Effects of essential amino acids supplementation in a low-protein diet on growth performance, intestinal health and microbiota of juvenile blotched snakehead (Channa maculata).

Developing a low-protein feed is important for the sustainable advancement of aquaculture. The aim of this study was to investigate the effects of essential amino acid (EAA) supplementation in a low-protein diet on the growth, intestinal health, and microbiota of the juvenile blotched snakehead, Channa maculata in an 8-week trial conducted in a recirculating aquaculture system. Three isoenergetic diets were formulated to include a control group (48.66 % crude protein (CP), HP), a low protein group (42.54 % CP, LP), and a low protein supplementation EAA group (44.44 % CP, LP-AA). The results showed that significantly lower weight gain (WG), specific growth rate (SGR), protein efficiency ratio (PER), and feed efficiency ratio (FER) were observed in fish that were fed LP than in the HP and LP-AA groups (P < 0.05). The HP and LP-AA groups exhibited a significant increase in intestinal villus length, villus width, and muscular thickness compared to the LP group (P < 0.05). Additionally, the HP and LP-AA groups demonstrated significantly higher levels of intestinal total antioxidant capacity (T-AOC), catalase (CAT), and superoxide dismutase (SOD) and lower levels of malondialdehyde (MDA) compared to the LP group (P < 0.05). The apoptosis rate of intestinal cells in the LP group was significantly higher than those in the LP and HP groups (P < 0.05). The mRNA expression levels of superoxide dismutase (sod), nuclear factor kappa B p65 subunit (nfκb-p65), heat shock protein 70 (hsp70), and inhibitor of NF-κBα (iκba) in the intestine were significantly higher in the LP group than those in the HP and LP-AA groups (P < 0.05). The 16s RNA analysis indicated that EAA supplementation significantly increased the growth of Desulfovibrio and altered the intestinal microflora. The relative abundances of Firmicutes and Cyanobacteria were positively correlated with antioxidant parameters (CAT and T-AOC), whereas Desulfobacterota was negatively correlated with sod and T-AOC. The genera Bacillus, Bacteroides, and Rothia were associated with the favorable maintenance of gut health. In conclusion, dietary supplementation with EAAs to achieve a balanced amino acid profile could potentially reduce the dietary protein levels from 48.66 % to 44.44 % without adversely affecting the growth and intestinal health of juvenile blotched snakeheads.

Lumpfish (Cyclopterus lumpus) Is Susceptible to Renibacterium salmoninarum Infection and Induces Cell-Mediated Immunity in the Chronic Stage.

Renibacterium salmoninarum is a Gram-positive, intracellular pathogen that causes Bacterial Kidney Disease (BKD) in several fish species in freshwater and seawater. Lumpfish (Cyclopterus lumpus) is utilized as a cleaner fish to biocontrol sea lice infestation in Atlantic salmon (Salmo salar) farms. Atlantic salmon is susceptible to R. salmoninarum, and it can transfer the infection to other fish species. Although BKD outbreaks have not been reported in lumpfish, its susceptibility and immune response to R. salmoninarum is unknown. In this study, we evaluated the susceptibility and immune response of lumpfish to R. salmoninarum infection. Groups of lumpfish were intraperitoneally (i.p.) injected with either R. salmoninarum (1×107, 1×108, or 1×109 cells dose-1) or PBS (control). R. salmoninarum infection kinetics and mortality were followed for 98 days post-infection (dpi). Transcript expression levels of 33 immune-relevant genes were measured in head kidney (n = 6) of fish infected with 1×109 cells/dose and compared to the control at 28 and 98 dpi. Infected lumpfish displayed characteristic clinical signs of BKD. Lumpfish infected with high, medium, and low doses had a survival rate of 65%, 93%, and 95%, respectively. Mortality in the high-dose infected group stabilized after 50 dpi, but R. salmoninarum persisted in the fish tissues until 98 dpi. Cytokines (il1ß, il8a, il8b), pattern recognition receptors (tlr5a), interferon-induced effectors (rsad2, mxa, mxb, mxc), and iron regulation (hamp) and acute phase reactant (saa5) related genes were up-regulated at 28 dpi. In contrast, cell-mediated adaptive immunity-related genes (cd4a, cd4b, ly6g6f, cd8a, cd74) were down-regulated at 28 dpi, revealing the immune suppressive nature of R. salmoninarum. However, significant upregulation of cd74 at 98 dpi suggests induction of cell-mediated immune response. This study showed that R. salmoninarum infected lumpfish in a similar fashion to salmonid fish species and caused a chronic infection, enhancing cell-mediated adaptive immune response.

