A new insight on copper: Promotion of collagen synthesis and myofiber growth and development in juvenile grass carp (Ctenopharyngodon idella).

Copper (Cu) is a trace element, essential for fish growth. In the current study, in addition to growth performance, we first explored the effects of Cu on collagen synthesis and myofiber growth and development in juvenile grass carp (Ctenopharyngodon idella). A total of 1080 fish (11.16 ± 0.01 g) were randomly divided into 6 treatments (3 replicates per treatment) to receive five doses of organic Cu, which were Cu citrate (CuCit) at 0.99 (basal diet), 2.19, 4.06, 6.15, and 8.07 mg/kg, and one dose of inorganic Cu (CuSO4·5H2O at 3.15 mg/kg), for 9 weeks. The results showed appropriate Cu level (4.06 mg/kg) enhanced growth performance, improved nutritional Cu status, and downregulated Cu-transporting ATPase 1 mRNA levels in the hepatopancreas, intestine, and muscle of juvenile grass carp. Meanwhile, collagen content in fish muscle was increased after Cu intake, which was probably due to the following pathways: (1) activating CTGF/TGF-ß1/Smads signaling pathway to regulate collagen transcription; (2) upregulating of La ribonucleoprotein domain family 6 (LARP6) mRNA levels to regulate translation initiation; (3) increasing proline hydroxylase, lysine hydroxylase, and lysine oxidase activities to regulate posttranslational modifications. In addition, optimal Cu group increased myofiber diameters and the frequency of myofibers with diameter >50 µm, which might be associated with upregulation of cyclin B, cyclin D, cyclin E, proliferating cell nuclear antigen, myogenic determining factor (MyoD), myogenic factor 5, myogenin (MyoG), myogenic regulatory factor 4 and myosin heavy chain (MyHC) and downregulation of myostatin mRNA levels, increasing protein levels of MyoD, MyoG and MyHC in fish muscle. Finally, based on percentage weight gain (PWG), serum ceruloplasmin (Cp) activity and collagen content in fish muscle, Cu requirements were determined as 4.74, 4.37 and 4.62 mg/kg diet (CuCit as Cu source) of juvenile grass carp, respectively. Based on PWG and Cp activity, compared to CuSO4·5H2O, the efficacy of CuCit were 131.80% and 115.38%, respectively. Our findings provide new insights into Cu supplementation to promote muscle growth in fish, and help improve the overall productivity of aquaculture.

Transcriptome-based nutrigenomics analysis reveals the roles of dietary taurine in the muscle growth of juvenile turbot (Scophthalmus maximus).

The present study explored transcriptomics and gene regulation variations in the muscle of turbot fed with dietary taurine. A 70-day feeding trial was conducted using turbot (initial body weight: 3.66 ± 0.02 g) fed with different levels of dietary taurine: 0 % (C), 0.4 % (T2), 1.2 % (T4) and 2.0 % (T6). Two methods were used to analyze and verify the taurine effects on muscle growth: (1) real-time quantitative PCR (qRT-PCR) for the key muscle growth-related genes and (2) transcriptomic analysis by next-generation sequencing (NGS). The results showed that 1.2 % of dietary taurine supplementation significantly increased the expression of muscle growth stimulatory genes, including TauT, myoD, Myf5, myogenin and follistatin. And also, the 1.2 % level significantly decreased the expression of the muscle growth-restricting gene (myostatin). Meanwhile, transcriptomics analysis found that 1.2 % dietary taurine supplementation significantly increased the number of up-regulated genes linked to metabolic pathways. In contrast, taurine significantly enriched the actin cytoskeleton and metabolic pathways in the T4 and T2 groups, respectively. These findings align with the gene ontology (GO) analysis, which indicated a higher number of cellular component (CC) gene expressions at a 1.2 % of dietary taurine compared to a 0.4 % of dietary taurine supplementation. In conclusion, dietary taurine had positive impacts on the growth-stimulatory genes. Moreover, 1.2 % of dietary taurine supplementation is important to the metabolic pathway enrichment.

Effects of dietary nucleotide and yeast cell wall on growth performance, feed utilization, anti-oxidative and immune response of grass carp (Ctenopharyngodon idella).

