversity indices were not affected, while dietary EOs promoted alterations in bacterial abundances in terms of phylum, class, and genus. Subtle, but significant alterations in microbiota composition, such as the decrease in Bacteroidia and Clostridia classes, were suggested to participate in the modulation of the intestine transcriptional immune profile observed in fish fed the EOs diet. Moreover, regarding microbiota functionality, increased bacterial sequences associated with glutathione and lipid metabolisms, among others, detected in fish fed the EOs supported the metabolic alterations suggested to potentially affect the observed immune-related transcriptional response. The overall results indicated that the tested dietary EOs may promote intestinal local immunity through the impact of the EOs on the host-microbial co-metabolism and consequent regulation of significant biological processes, evidencing the crosstalk between gut and microbiota in the inflammatory regulation upon administration of immunostimulant feed additives.

Unveiling the effect of dietary essential oils supplementation in Sparus aurata gills and its efficiency against the infestation by Sparicotyle chrysophrii.

A microencapsulated feed additive composed by garlic, carvacrol and thymol essential oils (EOs) was evaluated regarding its protective effect in gills parasitized by Sparicotyle chrysophrii in Sparus aurata. A nutritional trial (65 days) followed by a cohabitation challenge with parasitized fish (39 days) were performed. Transcriptomic analysis by microarrays of gills of fish fed the EOs diet showed an up-regulation of genes related to biogenesis, vesicular transport and exocytosis, leukocyte-mediated immunity, oxidation-reduction and overall metabolism processes. The functional network obtained indicates a tissue-specific pro-inflammatory immune response arbitrated by degranulating acidophilic granulocytes, sustained by antioxidant and anti-inflammatory responses. The histochemical study of gills also showed an increase of carboxylate glycoproteins containing sialic acid in mucous and epithelial cells of fish fed the EOs diet, suggesting a mucosal defence mechanism through the modulation of mucin secretions. The outcomes of the in vivo challenge supported the transcriptomic results obtained from the nutritional trial, where a significant reduction of 78% in the abundance of S. chrysophrii total parasitation and a decrease in the prevalence of most parasitic developmental stages evaluated were observed in fish fed the EOs diet. These results suggest that the microencapsulation of garlic, carvacrol and thymol EOs could be considered an effective natural dietary strategy with antiparasitic properties against the ectoparasite S. chrysophrii.

Reproductive performance of gilthead seabream (Sparus aurata) broodstock showing different expression of fatty acyl desaturase 2 and fed two dietary fatty acid profiles.

Previous studies have shown that it is possible to nutritionally program gilthead seabream offspring through fish oil (FO) replacement by vegetable oils (VO) in the broodstock diet, to improve their ability to grow fast when fed low fish meal (FM) and FO diets during grow-out phase. However, in those studies broodstock performance was reduced by the VO contained diet. Therefore, the present study aimed to determine if it is possible to replace FO by a mixture of FO and rapeseed oil (RO) with a specific fatty acid profile in broodstock diets, without altering gilthead seabream broodstock reproductive performance. Besides, the study also aimed to evaluate the reproductive performance of broodstock with different expression of fatty acid desaturase 2 gene (fads2) a key enzyme in synthesis of long chain polyunsaturated fatty acids. For that purpose, broodfish having either a high (HD) or low (LD) expression of fads2 were fed for three months during the spawning season with two diets containing different fatty acid profiles and their effects on reproductive hormones, fecundity, sperm and egg quality, egg biochemical composition and fads2 expression were studied. The results showed that blood fads2 expression in females, which tended to be higher than in males, was positively related to plasma 17ß-estradiol levels. Moreover, broodstock with high blood fads2 expression showed a better reproductive performance, in terms of fecundity and sperm and egg quality, which was correlated with female fads2 expression. Our data also showed that it is feasible to reduce ARA, EPA and DHA down to 0.43, 6.6 and 8.4% total fatty acids, respectively, in broodstock diets designed to induce nutritional programming effects in the offspring without adverse effects on spawning quality. Further studies are being conducted to test the offspring with low FM and FO diets along life span.