Dietary supplementation of fermented lemon peel enhances lysozyme activity and susceptibility to Photobacterium damselae for orange-spotted grouper, Epinephelus coioides.

The waste recycling of lemon peel, as a functional feed additive in aquafeed was evaluated by estimating the effects of fermented lemon peel (FLP) supplementation in diet on growth performance, innate immune responses, and susceptibility to Photobacterium damselae of grouper, Epinephelus coioides. A basal diet was added FLP at 0%, 1%, 3%, and 5%. Four tested diets were each fed to juvenile grouper (initial weight: 15.89 ± 0.10 g, triplicate groups) in a recirculation rearing system for eight weeks. Fish fed diets with 0%-3% FLP exhibited higher (p < 0.05) final weight, weight gain, and feed efficiency than fish fed the 5% FLP-diet. After challenge test, fish fed the 3% FLP-diet appeared the lowest mortality, followed by fish fed the 1% FLP-diet, and lowest in fish fed 0% and 5% FLP-diets. Plasma lysozyme activities were higher in fish fed diets with FLP than in fish fed the FLP-free control diet before challenge test. After challenge, fish fed diets with 1% and 3% FLP showed highest lysozyme activities, followed by fish fed the diet with 5% FLP, and lowest in fish fed the control diet. Hepatic malondialdehyde content was higher in fish fed the control diet than in fish fed diets with 1%-3% FLP. Results found that diets supplemented with 1%-3% fermented lemon peel can enhance lysozyme activity and resistance to pathogen P. damselae of grouper.

Activation of Autophagy Relieves Linoleic Acid-Induced Inflammation in Large Yellow Croaker (Larimichthys crocea).

High levels of soybean oil (SO) in fish diets enriched with linoleic acid (LA, 18:2n-6) could induce strong inflammation. However, the molecular mechanism underlying LA-induced inflammation in the liver of large yellow croaker (Larimichthys crocea) has not been elucidated. Based on previous research, autophagy has been considered a new pathway to relieve inflammation. Therefore, the present study was performed to investigate the role of autophagy in regulating LA-induced inflammation in the liver of large yellow croaker in vivo and in vitro. The results of the present study showed that activation of autophagy in liver or hepatocytes could significantly reduce the gene expression of proinflammatory factors, such as tumor necrosis factor α (TNFα) and interleukin 1ß (IL1ß). The results of the present study also showed that inhibition of autophagy could upregulate the gene expression of proinflammatory factors and downregulate the gene expression of anti-inflammatory factors in vivo and in vitro. Furthermore, autophagy could alleviate LA-induced inflammatory cytokine gene expression in vivo and in vitro, while inhibition of autophagy obtained the opposite results. In conclusion, our study shows that autophagy could regulate inflammation and alleviate LA-induced inflammation in the liver of large yellow croaker in vivo and in vitro for the first time, which may offer considerable benefits to the aquaculture industry and human health.

Seaweed dietary supplements enhance the innate immune response of the mottled rabbitfish, Siganus fuscescens.

Disease is one of the major bottlenecks for aquaculture development, costing the industry in excess of US $6 billion each year. The increase in pressure to phase out some traditional approaches to disease control (e.g. antibiotics) is pushing farmers to search for alternatives to treat and prevent disease outbreaks, which do not have detrimental consequences (e.g. antibiotic resistance). We tested the effects of eleven seaweed species and four established fish immunostimulants on the innate immune response (cellular and humoral immunity) of the rabbitfish Siganus fuscescens. All supplements including different seaweeds from the three groups (Chlorophyta, Phaeophyta and Rhodophyta) were included in the fish pellet at 3% (by weight) and had variably positive effects across the four innate immune parameters we measured compared to control fish. Diets supplemented with the red seaweed Asparagopsis taxiformis and the brown seaweed Dictyota intermedia led to the largest boosts in humoral and cellular innate immune defences, including particularly significant increases in haemolytic activity. Diets supplemented with Ulva fasciata also led to promising positive effects on the fish innate immune responses. We conclude that dietary seaweed supplements can boost the immune response of S. fuscescens and thus the top three species highlighted in this study should be further investigated for this emerging aquaculture species and other fish species.

Early immune response in large yellow croaker (Larimichthys crocea) after immunization with oral vaccine.

