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.

Effects of aqueous and ethanolic leaf extracts from drumstick tree (Moringa oleifera) on gilthead seabream (Sparus aurata L.) leucocytes, and their cytotoxic, antitumor, bactericidal and antioxidant activities.

Aqueous and ethanolic extracts of drumstick, Moringa oleifera, leaves were evaluated in vitro to ascertain their principal active components and determine their immunostimulant, cytotoxic, antitumoral, bactericidal and antioxidant activities. Phytochemical screening of M. oleifera leaf extracts showed a greater abundance of phenolic and cyanogenic glycosides in aqueous than in ethanolic extracts, characterized by several flavonoids, condensed tannins and saponins. No significant effects on gilthead seabream (Sparus aurata) head-kidney leucocyte activities (phagocytic ability and capacity, respiratory burst and peroxidase) were detected after incubation for 24 h with different concentrations (0.001/1 mg mL-1) of either extract. In addition, the aqueous extract showed a marked cytotoxic effect on both SAF-1 (at doses above 0.01 mg mL-1) and PLHC-1 (at doses above 0.25 mg mL-1) cell lines. The ethanolic extract improved the viability of SAF-1 cells and decreased the viability of PLHC-1 cells when used at higher concentrations. Both the ethanolic and, particularly, the aqueous extracts showed significant bactericidal activity on pathogenic Vibrio anguillarum and Photobacterium damselae strains. The antiradical activity of M. oleifera, as determined by the ABTS assay, increased in a linear dose-response with increasing extract concentrations. The results as a whole for the cytotoxic, bactericidal and antioxidant activities of M. oleifera leaf extracts point to their possible use as additives in functional diets for farmed fish.

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.

Effect of dietary supplementation with yeast Saccharomyces cerevisiae on skin, serum and liver of gilthead seabream (Sparus aurata L).

The effect of dietary supplementation with Saccharomyces cerevisiae on gilthead seabream (Sparus aurata L.) was studied. Four replicates of fish (n = 6) were fed with a commercial diet containing 0 (control, no yeast added) or 10 mg per kilogram of heat-killed (30 min, 60°C) S. cerevisiae. After 4 weeks, half of the fish (two replicates) were injured and continued with the same diet. At 3 and 7 days post-wounding, samples of blood, skin mucus, skin and liver were obtained from each fish. The results showed that calcium concentrations were significantly higher (with respect to control fish) in the serum from fish sampled at 3 days post-wounding, whereas antioxidant enzymes in the skin mucus were altered after wounding (at both 3 and 7 days). Histological analyses revealed oedema, signs of inflammation and white cell recruitment together with a reduction in the epidermis layer in the wounded regions of fish fed control diet. Yeast supplementation did not change growth performance and helped maintain the normal serum calcium concentrations in wounded fish. Furthermore, a reduction in inflammation around wounds in the animals fed yeast with respect to that fed control diet was evident in the histological study. Furthermore, increased levels of stress-related gene expression in liver and skin from wounded fish were obtained. Overall, yeast supplementation seemed to be a functional and appropriate dietary additive to improve skin recovery reducing the stress resulting from wounds.

Influence of dietary replacement of fish meal with fish soluble meal on growth and TOR signaling pathway in juvenile black sea bream (Acanthopagrus schlegelii).