Short-Term Supplementation of Dietary Arginine and Citrulline Modulates Gilthead Seabream (Sparus aurata) Immune Status.

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.

Skin wound healing in gilthead seabream (Sparus aurata L.) fed diets supplemented with arginine.

Dietary administration of arginine on the wound healing process of gilthead seabream was studied. Two replicates of fish (n = 8) were fed with either a commercial diet [control diet (CON), no arginine added] and the CON diet supplemented with 1% arginine (ARG1) or with 2% arginine (ARG2) for 30 days. Afterward, half of the fish were sampled while the other half were injured and continued to be fed the same diet for an extra week. Results by image analysis showed that the wound closure rate was significantly improved in fish that were fed the ARG1 diet, compared with those in the CON group. After seven days of wound healing, the aminotransferase and creatine kinase levels in the serum and the protease and peroxidase activities in the skin mucus were down-regulated, while the immunoglobulin M level in the skin mucus was up-regulated in the ARG1 group after wounding and in the CON group before wounding. Compared with the CON diet, the ARG1 diet remarkedly depressed the gene expression of mpo, il-8, and tnf-α, and enhanced the gene expression of tgf-ß1, igf-1, pcna, krt2, mmp9, fn1α, and colIα and the antioxidant enzyme cat in the skin tissues after wounding. Furthermore, compared with both the ARG1 and the CON groups, negative effects of the ARG2 diet on wound healing were demonstrated. In conclusion, a 1% arginine supplementation facilitates skin wound healing and prevents a systemic inflammation reaction by alleviating the inflammatory response and enhancing the re-epithelialization and ECM biosynthesis in skin wound sites.

Dietary supplementation of drumstick tree, Moringa oleifera, improves mucosal immune response in skin and gills of seabream, Sparus aurata, and attenuates the effect of hydrogen peroxide exposure.

In aquatic animals, the mucosal barrier is the first line of innate immune defence against external chemicals and pathogens. In this study, the effects of dietary Moringa oleifera leaf (MOL) supplementation on skin and gill mucosal immunity, antioxidants and stress responses were evaluated in seabream (Sparus aurata) fingerlings exposed to hydrogen peroxide (H2O2). A total of 144 specimens (10.11 ± 0.41 g) were divided into four treatments (three replicates per treatment contained 12 specimens each) and fed a non-supplemented control diet or a 1, 2.5 or 5% MOL-supplemented diet. After three weeks of feeding, six specimens from each aquarium were sampled for blood, mucus and tissues. The other six fish in each aquarium were subjected to H2O2 exposure. The results revealed that MOL did not negatively affect either cortisol or glucose levels. MOL supplementation significantly (P < 0.05) improved skin mucosal immunity-related characteristics, including phosphatase, peroxidase and lysozyme activity and IgM levels. Additionally, MOL upregulated the expression of antioxidant genes (sod and cat), an anti-inflammatory gene (tgf-ß), tight junction protein genes (occludin and zo-1), c3, and igm in both the skin and gills. However, H2O2 exposure significantly (P < 0.05) increased both cortisol and glucose levels and disrupted skin mucosal immune function by significantly (P < 0.05) decreasing phosphatase, peroxidase, protease, antiprotease and lysozyme activity and IgM levels. H2O2 exposure severely decreased the mRNA levels of the studied genes. MOL dietary supplementation at the 5% level successfully attenuated the negative effects of H2O2 on the mucosal immune response in both the skin and gills. In conclusion, dietary MOL supplementation at the 5% level is recommended to improve S. aurata mucosal immune function under both normal and stress conditions. Additionally, exposure to H2O2 disrupts the mucosal immunity of fish. This contributes knowledge on the routes involved in mucosal innate immunity and could help to understand the fish resistance against chemicals exposure. Graphical abstract.