Mesoporous silica nanoparticles (MSNs) have been used in the field of biomedicine as antigen carriers and adjuvants for protective antigens. In the present study, an oral nanovaccine against Vibrio alginolyticus was prepared employing MSNs as carriers. The uptake of the dihydrolipoamide dehydrogenase (DLDH) antigens in the intestine of large yellow croaker was evaluated using an immunohistochemistry assay. Additionally, the effects of the nanovaccine on the early immune response in large yellow croaker were investigated via oral vaccination. The presence of the antigens was detected in the mucosa and lamina propria of the foregut, midgut, and hindgut of large yellow croaker at 3 h following oral immunization. The expression levels of cytokines (i.e., lysozyme, IFN-γ, IFITM, TNF-α, IL-1ß, IL-2, IL-4, IL-10, and IL-13) in the intestine, spleen, and head kidney tissues of large yellow croaker before and after the immune challenge were determined via RT-qPCR assay. The obtained results revealed that the expression levels of lysozyme, IFN-γ, IFITM, TNF-α, IL-1ß, IL-2, IL-4, IL-10, and IL-13 in the intestine and head kidney of the vaccinated large yellow croaker, as well as the expression of lysozyme, IL-1ß, and IL-10 in the spleen, exhibited time-dependent oscillation regulation patterns. Notably, the nanovaccine immunization could induce early (6 h) and high expression of IFN-γ in the spleen and kidney tissues after the bacterial infection. The current study supplements the available data on the early immune response to fish nanovaccines. It also provides a valuable theoretical basis for the future development of large yellow croaker oral vaccines.

Effect of replacement of dietary fish oil with four vegetable oils on prostaglandin E2 synthetic pathway and expression of inflammatory genes in marine fish Larimichthys crocea.

As a lipid mediator with important immune function, prostaglandin E2 (PGE2) has been widely studied in mammals, whereas its synthetic pathway and immune function in fish have yet to be fully studied. To investigate the regulation of PGE2 synthetic pathway and inflammatory genes expression by dietary different oils and the underlying relationship, a 10-week feeding experiment and an immune challenge were carried out in marine fish Larimichthys crocea. Replacement of dietary fish oil (FO) with four vegetable oils (VO), including soybean oil, linseed oil, palm oil, and olive oil, all reduced PGE2 levels, and the decrease of arachidonic acid (ARA, substrate for PGE2) could account for this decline. Meanwhile, the expression of PGE2 synthesis related genes was basically upregulated, which seemed to be a feedback regulation, but it cannot compensate the deficiency of ARA. In addition, mRNA expression of inflammatory genes, including interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)α and interferon (IFN)γ was all upregulated in four VO groups compared with FO group, which was the opposite of PGE2 levels. To verify the inflammatory regulation of PGE2, an immune challenge was conducted, and PGE2 alleviated LPS-induced expression of inflammatory genes, including IL-6, TNFα and IFNγ, and the similar downregulation of toll-like receptor (TLR) genes expression revealed that TLR signaling pathway participated in the anti-inflammatory regulation of PGE2. In conclusion, replacement of dietary FO with four VO (lack of ARA) reduced the levels of PGE2 that could alleviate LPS-induced inflammatory genes expression via TLR signaling pathway, which could be one of the reasons that VO induced inflammation in marine fish.

The herbal extract deriving from aerial parts of Scutellaria baicalensis shows anti-inflammation and anti-hypoxia responses in cultured fin cells from rabbit fish.

A new cell line derived from dorsal fin of rabbit fish Siganus fuscescens was developed and characterized. The cell line was isolated from the dorsal fin, named as rabbit fish fin (RFF) cell line, and which was sub-cultured for 50 cycles since the development. This cell line was tested for growth in different temperatures and serum concentrations, and the best growing condition was at 20% serum at 28 °C. In cultured RFF cells, amplification of 18S rRNA from genomic DNA and immunostaining of cellular cytokeratin confirmed the proper identity of S. fuscescens fish. After 30th passage of cultures, the cells were exposed to challenge of inflammation, triggered by LPS, and hypoxia, mimicked by CoCl2. Cultured RFF cells showed robust sensitive responses to inflammation and hypoxia in directing the expressions of cytokines and hypoxia inducible factor-1α (HIF-1α). The water extract of aerial part of Scutellaria baicalensis (SBA) has been shown in rabbit fish to prevent inflammation. Here, we extended this notion of testing the efficacy of SBA extract in the developed cultured RFF cells. Application of SBA extract inhibited the expression of LPS-induced inflammatory cytokines, i.e. IL-1ß, IL-6, as well as the signaling of NF-κB. The application of CoCl2 in cultured RFF cells triggered the hypoxia-induced cell death and up regulation of HIF-1α. As expected, applied SBA extract in the cultures prevented the hypoxia-induced signaling. Our results show the established RFF cell line may be served as an ideal in vitro model in drug screening relating to inflammation and hypoxia. Additionally, we are supporting the usage of SBA herbal extract in fish aquaculture, which possesses efficacy against inflammation and hypoxia.