A 70-day feeding trial was conducted to study the effects of dietary nucleotide, yeast cell wall (containing 20% ß -glucan) and their combination on growth performance, feed utilization and immune response of grass carp (Ctenopharyngodon idella) with 69.97 ± 0.05 g of initial body weight. Four isonitrogenous (about 38% crude protein) and isolipidic (about 5% crude lipid) diets were established. Based on the control diet (CD), the other three experimental diets were prepared by adding 0.01% of nucleotide (NT), 0.1% of yeast cell wall (YCW) and NT (0.01%) +YCW (0.1%), respectively. Results showed that no significant difference was found in survival of grass carp ranging from 94.44% to 97.78% among all the groups (P > 0.05). Compared with the control group, weight gain rate, muscle crude protein content, serum protein, trypsin and chymotrypsin activities in midgut, lysozyme and immunoglobulin M in serum significantly increased in fish fed the YCW diet (P < 0.05). The significantly highest weight gain rate, villus height and digestive enzyme activities in midgut and innate immune parameters in serum were found in fish fed the NT + YCW diet (P < 0.05). The gene expressions of ß-defensin, hepcidin, il-10 and tgf-ß1 in the midgut, and tor and s6k1 in liver significantly increased in fish fed the NT + YCW diet. Meanwhile, the gene expressions of il-1ß and tnf-α in the midgut decreased significantly (P < 0.05). The liver histology showed the better development in dietary NT and/or YCW supplemented groups than those in the control group. In conclusion, combination of dietary NT and YCW had significantly synergetic improvements on the growth, feed utilization, digestive enzymes, innate immunity and histology of midgut and liver of grass carp.

Vitamin D3/VDR inhibits inflammation through NF-κB pathway accompanied by resisting apoptosis and inducing autophagy in abalone Haliotis discus hannai.

Vitamin D3 is believed to be a contributing factor to innate immunity. Vitamin D receptor (VDR) has a positive effect on inhibiting nuclear factor κB (NF-κB)-mediated inflammation. The underlying molecular mechanisms remain unclear, particularly in mollusks. Consequently, this study will investigate the process of vitamin D3/VDR regulating NF-κB pathway and further explore their functions on inflammation, autophagy, and apoptosis in abalone Haliotis discus hannai. Results showed that knockdown of VDR by using siRNA and dsRNA of VDR in vitro and in vivo led to more intense response of NF-κB signaling to lipopolysaccharide and higher level of apoptosis and autophagy. In addition, 1,25(OH)2D3 stimulation after VDR silencing could partially alleviate apoptosis and induce autophagy. Overexpression of VDR restricted the K48-polyubiquitin chain-dependent inhibitor of κB (IκB) ubiquitination and apoptosis-associated speck-like protein containing CARD (ASC) oligomerization. Besides, VDR silencing resulted in increase of ASC speck formation. In further mechanistic studies, we showed that VDR can directly bind to IκB and IKK1 in vitro and in vivo. In the feeding trial, H&E staining, TUNEL, and electron microscope results showed that vitamin D3 deficiency (0 IU/kg) could recruit more basophilic cells and increase more TUNEL-positive apoptotic cells and lipid droplets (LDs) than vitamin D3 supplement (1000 IU/kg and 5000 IU/kg). In summary, abalone VDR plays a negative regulator role in NF-κB-mediated inflammation via interacting with IκB and inhibiting ubiquitin-dependent degradation of IκB. Vitamin D3 in combination with VDR is essential to establish a delicate balance between autophagy and apoptosis in response to inflammation.

Role of creatine supplementation on the myofibre characteristics and muscle protein synthesis of grass carp (Ctenopharyngodon idellus).

To assess the role of dietary creatine on myofibre characteristics and protein synthesis in muscle, we fed grass carp (Ctenopharyngodon idellus, initial body weight: 88·47 ± 1·44 g) creatine-supplemented diets (1·84, 5·91, 8·48 and 15·44 g/kg diet) for 8 weeks. Creatine supplementation did not affect growth performance, but significantly increased creatine contents in muscle and liver. At 8·48 g/kg, creatine decreased the activities of alanine transaminase and aspartate aminotransferase in serum and improved hardness and chewiness of muscle due to shorter myofibre mean diameter, higher myofibre density and the frequencies of the diameters of classes I and III and collagen content, longer sarcomere length and upregulated mRNA levels of slow myosin heavy chains. Creatine supplementation upregulated the mRNA expressions of myogenic regulatory factors. The 8·48 g/kg creatine-supplemented diet significantly increased the contents of protein, total amino acids (AA), essential AA and free flavour AAs in muscle, the protein levels of insulin-like growth factor I, myogenic differentiation antigen and PPAR-γ coactlvator-1α in muscle and stimulated the phosphorylation of target of rapamycin (TOR) pathway in muscle. In summary, 8·48 mg/kg creatine improved fish health and skeletal muscle growth and increased hardness and protein synthesis in muscle of grass carp by affecting myofibre characteristics and the TOR signalling pathway. A second-order regression model revealed that the optimal dietary creatine supplementation of grass carp ranges between 8·48 and 12·04 g/kg.