Molecular genetic characterisation and expression profiling of calpain 3 transcripts in red sea bream (Pagrus major).

Calpains (CAPNs) belong to the papain superfamily of cysteine proteases, and they are calcium-dependent cytoplasmic cysteine proteases that regulate a variety of physiological processes. We obtained the sequence of CAPN3 from an NGS-based analysis of Pagrus major (PmCAPN3) and confirmed the conserved molecular biological properties in the predicted amino acid sequence. The amino acid sequence and predicted domains of CAPN3 were found to be highly conserved in all of the examined species, and one catalytic domain and four calcium binding sites were identified. In healthy P. major, the PmCAPN3 mRNA was most abundantly expressed in the muscle and skin, and ubiquitously expressed in the other tissues used in the experiment. After artificial infections with fish pathogens, significant changes in its expression levels were found in immune-related tissues, most of showed upregulation. In particular, the highest level of expression was found in the liver, a tissue associated with protease activity. Taken together, these results suggest a physiological activity for PmCAPN3 in P. major and reveal functional possibilities that have not yet been reported in the immune system.

Effects of Dietary Lipid Composition and Fatty Acid Desaturase 2 Expression in Broodstock Gilthead Sea Bream on Lipid Metabolism-Related Genes and Methylation of the fads2 Gene Promoter in Their Offspring.

Polyunsaturated fatty acids (PUFA) in parental diets play a key role in regulating n-3 LC-PUFA metabolism of the offspring. However, it is not clear whether this metabolic regulation is driven by the precursors presented in the diet or by the parental ability to synthesize them. To elucidate this, broodstocks of gilthead sea bream with different blood expression levels of fads2, which encodes for the rate-limiting enzyme in the n-3 LC-PUFA synthesis pathway, were fed either a diet supplemented with alpha-linolenic acid (ALA, 18:3n-3) or a control diet. The progenies obtained from these four experimental groups were then challenged with a low LC-PUFA diet at the juvenile stage. Results showed that the offspring from parents with high fads2 expression presented higher growth and improved utilization of low n-3 LC-PUFA diets compared to the offspring from parents with low fads2 expression. Besides, an ALA-rich diet during the gametogenesis caused negative effects on the growth of the offspring. The epigenetic analysis demonstrated that methylation in the promoter of fads2 of the offspring was correlated with the parental fads2 expression levels and type of the broodstock diet.

In vitro and in silico AHR assays for assessing the risk of heavy oil-derived polycyclic aromatic hydrocarbons in fish.

In the aftermath of the Great East Japan Earthquake of March 11, 2011, marine fish in Kesennuma Bay, Japan, have been contaminated with heavy oil containing polycyclic aromatic hydrocarbons (PAHs). To estimate the risk of six PAHs (benzo[α]pyrene, dibenzothiophene, phenanthrene, 2,3,5-trimethylnaphthalene, acenaphthene, and 1-methylphenanthrene), which have been detected at high levels in the tissues of fish from Kesennuma Bay, we attempted to evaluate the effects of these PAHs on the fish aryl hydrocarbon receptor (AHR) signaling pathway. We initially measured PAH concentrations and cytochrome P4501A catalytic activities (EROD: ethoxyresorufin-O-deethylase and MROD: methoxyresorufin-O-demethylase) as markers of AHR activation in greenlings (Hexagrammos otakii) collected from Kesennuma Bay in 2014. The results showed that alkylated PAH concentrations and EROD/MROD activities were higher in sites close to the oil-spilled sites than in the control site, suggesting AHR activation by spilled alkylated PAHs. We then investigated AHR-mediated responses to these PAHs in the in vitro reporter gene assay system where red seabream (Pagrus major) AHR1 (rsAHR1) or rsAHR2 expression plasmids were transiently transfected into COS-7 cells. The in vitro assay showed rsAHR isoform-, PAH-, and dose-dependent transactivation potencies. The relative effective concentrations of benzo[α]pyrene, dibenzothiophene, phenanthrene, 2,3,5-trimethylnaphthalene, acenaphthene, and 1-methylphenanthrene that induce 20% of the maximum benzo[α]pyrene response (REC20-BaP) for rsAHR1 activation were 0.052, 38, 79, 88, 270 nM, and no response, respectively, and those for rsAHR2 activation were 0.0049, 32, 53, 88, 60 nM, and no response, respectively. The results showed that the REC20-BaP values of benzo[α]pyrene for both the rsAHR1 and rsAHR2 isoforms were lower than the concentrations (0.041-0.20 nM) detected in the muscle tissue of fish from Kesennuma Bay, while the REC20-BaP values of other PAHs were higher than their tissue concentrations. In silico rsAHR homology modeling and subsequent ligand docking simulation analyses indicated that the rsAHR activation potencies of PAHs could be predicted from a rsAHR2 model. This study shows that in vitro and in silico rsAHR analyses may be a useful tool for assessing the risks to fish contaminated with PAHs.