The ameliorative effects of various fish protein hydrolysates in poultry by-product meal based diets on muscle quality, serum biochemistry and immunity in juvenile barramundi, Lates calcarifer.

In an effort to reduce the use of fishmeal (FM), the effect of using protein from poultry by product meal (PBM) along with the supplementation of three different fish protein hydrolysate (FPH) including yellowtail kingfish, carp and tuna hydrolysate (designated as KH, CH and TH, respectively) were evaluated in juvenile barramundi for growth performance, fillet quality, mucosal immunity, serum biochemistry, immune response and infection against Vibrio harveyi. Fish were fed a FM based control diet + three isonitrogenous and isolipidic diets containing 90% of PBM protein supplemented with different types of hydrolysates: 90% PBM +10% KH (90PBM + KH), 90% PBM + 10% CH (90PBM + CH) and 90% PBM + 10% TH (90PBM + TH). Growth performance and indices were unaffected by the hydrolysate supplemented diets when compared to the control. FPH supplemented PBM diets resulted in improved muscle quality by improving poly unsaturated fatty acids (PUFA), ∑n-3, ∑n-6 and ∑n-9, and health related lipid indexes were not affected. The internal architecture of spleen and kidney were not altered by test diets whilst FPH supplemented PBM modulated acidic mucins in intestine and skin of fish. Improved infection rate in response to two weeks post infection with V. harveyi in the FPH supplemented diets was further associated with an increased serum immune response and a concomitant regulation of proinflammatory and inflammatory cytokines in the head kidney. Serum biochemistry including alanine transaminase (ALT), glutamate dehydrogenase (GLDH) and total bilirubin (TB) showed a decreasing trend both in pre-challenge and post-challenge barramundi fed FPH supplemented diets whereas cholesterol level decreased significantly in post-challenge groups fed 90PBM + KH and 90PBM + TH than pre-challenge barramundi. This study signifies that supplementation of 10% with different three FPH, hydrolysed by an alcalase® enzyme in PBM-based diets for barramundi could be good strategies to overcome the negative consequences triggered by animal by-product ingredients.

Assessment of chemical, biological and immunological properties of "Damiana de California" Turnera diffusa Willd extracts in Longfin yellowtail (Seriola rivoliana) leukocytes.

In Mexican herbal medicines or natural remedies, Turnera diffusa (Turneraceae) known as "Damiana de California", has ethnopharmacological relevance, including aphrodisiac, diuretic, and antimicrobial activities. To explore the immunological effect of infusion and methanolic extracts from Damiana de California, this study investigated its chemical, biological, antimicrobial and immunological properties in Longfin yellowtail Seriola rivoliana leukocytes. The analysis of chemical compounds revealed a considerable level of total phenolic and flavonoid contents in the infusion compared with methanolic extract. Furthermore, the antioxidant activity showed high hydroxyl radical scavenging activity in infusion extract compared with BHT positive control. Superoxide radical scavenging activity and ion chelation were higher in methanolic extract followed by infusion treatment. Interestingly, notable antimicrobial activity was observed in both extracts of T. diffusa against Vibrio parahaemolyticus. An in vitro study was performed using leukocytes of S. rivoliana treated with infusion or methanolic extracts at 12.5, 25 and 50 µg/mL for 24 h. Remarkably, infusion extract induced proliferation at any concentration but not the methanolic extract, which was diminished in a dose-dependent fashion. The immunostimulation study demonstrated that the phagocytosis activity increased in those leukocytes stimulated with methanolic extract but diminished the respiratory burst activity, in contrast to the activity observed in those leukocytes stimulated with infusion treatment. Finally, leukocytes incubated with the extracts and confronted with V.parahaemolyticus up-regulated the transcription of proinflammatory cytokine IL-1ß gene in a dose response relationship. These findings suggest that the infusion treatment has potential therapeutic properties, promoting the antioxidant capacity and enhancing immune parameters in Longfin yellowtail S. rivoliana.

Immunomodulatory and antioxidant effects of Astragalus polysaccharide liposome in large yellow croaker (Larimichthys crocea).