Vitamin D3 deficiency induced intestinal inflammatory response of turbot through nuclear factor-κB/inflammasome pathway, accompanied by the mutually exclusive apoptosis and autophagy.

Vitamin D3 (VD3) participated widely in the nuclear factor-κB (NF-κB)-mediated inflammation, apoptosis, and autophagy through the vitamin D receptor (VDR). However, the molecular mechanisms remain not understood in teleost. The present study investigated the functions of VD3/VDR on intestinal inflammation, autophagy, and apoptosis of turbot in vivo and in vitro. Triple replicates of 30 fish were fed with each of three diets with graded levels of 32.0 (D0), 1012.6 (D1), and 3978.2 (D2) IU/kg VD3. Obvious intestinal enteritis was observed in the D0 group and followed with dysfunction of intestinal mucosal barriers. The intestinal inflammatory response induced by VD3 deficiency was regulated by the NF-κB/inflammasome signalling. The promotion of intestinal apoptosis and suppression of intestinal autophagy were also observed in the D0 group. Similarly, VD3 deficiency in vitro induced more intense inflammation regulated by NF-κB/inflammasome signalling. The mutually exclusive apoptosis and autophagy were also observed in the group without 1,25(OH)2D3 in vitro, accompanied by similar changes in apoptosis and autophagy increased apoptosis. The gene expression of VDRs was significantly increased with the increasing VD3 supplementation both in vivo and in vitro. Moreover, VDR knockdown in turbot resulted in intestinal inflammation, and this process relied on the activation of inflammasome mediated by NF-κB signalling. Simultaneously, intestinal apoptosis was promoted, whereas intestinal autophagy was inhibited. In conclusion, VD3 deficiency could induce intestinal inflammation via activation of the NF-κB/inflammasome pathway, intestinal apoptosis, and autophagy formed a mutually exclusive relation in teleost. And VDR is the critical molecule in those processes.

Effects of recombinant anti-lipopolysaccharide factor expressed by Pichia pastoris on the growth performance, immune response and disease resistance of Litopenaeusvannamei.

The codon-optimized anti-lipopolysaccharide factor (ALF) sequence was introduced into pPICZαA vector and transformed into Pichia pastoris GS115. The recombinant ALF yeast supernatant (rALF-mix) was freeze-dried and evaluated as a feed additive for Litopenaeus vannamei. It was found by antibacterial activity test in vitro that the rALF-mix had antibacterial activity under different pH and temperature conditions. The 0, 0.00375%, 0.0075%, 0.015%, 0.03% and 0.06% of rALF-mix were added respectively to make the six experimental diets. After a 10-week feeding trial with shrimps (2.36 ± 0.02 g), it was found that the weight gain rate (WGR) and protein efficiency ratio (PER) of shrimp in the groups with 0.0075%, 0.015% and 0.03% of dietary rALF-mix supplementation were significantly higher than those in the control group (P < 0.05). Dietary rALF-mix supplementation significantly increased the total haemocyte count, respiratory burst, phagocytic activity, total anti-oxidative capacity (T-AOC), phenol oxidase activity, nitric oxide synthase activity, lysozyme (LYZ) activity, serum antibacterial capacity in the hemolymph and the T-AOC, LYZ in the hepatopancreas of shrimps (P < 0.05). The malondialdehyde contents in hemolymph and hepatopancreas were significantly decreased (P < 0.05). Meanwhile, the expression levels of toll, immune deficiency, heat shock protein 70, crustin and lipopolysaccharide-ß-glucan binding protein in the gill of shrimps were significantly increased (P < 0.05). After the challenge test, it was showed that dietary rALF-mix supplementation significantly improved the resistance of L. vannamei to Vibrio parahaemolyticus (P < 0.05). In conclusion, the rALF-mix can be used as a functional feed additive to improve the growth, immunity and disease resistance of shrimp. Based on the quadratic regression analysis for WGR, the optimal supplemental level of rALF-mix in diet for shrimp was estimated to be 0.02813%.

Biotin alleviates hepatic and intestinal inflammation and apoptosis induced by high dietary carbohydrate in juvenile turbot (Scophthalmus maximus L.).