An 8-week feeding trial was conducted to evaluate the effect of replacement of fish meal (FM) with fish soluble meal (FSM) on growth performance, feed utilization and expression of genes involved in TOR signaling pathway for juvenile black sea bream (Acanthopagrus schlegelii). Six isonitrogenous (41%) and isolipidic diets were prepared to contain graded levels of FSM which replaced 0% (control diet), 10%, 20%, 30%, 40% and 60% protein from FM. Triplicate groups of 20 fish with initial weight 0.51 ± 0.01 g were fed with experimental diets twice daily to apparent satiation. The results showed significant differences in growth performance and feed utilization among all treatments, final body weight (FBW), percent weight gain (PWG), specific growth rate (SGR) and protein efficiency ratio (PER) significantly increased with dietary replacement levels of FM with FSM increasing from 0% to 40% (P < 0.05), PWG, SGR and PER were significantly reduced when replacement of FM with FSM further increased from 40% to 60%. Based on PWG against replacement levels of FM with FSM, A two-slope broken-line model analysis indicated that the optimal replacement of FM with FSM is to be 42.59%. Moreover, the lowest feed conversion ratio (FCR) was observed in fish fed the 40% FSM replacement diet. Muscle amino acid profile in muscle revealed that total essential amino acids, arginine and threonine were significantly influenced by replacement levels of FSM, while there was no significant difference in NEAA among all treatments. The hematological indices were not affected by the replacement levels of FM with FSM. The relative expression levels of irs-1, pi3k, akt, igf-1, s6k1 and tor were up-regulated when replacement levels of FM with FSM increased from 0% to 40%, and higher values were observed in fish fed with 40% FSM replacement diet compared to those fed the other diets. However, relative expression of 4e-bp2 was down-regulated when replacement levels of FM with FSM increased from 0% to 40% (P < 0.05). In summary, the results of present study indicated that FSM could be a viable alternative protein source for black sea bream, dietary FSM supplementation could improve growth and up-regulate the relative expression of irs-1, pi3k, akt, igf-1, s6k1 genes related to TOR signaling pathway in liver of juvenile black sea bream.

Effect of Jasonia glutinosa on immune and oxidative status of gilthead seabream (Sparus aurata L.).

Jasonia glutinosa (rock-tea, RT) has numerous biological activities. In the present work, the beneficial effects of dietary RT on gilthead seabream (Sparus aurata L.) were studied. Fish fed experimental diets containing 0 (control), 10 or 30% of RT for 15 and 30 days. Samples from skin mucus, blood, head kidney, liver and gut were obtained at 15 and 30 days. The antioxidant properties from RT were analysed such as the antioxidant capacity and phenolic content. The heat shock protein 70 level (HSP70) and the total oxidized proteins were evaluated on skin mucus as stress markers. Immune parameters, both humoral (peroxidase activity, immunoglobulin M levels and complement activity) and cellular (phagocytic, respiratory burst and peroxidase activities) were determined in skin mucus, serum or head-kidney leucocytes, respectively. Concomitantly, the expression of different genes related to inflammation and oxidative stress was studied both in liver and gut. Skin mucus peroxidase was significantly increased on fish fed 10% RT for 15 days with respect to the control group. In addition, Serum IgM levels were significantly increased while HSP70 levels and oxidized proteins were significantly decreased on skin mucus from fish fed 30% RT for 30 days, respectively. Besides, cellular immune parameters (phagocytosis, respiratory burst and peroxidase activity) were significantly higher in leucocytes from fish fed the RT diets for 15 days, but not for 30 days. Finally, the gene expression of antioxidant enzymes was up-regulated in liver at 15 and in liver and gut at 30 days. However, the expression of il1b and hsp70 was down-regulated in the liver of fish fed 30% RT for 30 days with respect to the values of control fish. The possible inclusion of RT in fish diets as an additive with antioxidant and/or immunostimulant activities is discussed.

Effects of dietary Origanum vulgare on gilthead seabream (Sparus aurata L.) immune and antioxidant status.

Origanum sp. is a very common genus of aromatic plants worldwide distributed around the Mediterranean area and O. vulgare (oregano) is the most important species of this genus throughout the world. Due the known medicinal properties of oregano, the effect of diets enriched with 0% (control), 0.5% and 1% oregano leaves powder was studied on the growth, immune and antioxidant status of gilthead seabream (Sparus aurata L.). Fish fed with oregano 0.5% and 1% enriched diets improved both humoral (IgM and bactericidal activity in skin mucus and protease activity in serum) and cellular (head kidney leucocytes phagocytic ability) immunity at 15 and 30 days. Furthermore, the addition of oregano did not provoke any significant effect neither in the growth promotion nor in the liver antioxidant enzymes activity studied in the serum and skin mucus. The possibility of using O. vulgare as a functional additive to fish diet is discussed.

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 supplementation of bovine lactoferrin on antioxidant status, immune response and disease resistance of yellowfin sea bream (Acanthopagrus latus) against Vibrio harveyi.