Astragalus polysaccharides (APS) have been widely used as immunopotentiators in aquaculture, however, the best way of their administration remains to be explored. In the present study, APS liposome (APSL) was prepared by film dispersion-ultrasonic method. The optimal conditions of APSL preparation were determined by response surface methodology, with a ratio of 10:1 (w/w) for soybean lecithin to APS and 8:1 (w/w) for soybean lecithin to cholesterol, and an ultrasound time of 15 min, which produced an encapsulation efficiency of 73.88 ± 0.88% of APSL. In vivo feeding experiments in large yellow croaker showed that both APS and APSL could enhance the contents of serum total protein (TP) and albumin (ALB), activities of serum non-specific immune enzymes such as acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM), and phagocytic activity of head kidney macrophages. Meanwhile, they both increased the activities of serum antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) and reduced the content of final lipid peroxidation product malondialdehyde (MDA) in serum, thus exhibiting the antioxidant effects. In vitro experiments on primary head kidney macrophages (PKM) showed that both APS and APSL inhibited ROS production, but obviously enhanced NO production and phagocytic activity of PKM. Furthermore, expression levels of pro-inflammatory cytokines (IL-1ß, IL-6, IL-8, and TNF-α), IFN-γ, and iNOS in PKM were significantly up-regulated after APS and APSL treatments, but no expression change of IFN-h was observed. Taken together, our results showed that both APS and APSL could improve several immune parameters and antioxidant ability of large yellow croaker either in vivo or in vitro, and the efficacy of APSL was markedly better than APS. These findings therefore indicated that the immunomodulatory and antioxidant activities of APS could be enhanced after encapsulated with liposome, and APSL may represent a potential drug delivery system of APS for development of immunoenhancers in aquaculture.

Dietary non-protein energy source regulates antioxidant status and immune response of barramundi (Lates calcarifer).

This study evaluates the effects of different dietary sources of non-protein energy on growth performance, histological structure, antioxidant status and immune response of barramundi Lates calcarifer. Fish were fed with isoenergetic diets (18 kJ/g) with two types of non-protein energy in the experimental groups and a regular diet was used as the control for 56 days. The specific growth rate and survival of fish were not significantly different between experimental diets. Hepatic histology did not reveal significant differences between dietary treatments at cellular level. The activity of most antioxidant enzymes in the lipid group significantly increased, and the antioxidant capacity in the carbohydrate group was significantly higher than that in other treatments. In the TOR pathway, LST8 homolog (mLST8) expression in the high lipid group was downregulated, and the mechanistic target of rapamycin (mTOR) expression in the high carbohydrate group was downregulated and eIF4E expression was upregulated. The C-reactive protein (CRP) expression in the high lipid and high carbohydrate groups was upregulated. The expression levels of heat shock protein genes in the high lipid group and the high carbohydrate group were significantly downregulated. This study indicates that the lipid diet have less effect in fish immunity but is more suitable as a non-protein ingredient for energy supply for barramundi.

Study on Schizochytrium sp. improving the growth performance and non-specific immunity of golden pompano (Trachinotus ovatus) while not affecting the antioxidant capacity.

A feeding experiment was conducted to determine the effects of Schizochytrium sp. on growth performance, antioxidant capacity and non-specific immunity in golden pompano (Trachinotus ovatus).Two diets were formulated with or without Schizochytrium sp. supplemented (D1:0% and D2: 3%) to feed fish for 8 weeks. Results showed that growth performance, feed intake and survival rate increased significantly with Schizochytrium sp. supplemented (P < 0.05). Feed coefficient rate (FCR) of golden pompano fed the diet supplemented with Schizochytrium sp. was significantly lower than that of fish fed the control diet (P < 0.05). No significant differences were found in antioxidant capacity both in transcriptional level, including nclear factor erythroid-2-related factor-2 (Nrf2), Kelch-like-ECH-associated protein (keap1), catalase (CAT), glutathione peroxidase (GSH-PX) and heme oxygenase 1 (HO-1) and enzyme activity, such as total antioxidant capacity (T-AOC), malondialdehyde (MDA) and superoxide dismutase (SOD) (P > 0.05). Gut amylase and lipase were significantly higher in dietary Schizochytrium sp. supplemented treatment than that in control group (P < 0.05). The relative peroxisome proliferator-activated receptor-α (PPARα) expression level in liver was significantly higher in Schizochytrium sp supplemented treatment than that in control one (P < 0.05). The mRNA expression of myeloid differentiation factor 88 (MyD88), IL-1R-associated kinases 4 (IRAK4), interferon regulating Factor 3 (IRF3), interferon regulating Factor 3(IRF7) and heat shock protein 70 (HSP70) were significantly lower in Schizochytrium sp. supplemented treatment than that in control one (P < 0.05). In Schizochytrium sp. supplemented diet, golden pompano had significantly longer villi length than that in control diet (P < 0.05); muscle thickness in Schizochytrium sp. supplemented diet was thicker than that in control one (P < 0.05) and there were more goblet cells in Schizochytrium sp. treatment (P < 0.05). After the rearing trial, an air exposure trial was conducted. Results showed that the air-exposure mortality (AEM) and mRNA expression level of Nrf2, keap1, CAT, GSH-PX and HO-1 showed no significant difference (P > 0.05). These results indicated that dietary Schizochytrium sp. improved the growth performance and non-specific immunity of golden pompano while made no difference to antioxidant capacity.