Fatty Acid Composition and Fatty Acid Associated Gene-Expression in Gilthead Sea Bream (Sparus aurata) are Affected by Low-Fish Oil Diets, Dietary Resveratrol, and Holding Temperature.

To sustainably produce marine fish with a high lipid quality rich in omega-3 fatty acids, alternative sources of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are being identified. Moreover, the use of bioactive compounds that would stimulate the in vivo fatty acid synthesis, such as resveratrol (RV), would reduce the dependence on fish oil in aquafeeds. Gilthead sea bream (Sparus aurata) were fed four experimental diets combining two fish oil levels (6% dry matter (DM); 2% DM) with or without 0.15% DM resveratrol supplementation (F6, F2, F6 + RV, F2 + RV) for two months. Additionally, the fish were challenged either at 19 °C or 23 °C. A higher water temperature promoted their feed intake and growth, resulting in an increased crude lipid content irrespective of dietary treatment. The fatty acid composition of different tissues was significantly affected by the holding temperature and dietary fish oil level. The dietary RV significantly affected the hepatic EPA and DHA content of fish held at 19 °C. The observed effect of RV may be partly explained by alterations of the mRNA steady-state levels of ∆6-desaturase and ß-oxidation-related genes. Besides the relevant results concerning RV-mediated regulation of fatty acid synthesis in marine fish, further studies need to be conducted to clarify the potential value of RV to enhance fillet lipid quality.

Nano-delivery of trace minerals for marine fish larvae: influence on skeletal ossification, and the expression of genes involved in intestinal transport of minerals, osteoblast differentiation, and oxidative stress response.

Currently, the larviculture of many marine fish species with small-sized larvae depends for a short time after hatching, on the supply of high-quality live zooplankton to ensure high survival and growth rates. During the last few decades, the research community has made great efforts to develop artificial diets, which can completely substitute live prey. However, studies aimed at determining optimal levels of minerals in marine larvae compound feeds and the potential of novel delivery vectors for mineral acquisition has only very recently begun. Recently, the agro-food industry has developed several nano-delivery systems, which could be used for animal feed, too. Delivery through nano-encapsulation of minerals and feed additives would protect the bioactive molecules during feed manufacturing and fish feeding and allow an efficient acquisition of active substances into biological system. The idea is that dietary minerals in the form of nanoparticles may enter cells more easily than their larger counterparts enter and thus speed up their assimilation in fish. Accordingly, we evaluated the efficacy of early weaning diets fortified with organic, inorganic, or nanoparticle forms of trace minerals (Se, Zn, and Mn) in gilthead seabream (Sparus aurata) larvae. We tested four experimental diets: a trace mineral-deficient control diet, and three diets supplemented with different forms of trace minerals. At the end of the feeding trial, larvae growth performance and ossification, and the level of expression of six target genes (SLC11A2ß, dmt1, BMP2, OC, SOD, GPX), were evaluated. Our data demonstrated that weaning diets supplemented with Mn, Se, and Zn in amino acid-chelated (organic) or nanoparticle form were more effective than diets supplemented with inorganic form of minerals to promote bone mineralization, and prevent skeletal anomalies in seabream larvae. Furthermore, nanometals markedly improved larval stress resistance in comparison to inorganic minerals and upregulated mRNA copy number of OC gene. The expression of this gene was strongly correlated with mineralization degree, thus confirming its potency as a good marker of bone mineralization in gilthead seabream larvae.