Excessive dietary carbohydrate commonly impairs the functions of liver and intestine in carnivorous fish. In the present study, a 10-week feeding trial was carried out to explore the regulation of biotin on the hepatic and intestinal inflammation and apoptosis in turbot (Scophthalmus maximus L.) fed with high carbohydrate diets. Three isonitrogenous and isolipidic experimental diets were designed as follows: the CC diet with 18.6% of carbohydrate and 0.04 mg/kg of biotin, the HC diet with 26.9% of carbohydrate and 0.05 mg/kg of biotin, and the HCB diet with 26.9% of carbohydrate and 1.62 mg/kg of biotin. Results showed that high dietary carbohydrate (HC diet) impaired the morphology of liver and intestine, however, inclusion of dietary biotin (HCB diet) normalized their morphology. Inflammation-related gene expression of nuclear factor κB p65 (nf-κb p65), tumor necrosis factor α (tnf-α), interleukin-1ß (il-1ß), il-6 and il-8, and the protein expression of NF-κB p65 in the liver and intestine were significantly up-regulated in the HC group compared to those in the CC group (P < 0.05), the HCB diet decreased their expression compared to the HC group (P < 0.05). The gene expression of il-10 and transforming growth factor-ß (tgf-ß) in the liver and intestine were significantly decreased in the HC group compared to the CC group (P < 0.05), and inclusion of dietary biotin increased the il-10 and tgf-ß expression in the liver and intestine (P < 0.05). Moreover, compared to the CC group, the HC group had a stronger degree of DNA fragmentation and more TUNEL-positive cells in the liver and intestine, and the HCB group had a slighter degree of DNA fragmentation and fewer TUNEL-positive cells compared to the HC group. Meanwhile, the gene expression of B-cell lymphoma protein-2-associated X protein (bax) and executor apoptosis-related cysteine peptidase 3 (caspase-3) were significantly up-regulated and the gene expression of B-cell lymphoma-2 (bcl-2) was significantly down-regulated both in the liver and intestine in the HC group compared with those in the CC group (P < 0.05). Inclusion of dietary biotin significantly decreased the bax and caspase-3 mRNA levels and increased bcl-2 mRNA level in the liver and intestine (P < 0.05). In conclusion, high dietary carbohydrate (26.9% vs 18.6%) induced inflammation and apoptosis in liver and intestine. Supplementation of biotin (1.62 mg/kg vs 0.05 mg/kg) in diet can alleviate the high-dietary-carbohydrate-induced hepatic and intestinal inflammation as well as inhibit apoptosis in turbot. The present study provides basic data for the application of biotin into feed, especially the high-carbohydrate feed for turbot.

Intestinal Microbiota Mediates Gossypol-Induced Intestinal Inflammation, Oxidative Stress, and Apoptosis in Fish.

Gossypol, the main antinutritional factor in cottonseed protein concentrate (CPC), could affect the growth conditions of fish, but the underlying mechanism remains unclear. In this study, an 8-week feeding trial was carried out to investigate the effects of gossypol on Nile tilapia (Oreochromis niloticus). Three experimental diets were designed, including control diet (CON), control diet supplemented with 150 mg/kg gossypol (ML), and 300 mg/kg gossypol (MH). 16S rRNA gene sequencing showed that gossypol significantly reduced the richness and diversity of the gut microbiota. Untargeted metabolite analysis revealed that most metabolites were down-regulated by gossypol, and riboflavin was the key metabolite with significant difference between CON-treated and gossypol-treated groups. Gossypol caused intestinal inflammation, oxidative stress, and apoptosis. Through fecal bacteria transplantation experiments, we demonstrated that intestinal microbiota mediated gossypol-induced negative effects, suggesting that intestinal microbiota and its metabolite may account for the harmful effects of gossypol.

Dietary recombinant human lysozyme improves the growth, intestinal health, immunity and disease resistance of Pacific white shrimp Litopenaeus vannamei.