Probiotic properties and fatty acid composition of the yeast Kluyveromyces lactis M3. In vivo immunomodulatory activities in gilthead seabream (Sparus aurata).

The aim of this study was to analyze the probiotic potential, fatty acid composition and immunostimulant activities of Kluyveromyces lactis M3 isolated from a hypersaline sediment. For this purpose, K. lactis M3 resistance to different pH, salinities and bile, as well as its antioxidant capability were assayed. Furthermore, total fatty acid composition of the yeast was determined where the dominant fatty acids were palmitic, palmitoleic, oleic and linoleic acids. K. lactis M3 showed no cytotoxic effects on peripheral blood leukocytes. During an in vivo experiment in gilthead seabream (Sparus aurata), dietary K. lactis M3 supplemented at 0.55 or 1.1% of the basal diet enhanced bactericidal activity against Vibrio parahaemolyticus N16, V. harveyi Lg 16/00, and V. anguillarum CECT 43442 compared to fish fed commercial diet (control group). Finally, nitric oxide production, peroxidase activity and skin mucus lectin union levels strongly increased in fish fed K. lactis M3 with respect to the control group. The results suggested that the yeast K. lactis M3 had exhibited high antioxidant capability, and its dietary administration at 0.55 or 1% basal diet had immunostimulant activity for gilthead seabream. For all these reasons, it should be considered an appropriate probiotic candidate for the aquaculture fish industry.

Effects of dietary lipid sources on the intestinal microbiome and health of golden pompano (Trachinotus ovatus).

Replacement of fish oil (FO) with vegetable oils (VO) in diets is economically desirable for the sustainable development of the aquaculture industry. However, inflammation provoked by FO replacement limited its widely application in fish industry. In order to understand the mechanism of VO-induced inflammation, this study investigated the impact of different dietary vegetable oils on the intestinal health and microbiome in carnivorous marine fish golden pompano (Trachinotus ovatus). Three diets supplemented with fish oil (FO, rich in long-chain polyunsaturated fatty acids), soybean oil (SO, rich in 18:2n-6) and linseed oil (LO, rich in 18:3n-3), respectively, were fed on juvenile golden pompano for 8 weeks, and the intestinal histology, digestive enzymes activities, immunity and antioxidant indices as well as intestinal microbiome were determined. The results showed that dietary SO significantly impaired intestinal health, and decreased the number and height of intestinal folds, and muscle thickness, as well as the zonula occludens-1 (zo-1) mRNA expression in intestine. Moreover, the two dietary VO significantly decreased the amylase and lipase activities in intestine, and reduced the trypsin activity in the dietary SO group. Furthermore, the two VO diets increased intestinal acid phosphatase (ACP) activity, while intestinal lysozyme (LZM) activity and serum diamine oxidase (DAO) activity in the SO group were also significantly increased (P < 0.05). Analysis of the intestinal microbiota showed that the two VO diets significantly increased the abundance of intestinal potentially pathogenic bacteria (Mycoplasma and Vibrio) and decreased proportions of intestinal probiotics (Bacillus and Lactococcus), especially in the dietary SO group. These results indicate that complete replacement of FO with VO in diets would induce intestinal inflammation and impair intestinal function, which might be due to changes in intestinal microbiota profiles, and that dietary SO would have a more negative effect compared to dietary LO on intestinal health in T. ovatus.

Effects of prebiotic mixtures on growth performance, intestinal microbiota and immune response in juvenile chu's croaker, Nibea coibor.