Identification of a fish short-chain dehydrogenase/reductase associated with bone metabolism.

Although human and mouse genetics have largely contributed to the better understanding of the mechanisms underlying skeletogenesis, much more remains to be uncovered. In this regard alternative and complementary systems have been sought and cell systems capable of in vitro calcification have been developed to study the mechanisms underlying bone formation. In gilthead seabream (Sparus aurata), a gene coding for an unknown protein that is strongly up-regulated during extracellular matrix (ECM) mineralization of a pre-osteoblast cell line was recently identified as a potentially important player in bone formation. In silico analysis of the deduced protein revealed the presence of domains typical of short-chain dehydrogenase/reductases (SDR). Closely related to carbonyl reductase 1, seabream protein belongs to a novel subfamily of SDR proteins with no orthologs in mammals. Analysis of gene expression by qPCR confirmed the strong up-regulation of sdr-like expression during in vitro mineralization but also revealed high expression levels in calcified tissues. A possible role for Sdr-like in osteoblast and bone metabolism was further evidenced through (i) the localization by in situ hybridization of sdr-like transcript in pre-osteoblasts of the operculum and (ii) the regulation of sdr-like gene transcription by Runx2 and retinoic acid receptor, two regulators of osteoblast differentiation and mineralization. Expression data also indicated a role for Sdr-like in gastrointestinal tract homeostasis and during gilthead seabream development at gastrulation and metamorphosis. This study reports a new subfamily of short-chain dehydrogenases/reductases in vertebrates and, for the first time, provides evidence of a role for SDRs in bone metabolism, osteoblast differentiation and/or tissue mineralization.

Molecular and functional characterisation of two elovl4 elongases involved in the biosynthesis of very long-chain (>C24) polyunsaturated fatty acids in black seabream Acanthopagrus schlegelii.

Elongation of very long-chain fatty acid (Elovl) 4 proteins are important fatty acyl elongases that participate in the biosynthesis of long-chain (C20-24) and very long-chain (˃C24) polyunsaturated fatty acids (LC-PUFA and VLC-PUFA, respectively) in teleost fish, especially in marine species. Moreover, knowledge of Elovl4 and other elongases such as Elovl2 has contributed to an advanced understanding of the LC-PUFA biosynthetic pathway in marine fish. In the present study, elovl4a and elovl4b were cloned from black seabream Acanthopagrus schlegelii and functionally characterised using recombinant expression in yeast. The elovl4a and elovl4b cDNA sequences included open reading frames (ORF) of 969 and 918 base pairs (bp), encoding proteins of 322 and 315 amino acids (aa), respectively. The functional characterisation of A. schlegelii Elovl4 proteins showed they were able to utilise all assayed C18-22 PUFA substrates except 22:6n-3. Moreover, it was particularly noteworthy that both A. schlegelii Elovl4a and Elovl4b proteins had the ability to elongate 20:5n-3 and 22:5n-3 to 24:5n-3, which can be potentially desaturated and ß-oxidised to 22:6n-3. Tissue transcript abundance analysis showed the highest expression of elovl4a and elovl4b in brain and eye, respectively, suggesting these tissues were major sites for VLC-PUFA biosynthesis in black seabream. The functions of the A. schlegelii Elovl4-like elongases, Elovl4a and Elovl4b, characterised in the present study, along with those of the Elovl5 and fatty acyl desaturase (Fads2) proteins of A. schlegelii characterised previously, provided evidence of the biosynthetic pathways of LC-PUFA and VLC-PUFA in this teleost species.