The present study was conducted to investigate the effects of dietary recombinant human lysozyme (RHL) on the growth, immune response, anti-oxidative activity, intestinal morphology, intestinal microflora and disease resistance of shrimp Litopenaeus vannamei. Shrimps with an initial body weight of 2.36 ± 0.02 g were fed diets supplemented with 0 (control group, R0), 0.0025% (R1), 0.005% (R2), 0.01% (R3), 0.02% (R4) and 0.04% (R5) of RHL, respectively. After a 10-week feeding trial, the final body weight, survival rate, weight gain ratio and protein efficiency rate of the shrimps in dietary RHL supplemented groups were significantly higher than that in the control group, while feed conversion ratio was significantly lower (P < 0.05). The total haemocyte count, total anti-oxidative capacity, respiratory burst, activities of phagocytosis, nitric oxide synthase, phenol oxidase and lysozyme in serum were significantly higher in dietary RHL supplemented groups than those in the control group (P < 0.05). Meanwhile, the intestinal pile height and wall thickness were significantly higher in dietary RHL supplemented groups than those in the control group (P < 0.05). Dietary RHL significantly improved the expressions of immune-related genes in gill, such as lipopolysaccharide-ß-glucan binding protein, Toll, immune deficiency, heat shock protein 70 and Crustin (P < 0.05). The abundance of proteobacteria and bacteroidetes in intestine was higher, while the abundance of firmicutes and cyanobacteria was lower than those in the control group at the phylum level. In addition, dietary RHL supplementation significantly improved the protective ability of shrimp against V. parahaemolyticus infection (P < 0.05). Based on the broken-line model analysis for weight gain ratio after the feeding trial, the optimal level of dietary RHL supplementation for shrimp was estimated to be 0.006375%.

Arginine Regulates TOR Signaling Pathway through SLC38A9 in Abalone Haliotis discus hannai.

Arginine plays an important role in the regulation of the target of the rapamycin (TOR) signaling pathway, and Solute Carrier Family 38 Member 9 (SLC38A9) was identified to participate in the amino acid-dependent activation of TOR in humans. However, the regulations of arginine on the TOR signaling pathway in abalone are still unclear. In this study, slc38a9 of abalone was cloned, and the slc38a9 was knocked down and overexpressed to explore its function in the regulation of the TOR signaling pathway. The results showed that knockdown of slc38a9 decreased the expression of tor, ribosomal s6 protein kinase (s6k) and eukaryotic translation initiation factor 4e (eif4e) and inhibited the activation of the TOR signaling pathway by arginine. Overexpression of slc38a9 up-regulated the expression of TOR-related genes. In addition, hemocytes of abalone were treated with 0, 0.2, 0.5, 1, 2 and 4 mmol/L of arginine, and abalones were fed diets with 1.17%, 1.68% and 3.43% of arginine, respectively, for 120 days. Supplementation of arginine (0.5-4 mmol/L) increased the expressions of slc38a9, tor, s6k and eif4e in hemocytes, and abalone fed with 1.68% of dietary arginine showed higher mRNA levels of slc38a9, tor, s6k and eif4e and phosphorylation levels of TOR, S6 and 4E-BP. In conclusion, the TOR signaling pathway of abalone can be regulated by arginine, and SLC38A9 plays an essential role in this regulation.

Ascorbic Acid Regulates the Immunity, Anti-Oxidation and Apoptosis in Abalone Haliotis discus hannai Ino.

The present study was conducted to investigate the roles of ascorbic acid (AA) in immune response, anti-oxidation and apoptosis in abalone (Haliotis discus hannai Ino). Seven semi-purified diets with graded levels of AA (0, 50, 100, 200, 500, 1000 and 5000 mg/kg) were fed to abalone (initial weight: 12.01 ± 0.001 g, initial shell length: 48.44 ± 0.069 mm) for 100 days. The survival, weight gain rate and daily increment in shell length were not affected by dietary AA. The AA content in the gill, muscle and digestive glands of abalone was significantly increased by dietary AA. In terms of immunity, dietary AA significantly improved the total hemocyte count, respiratory burst and phagocytic activity in hemolymph, and lysozyme activity in cell-free hemolymph (CFH). In the digestive gland, the TLR-MyD88-dependent and TLR-MyD88-independent signaling pathways were suppressed by dietary AA supplementation. The mRNA levels of ß-defensin and arginase-I in the digestive gland were significantly increased by dietary AA. In the gill, only the TLR-MyD88-dependent signaling pathway was depressed by dietary AA to reduce inflammation in abalone. The level of mytimacin 6 in the gill was significantly upregulated by dietary AA. After Vibrio parahaemolyticus infection, the TLR signaling pathway in the digestive gland was suppressed by dietary AA, which reduced inflammation in the abalone. In terms of anti-oxidation, superoxide dismutase, glutathione peroxidase and catalase activities, as well as total anti-oxidative capacity and reduced glutathione content in CFH, were all significantly upregulated. The malondialdehyde content was significantly downregulated by dietary AA. The anti-oxidative capacity was improved by triggering the Keap1-Nrf2 pathway in abalone. In terms of apoptosis, dietary AA could enhance the anti-apoptosis ability via the JNK-Bcl-2/Bax signaling cascade in abalone. To conclude, dietary AA was involved in regulating immunity, anti-oxidation and apoptosis in abalone.