Prebiotics has been known to be growth promoter and immunostimulant in aquatic animals. In this study, we investigated the effects of prebiotics on growth performance, intestinal microbiota, short-chain fatty acids (SCFAs) production and immune response of the marine fish, juvenile chu's croaker (Nibea coibor). The fish were fed IG (including 0.5% inulin and 0.5% GOS), GS (0.5% GOS and 0.5% D-sorbitol), IGS (0.33% inulin, 0.33% GOS and 0.33% D-sorbitol) or control diets for 8 weeks. The results showed that the growth performance of the fish was promoted by IG and GS, but not by IGS. The intestinal microbiota in NDC (non-digestible carbohydrates, NDC)-supplemented groups was clearly separated from that of the control, and the highest Shannon and Simpson diversity indices were observed in the IGS group. In the intestine of the croaker, Proteobacteria, Firmicutes, and Bacteroidetes were dominant; among them, 24 taxa revealed a significant difference among groups. Most of these bacteria are able to produce SCFAs, which were significantly increased in all NDC-supplemented groups. Moreover, NDCs were found to activate the immune system of the fish by modulating the serum complements, cytokine levels, lysozyme activities and antioxidant capacity. Furthermore, the results of this study revealed correlations among intestinal microbiota, SCFAs production, innate immunity, antioxidant capacity and digestive enzymes in the croaker fed NDCs. Taken together, our results demonstrated that NDC mixtures might promote growth performance, antioxidant capacity and immune responses of the croaker through modulating the composition of intestinal microbiota and the subsequent SCFAs production, which suggest that NDCs were efficient feed additives for marine fish.

Beneficial effects of tuna hydrolysate in poultry by-product meal diets on growth, immune response, intestinal health and disease resistance to Vibrio harveyi in juvenile barramundi, Lates calcarifer.

This study was conducted to investigate the effects that tuna hydrolysate (TH) supplementation in poultry by-product meal (PBM) diets would have on growth, immunity and resistance to Vibrio harveyi infection in juvenile barramundi, Lates calcarifer. Five isonitrogenous and isocaloric diets containing fishmeal (FM) without TH supplementation (control) and four diets with 10% TH supplementation viz. a FM protein diet (FMBD + TH), a 75% PBM protein diet (LPBM + TH) and two 90% PBM protein diets, either bioprocessed (BPBM + TH) or unprocessed (HPBM + TH), were formulated for juvenile barramundi, Lates calcarifer. The diets were fed to triplicate groups of juvenile barramundi (average pool weight 12.63 ±â€¯0.11 g) for 10 weeks. Significantly (P < 0.05) higher final body weights and specific growth rates were noted in fish fed with FMBD + TH and BPBM + TH diets when compared to the control. Transmission electron microscopy observation of fish distal intestines revealed a significant enhancement of microvilli length in fish fed FMBD + TH and BPBM + TH whereas scanning electron microscopy analysis found no significant difference in microvilli density. A bacterial challenge with Vibrio harveyi was conducted for 14 days after the growth trial to test the immune response and survival of barramundi. In the pre-challenge condition, a significant reduction in blood glucose was found in BPBM + TH compared to the control, and fish in the post-challenge at 24 h had higher glucose levels compared to fish in the pre- and post-challenge conditions at 72 h. The serum lysozyme activity was significantly higher in FMBD + TH and BPBM + TH compared to the control and fish at 72 h post-challenge exhibited higher lysozyme activity in each treatment compared to all dietary groups in the post-challenge condition at 24 h and to HPBM + TH and BPBM + TH in the pre-challenge condition. Fish fed FMBD + TH, LPBM + TH and BPBM + TH diets had significantly higher survival to the bacterial challenge than fish in the control and HPBM + TH. These results showed that PBM supplemented with TH could successfully replace FM without compromising growth, however, bioprocessed PBM supplemented with TH (BPBM + TH) may significantly improve growth performance, immune response, intestinal health and disease resistance in juvenile barramundi.

High percentage of dietary palm oil suppressed growth and antioxidant capacity and induced the inflammation by activation of TLR-NF-κB signaling pathway in large yellow croaker (Larimichthys crocea).

A 70-day feeding trial was conducted to investigate the effects of dietary fish oil (FO) replaced by palm oil (PO) on growth, biochemical and antioxidant response as well as inflammatory response in the liver of large yellow croaker (initial weight 15.87 ±â€¯0.14 g). Four iso-proteic and iso-lipidic experimental diets were formulated with 0% (the control group), 33.3%, 66.7% and 100% FO replaced by PO. Fish fed the diet with 100% PO showed significantly lower growth performance than the control group. As expected, the contents of C16:0, C18:1n-9 and C18:2n-6 were increased with increasing dietary PO levels. There were remarkable increases in total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C) levels in fish fed the diet with 100% PO compared to the control group. Moreover, dietary PO significantly increased activities of plasma alanine transaminase (ALT) and aspartate aminotransferase (AST) in fish fed the diet with 100% PO compared to the control group. The total antioxidant capacity (T-AOC) and the activity of catalase (CAT) in plasma were significantly decreased in fish fed the diet with 100% PO compared to the control group, and meanwhile no significant differences were found in T-AOC and CAT activity in fish fed diets with no more than 66.7% PO. Fish fed the diet with 100% PO exerted significantly higher toll like receptors (TLRs) and myeloid differentiation factor (MyD88) mRNA expression levels than the control group. The IFNγ, IL-1ß and TNFα mRNA expressions were increased with increasing dietary PO levels. The increase of pro-inflammatory gene expression may be due to the activation of NF-κB signaling as the ratio of nucleus p65 to total p65 protein was elevated with the increase of dietary PO levels. These results showed that relatively higher PO levels in diets suppressed the growth and antioxidant capacity as well as induced the inflammatory response by activating TLR-NF-κB signaling pathway in juvenile large yellow croaker.