This study investigated the effect of the dietary supplementation of bovine lactoferrin (LF) on growth performance, hematological and immunological parameters, antioxidant enzymes activity and disease resistance against Vibrio harveyi in yellowfin sea bream (Acanthopagrus latus) fingerling. The fish with initial body weight 10 ±â€¯0.3 g were randomly distributed at 10 fish per each 250 L fiberglass tank, and fed with four experimental diets (a control basal diet and three supplemented diets with 400, 800 and 1200 mg LF kg-1 diet) for 8 weeks. The obtained results showed that fish fed with LF supplemented diets had significantly higher final body weight as compared to control diet (P < 0.05). There were no significant differences between LF-treatments and the control group in white blood cell counts, red blood cell counts, hemoglobin and hematocrit. Total protein and complement activity (ACH50) in the serum of yellowfin sea bream were enhanced with increasing the dietary LF supplementation level (P < 0.05). The mucus lysozyme activity in fish fed on 800 and 1200 mg LF kg-1 was significantly higher than those fed on 400 mg LF kg-1 and control fish (P < 0.05). None of the antioxidant enzymes (catalase, glutathione reductase, glutathione S-transferase) was affected by LF supplementation (P > 0.05). Fish fed with dietary LF had a significantly higher survival rate than those fed with the control diet after challenge with Vibrio harveyi (P < 0.05). These results revealed that diet supplementation in A. latus especially with 1200 mg LF kg-1 improve fish growth performance and immune parameters, as well as survival rate against Vibrio harveyi.

Dietary tryptophan supplementation induces a transient immune enhancement of gilthead seabream (Sparus aurata) juveniles fed fishmeal-free diets.

European aquaculture is an industry with a high sustainability profile contributing to the supply of safe seafood. However, several diseases can affect farmed fish and it is imperative to find alternatives for chemotherapeutic treatments when disease outbreaks occur. Maintenance of health through nutrition is well-establish in modern animal farming, and amino acids (AA) are promising candidates as functional additives to improve fish health. Therefore, the goal of this research is to provide a better understanding of the influence of tryptophan supplementation on nutritional condition and immune mechanisms in fish. Triplicate groups of fish (13.3 ±â€¯0.3g) previously fed with a fishmeal-based diet were either fed a control diet with an extreme formulation (0% fishmeal) but meeting the AA requirements (CTRL), or the SUP diet, formulated as the CTRL with an increase in tryptophan (TRP) content. After 2 and 13 weeks of feeding, head-kidney (HK), liver (L) and white skeletal muscle (WSM) were collected for gene expression, whereas plasma was suited for humoral immune parameters. A holistic approach using transcriptomic, humoral and zootechnical parameters was undertaken. The expression of 29-31 genes for WSM, L or HK confirms an effect due to the treatment across time. A two-way ANOVA analysis revealed that 15-24 genes varied significantly depending on the tissue, and the multivariate analysis by means of PLS-DA explained (R2) and predicted (Q2) with four components up to 93% and 78% of total variance, respectively. Component 1 (R2 = 50.06%) represented the time effects, whereas components 2 (24.36%) and 3 (13.89%) grouped fish on the basis of dietary treatment, at early sampling. The HK results in particular suggest that fish fed SUP diet displayed an immunostimulated state at 2 weeks. No major differences were observed in plasma humoral parameters, despite an increase in antiprotease and peroxidase activities after 13 weeks regardless of dietary treatment. These results suggest that tryptophan supplementation may improve the seabream immune status after 2 weeks. Hence, the use of functional feeds is especially relevant during a short-term feeding period before a predictable stressful event or disease outbreak, considering that these putative advantageous effects seem to disappear after a 13 weeks feeding period.

The effect of dietary fucoidan on growth, immune functions, blood characteristics and oxidative stress resistance of juvenile red sea bream, Pagrus major.