Creatine improves the flesh quality of Pacific white shrimp (Litopenaeus vannamei) reared in freshwater.

Creatine improves flesh quality on mammalian but studies on crustaceans are scarce. In the present study, diets with six levels of creatine (1.23, 2.58, 5.12, 8.28, 14.12, 24.49 g kg-1 diet) were hand-fed to juvenile Litopenaeus vannamei (IBW: 1.50 ± 0.02 g) reared in freshwater for 46 days. Results showed creatine supplementation did not affect the growth performance (FBW: 17.04 ± 1.28 g) or the content of guanidinoacetic acid in muscle and hepatopancreas whereas significantly increased muscular creatine content. Diet with 8.28 g kg-1 creatine significantly increased muscular hardness and chewiness by decreasing myofiber diameter and increasing myofiber density. Additionally, creatine downregulated the mRNA expression of fast sMyHC1, sMyHC2, sMyHC6a and upregulated slow sMyHC5 and sMyHC15 mRNA expression. Muscular protein, collagen, total amino acid and flavor amino acid contents increased with creatine supplementation. In conclusion, the diet with 8.28 g kg-1 creatine improved the flesh quality of L. vannamei.

Dietary taurine modulates hepatic oxidative status, ER stress and inflammation in juvenile turbot (Scophthalmus maximus L.) fed high carbohydrate diets.

This study was conducted to explore the beneficial role of taurine against chronic high carbohydrate diet-induced oxidative stress, endoplasmic reticulum (ER) stress and inflammation, and to understand the underlying molecular mechanisms in turbot. Two 10-week feeding trials were simultaneously conducted. For the one, six experimental diets with graded levels of taurine supplementation (0, 0.4%, 0.8%, 1.2%, 1.6% and, 2.0%, respectively) and 15% of carbohydrate were used. For the other one, three graded levels of dietary taurine supplementation (0.4%, 1.2% and 2.0%, respectively) with 21% of carbohydrate were used. The results showed that higher expression level of inflammation cytokines and ER stress related genes were detected in higher dietary carbohydrate group. In both feeding trials, 1.2% of dietary taurine supplementation improved anti-oxidative status by decreasing the content of malondialdehyde, increasing the catalase activity and total anti-oxidative capacities. In feeding trial 1, appropriate taurine supplementation lowered contents of tumour necrosis factor-a, interleukin-6, aspartate aminotransferase and alkaline phosphatase in plasma, and decreased the expressions of pro-inflammatory cytokines, such as interleukin-8 (il-8) and interferon-γ (ifn-γ). Furthermore, dietary taurine reduced ER stress by decreasing the mRNA levels of activating transcription factor 6, protein kinase R-like endoplasmic reticulum kinase and G protein-coupled receptor 78. The optimal dietary taurine content was estimated as 1.40% based on the analysis of specific growth rate. In feeding trial 2, dietary taurine supplementation attenuated liver inflammation partly referring to significantly down-regulated mRNA levels of nuclear transcription factor-κB p65, ifn-γ, interleukin1ß and up-regulate the transcript of ribosomal protein S6 kinase 1. Dietary taurine supplementation in feeding trial 2 significantly increased the Nrf2-related factor 2 protein level and decreased the NFκB p65 protein level only at 21% of dietary carbohydrate level. Taurine can alleviate the oxidative damage and inflammation caused by 21% of dietary carbohydrate to a certain degree. Overall, the present study confirmed that dietary taurine supplementation improved growth performance and anti-oxidative response, and reduced liver inflammatory and ER stress processes induced by high dietary carbohydrate in turbot.

The effect of dietary cecropin AD on intestinal health, immune response and disease resistance of juvenile turbot (Scophthalmus maximus L.).