Adipose tissue contributes to hepatic pro-inflammatory response when dietary fish oil is replaced by vegetable oil in large yellow croaker (Larimichthys crocea): An ex vivo study.

The shortage of fish oil (FO) leads to the extensive use of vegetable oil (VO) in marine fish diets. High replacement percentage of dietary FO by VO induced pro-inflammatory response of adipose tissue (AT) and liver tissue (LT) in large yellow croaker (Larimichthys crocea). Mammalian studies showed that the secretion of cytokines by AT affected the immune response of LT. To investigate whether or not the inflammation response of LT is related to AT in large yellow croaker, LT and AT cells from fish fed FO diet (FOL and FOA) and VO diet (VOL and VOA) were co-cultured in a trans-well system, which resulted in an assembly of the two cells types sharing the culture medium but being separated by the membrane of the insert. Co-culture of FOL and FOA was selected as the control group (FOL-FOA). Results indicated that, when compared with the control group, the expression of pro-inflammatory genes (toll like receptors [TLRs], tumour necrosis factor α [TNFα], interleukin 1ß [IL1ß], suppressor of cytokine signalling 3 [SOCS3] and cyclooxygenase 2 [COX2]) in FOL was significantly increased in the co-culture group of FOL and VOA (FOL-VOA), while the expression of anti-inflammatory genes (arginase I [ArgI] and transforming growth factor ß1 [TGFß1]) in FOL was significantly depressed. On the contrary, a significantly depressed expression of pro-inflammatory genes (TLRs, TNFα, IL1ß and COX2) and increased expression of anti-inflammatory genes (interleukin 10 [IL10]) in VOL was observed in the co-culture group of VOL and FOA (VOL-FOA) when compared with the co-culture group of VOL and VOA (VOL-VOA). The change of immune-related gene expressions in LT cells was attributed to nuclear factor κB (NF-κB) signalling since the expression of the p65 protein was observed to show a similar trend to the expression of pro-inflammatory genes. It is speculated that dietary VO increased the secretion of cytokines, which induced pro-inflammatory response in LT cells. These ex vivo results indicate that AT plays a vital role in LT pro-inflammatory response in fish fed VO diet.

Antioxidant, intestinal immune status and anti-inflammatory potential of Chenopodium ambrosioides L. in fish: In vitro and in vivo studies.

Chenopodium ambrosioides L. has been used for centuries as traditional medicine in many clinical situations. The objectives of this study were first to assess the nutraceutical potential of C. ambrosioides L. extract through analyses of its chemical composition and antioxidant properties, followed by assessing toxicity and antioxidative activities on fish splenocytes. The second one was to perform an in vivo study using dietary C. ambrosioides L. extract (0.0, 0.5, 1.0 and 2.0%; w/w) for 15 and 30 days (2-week and 4-week treatments) to assess associated-intestine health status by short-chain fatty production, antioxidant enzyme activities and anti-inflammatory effects on Pacific red snapper (Lutjanus peru). Non-polar and polar fractions were detected by gas chromatography/mass spectrometry (GC-MS) in C. ambrosioides, of which the most abundant compounds were carvacrol, phytol, squalene, vitamin E and sucrose. The extract of C. ambrosioides L. enhanced a considerable antiradical and reducing power; fish splenocytes responded positively with higher (88%) cell viability than control. The production of nitric oxide and superoxide anion, as well as superoxide dismutase and catalase activities, were also enhanced in splenocytes treated with C. ambrosioides L. The in vivo study results showed that acetate was the major short-chain fatty acid found in fish receiving C. ambrosioides L. after week four. Pro-inflammatory cytokine gene expression in intestine was modulated in fish fed with C. ambrosioides L. at any time of the experimental trial. In addition, the histological findings suggested that its extract did not cause inflammatory damage in intestine. Overall, the results suggest that C. ambrosioides L. is safe for immune cells and promoting intestinal health status of fish through antioxidant and anti-inflammatory effects, making it an interesting additive in functional diets.