We determined the supplementation effects of dietary fucoidan on growth, immune responses, blood characteristics, and oxidative stress resistance of juvenile red sea bream. A fishmeal (FM)-based basal diet supplemented with 0% (D1, control), 0.05% (D2), 0.1% (D3), 0.2% (D4), 0.4% (D5), and 0.8% (D6) mozuku fucoidan to formulate six experimental diets. Each diet was randomly allocated to triplicate groups of fish (3.8 g) for 60 days. Results showed that fish-fed diet D5 showed significantly higher (P < 0.05) growth performance compared to the control (D1). Diet groups D2 to D4 also showed intermediate values compared to D1. Feed conversion efficiency and protein efficiency ratio were significantly higher in diet group D5, which was not significantly different with D3. Fucoidan supplementation increased whole-body lipid, which was significantly higher in the D5 group. Condition factor (CF) was significantly higher in fish fed ≥ 0.2% fucoidan-supplemented diet groups. Diet group D5 and D4 showed significantly lower blood urea nitrogen (BUN) and aspartate aminotransferase (AST) level, respectively. Dietary fucoidan reduced the oxidative stress of fish. Among the measured nonspecific immune parameters, only peroxidase activity (PA) and total serum protein (TSP) were significantly influenced by dietary supplementation and it was higher in D4 group. Fucoidan supplementation reduces thiobarbituric acid reactive substance (TBARS) values numerically and it was lowest in fish-fed diet group D5. Under the present experimental condition, finally, we concluded that 0.3-0.4% dietary fucoidan supplementation enhanced the growth and health performance of red sea bream by increasing growth, immune response, blood characteristics, and oxidative stress resistance.

Dietary supplementation of selenium nanoparticles modulated systemic and mucosal immune status and stress resistance of red sea bream (Pagrus major).

Dietary supplementation of selenium nanoparticles (Se-NPs) at different levels (0, 0.5, 1, and 2 mg kg-1 diet) was evaluated to find out the effects on serum and skin immune responses as well as stress resistance in the red sea bream (Pagrus major). After 45 days of experimental trial, serum and mucosal immune responses were significantly high in fish fed 1 mg Se-NPs kg-1 diet (P < 0.05). In this group, alternative complement pathway, total serum protein, antioxidant activity of catalase enzyme, serum bactericidal activity, serum lysozyme activity, and amounts of skin mucus secretions as well as stress resistance against low salinity stress increased significantly, when compared to fish fed Se-NP-free diet (P < 0.05). Furthermore, fish fed Se-NPs at 2 mg kg-1 diet exhibited higher alternative complement pathway, total serum protein, mucus lysozyme activity, serum and mucus peroxidases, amount of mucus secreted, and tolerance against low salinity stress than the fish fed Se-NP-free diet (P < 0.05). Interestingly, the nitro blue tetrazolium activity in all groups fed with diets supplemented with Se-NPs are significantly higher than Se-NP-free diet (P < 0.05). The present results demonstrate that the dietary supplementation with Se-NPs (mainly from 1 to 2 mg kg-1 level) could be useful for maintaining the overall health status of red sea bream.

Influence of dietary inosine and vitamin C supplementation on growth, blood chemistry, oxidative stress, innate and adaptive immune responses of red sea bream, Pagrus major juvenile.

Both inosine (INO) and vitamin C (l-ascorbic acid, AsA) play important roles in growth performance, feed utilization and health status of fish. Therefore, a 56 days feeding trial was conducted to determine the interactive effects of dietary INO and AsA on growth performance, oxidative status, innate and adaptive immune responses of red sea bream. Fish growth performance and fed utilization parameters were significantly affected by dietary INO supplementation but not by AsA. Fish fed diets with INO at 4 g kg-1 diet in combination of high and low levels of AsA (3.1 g kg-1 and 9.3 g kg-1) produced the highest growth and feed utilization performances. In terms of growth and feed utilization performances no significant interaction effects were observed between INO and AsA. Dietary INO significantly influenced hematocrit, glucose and glutamyl oxaloacetic transaminase (GOT) content of red sea bream meanwhile AsA also significantly influenced hematocrit, glucose, total cholesterol, blood urea nitrogen (BUN) and glutamic-pyruvate transaminase (GPT) content of the test fish. No significant interaction effects was also observed between INO and AsA on measured hematological parameters. Reactive oxygen metabolites (d-ROMs) significantly influenced by both INO and AsA. Fish fed diet groups D1, D4 and D6 showed best oxidative stress resistance. Only INO was a significant factor on nitro-blue-tetrazolium activity (NBT) and bactericidal activity (BA). Neither INO nor AsA was a significant factor on serum catalase activity (CAT), total serum protein (TSP), peroxidase activity (PA) and lysozyme activity (LA). No significant interaction effects was observed between INO and AsA on measured innate immune parameters. Agglutination antibody titer was significantly influenced by dietary supplementation, after 15 days of vaccination but not in 21 days. In the day 15th fish and diet group D3 and D5 showed significantly higher values compared to diet groups control and D1. INO was the only significant factor of increasing agglutination antibody titer in 15 t h day. While AsA was not a significant factor on agglutination antibody titer values in day 15 t h, there was an interaction between dietary INO and AsA levels. Finally under the experimental conditions, fish fed high INO and low AsA levels (4 g kg-1 and 0.31 g kg-1 diet, respectively) showed best growth and feed utilization performance. Simultaneously, low level of INO and high level of AsA (2 g kg-1 and 0.93 g kg-1 diet, respectively) improved blood chemistry and immunological parameters. Furthermore, combined use of INO and AsA is possible to improve hemato-immunological responses of red sea bream.