Cecropin AD (CAD) is a commercial cationic antimicrobial peptide that has been seldom studied in marine fish. This study investigated the effects of dietary CAD on intestinal health, immune response, disease resistance, and growth performance of turbot. A diet using fishmeal and plant protein as the main protein resources was used as the control (crude protein 53%, crude lipid 12%). CAD was supplemented into the control diet at the level of 250, 500, 750, and 1000 mg kg-1 to formulate four experimental diets, C1, C2, C3, and C4, respectively. No significant difference was observed in fish growth performance, feed utilization efficiency and whole-body composition among all groups. Dietary CAD significantly increased the activity of lysozyme and complement component 3 level in both serum and distal intestine (DI), as well as the immunoglobulin M content in DI. The gene expression of immune cytokines such as IFN-γ, IL-1ß, and chemokine SmCCL19, and the goblet cell number in DI were also significantly increased by dietary CAD supplementation. Compared with the control group, the microbiota analysis indicated group C4 showed significantly decreased α-diversity, obvious alternation in dominant bacteria composition at phylum level, different clustering, and significantly decreased relative abundance of Lactobacillus. Besides, the relative abundance of Bacteroides was significantly decreased in groups C1, C3, and C4. In addition, the lowest mortality of turbot challenged with Edwardsiella tarda was observed in fish fed diets C2 and C3. In conclusion, moderate levels of CAD in diet of turbot improved the intestinal immune response without disrupting the intestinal bacterial community, and enhanced the disease resistance. However, dietary CAD at 1000 mg kg-1 greatly affected the intestinal bacterial composition and showed potentially inhibitory effects towards Lactobacillus.

Effects of Yeast Autolysate in the Practical Diet on the Growth Performance, Immune Response, and Disease Resistance of Pacific White Shrimp.

The present study was conducted to investigate the effects of supplementing the practical diet with yeast autolysate (YA) on the growth performance, immunity, and disease resistance of Pacific white shrimp Litopenaeus vannamei. Four isonitrogenous and isolipidic practical diets were formulated. The relatively high-fish-meal control diet contained 25% fish meal without YA supplementation (E1). The other control diet contained 20% fish meal without YA (E2). With the E2 diet as the basis, two additional experimental diets were created by further supplementation with 1% YA (E3) and 2% YA (E4). The shrimp (initial weight: 0.30 ± 0.02 g) were fed with the four experimental diets for 8 weeks and then challenged with Vibrio parahaemolyticus. The results indicated that there were no significant differences in survival rate (SR) or feed intake (FI) among these groups. The weight gain rate (WGR) of group E1 was not significantly different from that of groups E3 and E4. The feed conversion ratio (FCR) in group E4 was lower than that of group E2, and group E4 had the highest protein efficiency ratio (PER). The total hemocyte counts (THC) and lysozyme activities in group E3 and group E4 were significantly higher than those of the other groups. Group E3 had the highest respiratory burst (RB). After V. parahaemolyticus administration, group E3 and group E4 had significantly lower cumulative mortalities than group E1 did. In conclusion, the 20% fish meal diet without YA supplementation (E2) yielded a significantly lower growth rate than the 25% fish meal diet without YA supplementation (E1) did. Furthermore, the Pacific white shrimp that received dietary supplementation with 1% YA demonstrated improved growth rate, immune response, and resistance to the V. parahaemolyticus challenge compared with those that were fed the 20% fish meal diet without YA supplementation (E2).

Impacts of replacement of dietary fish oil by vegetable oils on growth performance, anti-oxidative capacity, and inflammatory response in large yellow croaker Larimichthys crocea.

A 12-week feeding trial was conducted to evaluate the effects of replacement of dietary fish oil by palm and linseed oils on the growth performance, anti-oxidative capacity, and inflammatory responses of large yellow croaker (initial body weight: 36.82 ± 0.29 g). The control diet was designed to contain 6.5% of fish oil, and named as FO. On the basis of the control diet, the fish oil was 100% replaced by palm and linseed oils, and these two diets were named as PO and LO, respectively. Results showed that the specific growth rate significantly reduced in the PO and LO groups. Crude lipid content in liver of fish fed FO was significantly lower than that in the PO and LO groups. Fatty acid composition in liver reflected the dietary input. Compared with the FO group, palm oil inclusion significantly decreased expressions of superoxide dismutase 1, catalase, and nuclear factor erythroid 2-related factor 2 in liver, while linseed oil inclusion significantly increased expressions of above genes. However, both of the PO and LO groups had a significantly lower total anti-oxidative capacity in liver than the fish fed FO. Dietary palm and linseed oils significantly decreased expressions of arginase I and interleukin 10, and increased expressions of tumor necrosis factor α, interleukin 1ß, toll-like receptor 22, and myeloid differentiation factor 88 in liver. In conclusion, total replacement of dietary fish oil by palm and linseed oils could suppress growth performance and liver anti-oxidative capacity, and induce inflammatory responses of large yellow croaker.

Dietary daidzein improved intestinal health of juvenile turbot in terms of intestinal mucosal barrier function and intestinal microbiota.