The effect of starvation and re-feeding on vasotocinergic and isotocinergic pathways in immature gilthead sea bream (Sparus aurata).

This study describes the responses of the vasotocinergic and isotocinergic systems to food deprivation and re-feeding processes in immature gilthead sea bream (Sparus aurata). The animals were subjected to the following experimental treatments: (1) normal feeding (control), (2) food deprivation for 21 days; and (3) re-feeding for 7 days, beginning 14 days after starvation. The animals were sampled at 0, 7, 14 and 21 days from the beginning of the trial. The pituitary and plasma arginine vasotocin (AVT) and isotocin (IT) levels and the hypothalamic pro-vasotocin and pro-isotocin mRNA expression levels were measured. In addition, the mRNA levels of three receptors, avtr v1, avtr v2 and itr, were analyzed in target organs associated with (1) the integration and control of different physiological pathways related to stress and food intake (i.e., the hypothalamus), (2) hormonal release into the bloodstream (i.e., the pituitary), and (3) metabolism and its control (i.e., the liver). The metabolic parameters in the liver were also determined. The hepatosomatic index decreased, and hepatic metabolites were mobilized beginning in the early stages of starvation. Moreover, an over-compensation of these parameters occurred when the fish were re-fed after starvation. In terms of the vasotocinergic and isotocinergic systems, feed restriction induced a clear time-dependent regulation among metabolic organization, stress regulation and orexigenic processes in the mature hormone concentration and pro-peptide and receptor mRNA expression. Our results reveal the important role of the AVT/IT endocrine systems in the orchestration of fish physiology during starvation and re-feeding and indicate their involvement in both central and peripheral organs.

Effects of dietary administration of fenugreek seeds, alone or in combination with probiotics, on growth performance parameters, humoral immune response and gene expression of gilthead seabream (Sparus aurata L.).

The use of immunostimulants is considered a promising preventive practice that may help to maintain animal welfare and a healthy environment, while increasing production and providing higher profits. The purpose of this study was to evaluate the effects on gilthead seabream (Sparus aurata L.) of the dietary administration of fenugreek (Trigonella foenum graecum) seeds, alone or combined with one of the following probiotic strains: Bacillus licheniformis (TSB27), Lactobacillus plantarum or Bacillus subtilis (B46). Gilthead seabream were fed a control or one of the supplemented diets for 3 weeks. The effects of these supplemented diets on growth performance parameters and the humoral immune response (natural haemolytic complement, peroxidase, total IgM levels, proteases and antiproteases activities) were evaluated after 2 and 3 weeks of feeding. Simultaneously, the expression levels of some immune-relevant genes (igm, tcr-ß, csfr1 and bd) were measured in the head-kidney. Interestingly, all probiotic supplemented diets increased seabream growth rates, especially the B. licheniformis supplemented diet. Generally, humoral immune parameters were enhanced by the dietary supplementation at the different time points measured. The results showed a significant increases in the immune parameters, principally in fish fed only fenugreek or fenugreek combined with B. subtilis. Furthermore, real time qPCR revealed that dietary supplementation significantly enhances the expression of immune-associated genes in the head-kidney, particularly igm gene expression. These results suggest that fenugreek alone or combined with one of the probiotic strains mentioned enhances the immune response of gilthead seabream, a species with one of the highest rates of production in marine aquaculture.

Dietary Butyrate Helps to Restore the Intestinal Status of a Marine Teleost (Sparus aurata) Fed Extreme Diets Low in Fish Meal and Fish Oil.

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 ®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.