Effects of dietary inclusion of Moringa oleifera leaves on growth and some systemic and mucosal immune parameters of seabream.

The effect of the dietary incorporation of drumstick, Moringa oleifera, leaf meal (MOL; 0, 5, 10 and 15%) on the growth, feed utilization, some skin mucus and systemic immune parameters and intestinal immune-related gene expression in gilthead seabream (Sparus aurata) specimens. The experiment lasted 4 weeks. The results revealed that MOL can be incorporated in S. aurata diet up to 10% with no significant negative effect on growth and feed utilization. However, there was a significant decrease with MOL at a level of 15% after 2 weeks of feeding. The systemic immune status of fish fed with the different levels of MOL showed an improvement in head kidney leucocyte phagocytosis, respiratory burst and peroxidase activities. Also, serum humoral components, including protease, ACH50 and lysozyme activities and IgM level, increased with MOL inclusion especially at the 5% level. MOL at 5% improved skin-mucosal immunity such as protease, antiprotease, peroxidase and lysozyme activities. Moreover, the feeding of MOL revealed an upregulation of the intestinal mucosal immunity genes (lyso and c3), tight junction proteins (occludin and zo-1) and anti-inflammatory cytokines (tgf-ß) with a downregulation of pro-inflammatory cytokine (tnf-α). Therefore, it is recommended to incorporate MOL in S. aurata diets at a level of 5% for the best immune status or 10% for the high growth performance and acceptable immune surveillance. Graphical abstract ᅟ.

In vitro effects of Origanum vulgare leaf extracts on gilthead seabream (Sparus aurata L.) leucocytes, cytotoxic, bactericidal and antioxidant activities.

Origanum vulgare is a well-known medicinal plant that has been used since ancient times as an additive in foods and cosmetic preparations. The possible application of O. vulgare extracts in fish was assessed by using gilthead seabream (Sparus aurata) as a marine fish model due to its importance in aquaculture. The in vitro effects of aqueous and ethanolic leaf extracts of O. vulgare were tested in order to observe any immunostimulant, cytotoxic, bactericidal or antioxidant properties. The results showed that medium or high concentration of aqueous extracts and low concentrations of ethanolic extract, increased head kidney leucocyte activities as well as the number of SAF-1 cells. However, moderate to high concentrations of ethanolic extracts decreased both leucocyte activities and the number of viable SAF-1 cells, suggesting some possible toxic effect towards them. Only the highest concentration of the aqueous extract and medium to high concentrations of the ethanolic extracts showed cytotoxic activity against the tumor PLHC-1 cell line. Bactericidal activity was only detected against Vibrio harveyi, V. anguillarum and Photobacterium damselae when using the highest concentration of aqueous extract and moderate to high concentrations of ethanolic extract. Finally, both plant extracts presented antioxidant activity particularly the aqueous extract. Overall, the results suggest that both extracts (when used at the appropriate concentration) have immunostimulant, cytotoxic, bactericidal and antioxidant properties, making O. vulgare an interesting candidate for incorporation as additive in functional diets for farmed fish.