A 12-week feeding trial was conducted to investigate the effect of dietary daidzein on the intestinal mucosal barrier function and the intestinal microbiota profile of juvenile turbot (Scophthalmus maximus L.). Three isonitrogenous and isolipidic experimental diets were formulated to contain 0 (FM), 40 (D.40) and 400 (D.400) mg kg-1 daidzein, respectively. Fish fed D.400 had significantly lower growth performance than fish fed D.40. Dietary daidzein significantly increased the feed efficiency, while significantly decreased the feed intake. Daidzein supplementation increased the activity of total anti-oxidative capacity and the gene expression of anti-inflammatory cytokine transforming growth factor-ß1, Mucin-2 and tight junction proteins (Tricellulin, Zonula occludens-1 transcript variant 1, Zonula occludens-1 transcript variant 2 and Claudin-like and Occludin), and down-regulated the gene expression of pro-inflammatory cytokines interleukin-1ß and tumor necrosis factor-α in the intestine of turbot. Dietary daidzein increased intestinal microbial diversities, the abundance of several short chain fatty acids producers, and decreased the abundance of some potential pathogenic bacteria. However, D.400 had dual effects on lactic acid bacteria and increased the abundance of potential harmful bacterium Prevotella copri. Collectively, dietary daidzein at the levels of 40 and 400 mg kg-1 could enhance the intestinal mucosal barrier function and alter the intestinal microbiota of turbot. However, high dose of daidzein must be treated with caution for its unclear effects on intestinal microbiota of turbot in the present study.

[Ultrasound-guided needle-knife for trigger finger].

OBJECTIVE: To explore the efficacy of ultrasound-guided needle-knife with precise three-dimensional stereotactic localization of ashi points for stenosing tenosynovitis of flexor tendon (trigger finger). METHODS: A total of 74 patients were randomly divided into an observation group and a control group, 37 cases in each group. The patients in the observation group were treated with ultrasound-guided intrathecal injection and releasing method of needle-knife, while the patients in the control group were treated with ultrasound-guided intrathecal injection. The self-made 9-score scale of trigger finger was recorded before treatment, immediately after treatment, 1 month and 3 months after treatment; the curative effect of the two groups was evaluated. RESULTS: The results of self-made 9-score scale in the observation group immediately after treatment, 1 month and 3 months after treatment were lower than that before treatment (all P<0.01); the scores in the observation group were lower than those in the control group at each time point after treatment (all P<0.01). The excellent and good rate immediately after treatment was 100.0% (37/37) in the observation group, which was superior to 8.1% (3/37) in the control group (P<0.05); the cured rates in the observation group were 100.0% (37/37) 1 month after treatment and 97.3% (36/37) 3 months after treatment, which were superior to 13.5% (5/37) and 10.8% (4/37) in the control group, respectively (P<0.05). CONCLUSION: The needle-knife with three-dimensional stereotaxic location of ashi point could significantly improve the symptoms of trigger finger, with superior immediate and long-term efficacy.

Citric acid mitigates soybean meal induced inflammatory response and tight junction disruption by altering TLR signal transduction in the intestine of turbot, Scophthalmus maximus L.

A 12-week feeding trial was conducted to investigate the effect of citric acid on the involvement of TLRs in the soybean meal induced inflammatory response and tight junction disruption in the distal intestine of juvenile turbot (Scophthalmus maximus L.). Four isonitrogenous and isolipidic practical diets were formulated: fish meal-based diet (FM); 40% fish meal protein in FM replaced with soybean meal protein (SBM); SBM + 1.5% citric acid and SBM + 3% citric acid. Compared to the FM, diet SBM significantly increased the gene expression of TLRs (TLR2, TLR3, TLR5b, TLR9, TLR21, TLR22) and MyD88, as well as TLR related molecules (NF-κB, IRF-3, p38 and JNK), which were remarkably reduced by dietary citric acid. Similarly, citric acid supplementation in SBM markedly depressed gene expression of pro-inflammatory cytokines (TNF-α and IFN-γ) and pore-forming tight junction protein Claudin-7, and enhanced gene expression of the anti-inflammatory cytokine TGF-ß1 and TJ proteins related to the decrease in paracellular permeability (Claudin-3, Claudin-4, Occludin, Tricellulin and ZO-1). Compared to the SBM, the concentration of IgM and C4 in serum was significantly reduced by dietary citric acid. In brief, dietary citric acid could synchronously inhibit TLRs-dependent inflammatory response regulated by NF-κB and IRF3, as well as cause TLRs-dependent tight junction disruption modulated by p38 and JNK. Therefore, citric acid could function on mitigating soybean meal induced enteropathy in the distal intestine of juvenile turbot.