Momordica charantia saponins administration in low-protein-high-carbohydrate diet improves growth, blood biochemical, intestinal health and microflora composition of juvenile common carp (Cyprinus carpio).

An 8-week feeding trial was conducted to explore the feasibility of Momordica charantia saponins (MCS) administration to facilitate the protein-sparing action of high carbohydrate in diets for juvenile common carp (Cyprinus carpio) with initial mass of 5.41 ± 0.02 g. Based on our previous study, four diets with different the ratio of protein and carbohydrate (P/C ratio) were designed: 32%P/40%C, 30%P/43%C, 28%P/46%C, 28%P/46%C supplemented with 0.16% MCS (28%P/46%C + MCS). Each diet treatment was divided into 3 replicates. Results revealed that 30%P/43%C group increased growth performance and intestinal digestion, decreased intestinal inflammation, and optimized the intestinal microbiota compared to 32%P/40%C group, which presented the stronger protein-sparing action of high carbohydrate. But if the P/C ratio reduced to 28%P/46%C or less, the saving action would be restrained. However, compared to the 30%P/43%C and 28%P/46%C groups, 28%P/46%C + MCS group significantly elevated growth performance and activities of digestive enzymes and antioxidative enzymes, whilst the opposite trend occurred in the contents of glucose, triglyceride, total cholesterol, low density lipoprotein cholesterol, blood urea nitrogen, glutamic oxalacetic transaminase, glutamic-pyruvic transaminase and malondialdehyde. In addition, 28%P/46%C + MCS group markedly upregulated the expressions of GH/IGF axis genes, genes involved in protein synthesis, antioxidant genes and anti-inflammatory cytokine, whilst the opposite trend occurred in the expressions of pro-inflammatory cytokines. Moreover, 28%P/46%C + MCS group obtained the remarkably higher Enterococcus proportion and lower Lactococcus proportion compared to the 30%P/43%C and 28%P/46%C groups, whereas the opposite occurred in 30%P/43%C group, which indicated that there existed differences in the improvement mechanism on intestinal microflora composition between MCS and appropriate P/C ratio. Combined with the above mentioned changes in our research, we concluded that 0.16% MCS administration in a 28%P/46%C diet could facilitate the protein-sparing action of high carbohydrate in diets for common carp, which could decrease the 5% dosage of soybean meal and synchronously reduce the 4% crude protein of diets without affecting the growth and immune ability for common carp.

Salinity levels affect the lysine nutrient requirements and nutrient metabolism of juvenile genetically improved farmed tilapia (Oreochromis niloticus).

This 62-d research aimed to evaluate the effects of dietary lysine levels (DLL) and salinity on growth performance and nutrition metabolism of genetically improved farmed tilapia (GIFT) juveniles (Oreochromis niloticus). Six diets with lysine supplementation (1·34, 1·70, 2·03, 2·41, 2·72 and 3·04 % of DM) were formulated under different cultured salinities in a two-factorial design. The results indicated that supplemental lysine improved the specific growth rate (SGR) and weight gain (WG) and decreased the feed conversion ratio (FCR). Meanwhile, the fish had higher SGR and WG and lower FCR at 8 ‰ salinity. Except for moisture, the whole-body protein, lipid and ash content of GIFT were increased by 8 ‰ salinity, which showed that DLL (1·34 %) increased the whole-body fat content and DLL (2·41 %) increased whole-body protein content. Appropriate DLL up-regulated mRNA levels of protein metabolism-related genes such as target of rapamycin, 4EBP-1 and S6 kinase 1. However, 0 ‰ salinity reduced these protein metabolism-related genes mRNA levels, while proper DLL could improve glycolysis and gluconeogenesis mRNA levels but decrease lipogenesis-related genes mRNA levels in liver. 0 ‰ salinity improved GLUT2, glucokinase and G6 Pase mRNA levels; however, sterol regulatory element-binding protein 1 and fatty acid synthase mRNA levels were higher at 8 ‰ salinity. Moreover, 8 ‰ salinity also increased plasma total protein and cholesterol levels and decreased glucose levels. These results indicated that the recommended range of lysine requirement under different salinity was 2·03-2·20 % (0 ‰) and 2·20-2·41 % (8 ‰) and 8 ‰ salinity resulted in higher lysine requirements due to changes in the related nutrient metabolism, which might provide useful information for designing more effective feed formulations for GIFT cultured in different salinity environment.

The role of dietary chromium supplementation in relieving heat stress of juvenile blunt snout bream Megalobrama amblycephala.

The present study assessed the role of dietary chromium (Cr) supplementation in relieving heat stress (HS) of juvenile blunt snout bream Megalobrama amblycephala. The supplemented Cr contents by chromium picolinate (Cr-Pic) was 0 mg/kg (control group), 0.4 mg/kg, 1.6 mg/kg and 12.0 mg/kg, respectively. The fish continued to be fed four diets at suitable temperatures (26 °C) for 2 weeks, and then the temperature was then heated up to 33 °C through thermo-regulated system. The results showed that Cr supplementation had no significant effect on the immune indices and antioxidant indices before HS (P > 0.05). However, Cr supplementation played an important role in relieving HS. After HS, compared with the control group, 1.6 mg/kg and 12.0 mg/kg Cr supplementation groups significantly lowered the plasma glucose level and aspartate transaminase (AST) activity (P < 0.05), and 0.4 mg/kg and 1.6 mg/kg Cr supplementation groups significantly lowered alanine aminotransferase (ALT) activity (P < 0.05). 0.4 mg/kg and 1.6 mg/kg supplementation groups significantly improved hepatic total superoxide dismutase (T-SOD) activity (P < 0.05). Furthermore, 0.4mg/kg-12.0 mg/kg Cr supplementation groups significantly improved the activities of hepatic glutathione peroxidase (GPx) and catalase (CAT) and lowered hepatic malondialdehyde (MDA) level in liver (P < 0.05). The mRNA levels of hepatic copper zinc superoxide dismutase (Cu/Zn-SOD), CAT and GPx were significantly improved in 0.4mg/kg-12.0 mg/kg supplementation Cr groups (P < 0.05), however, there was no significant variation of hepatic manganese superoxide dismutase (Mn-SOD) mRNA levels under different levels of supplementation (P > 0.05). Significantly lower mRNA levels of hepatic pro-inflammatory cytokines observed in 0.4mg/kg-12.0 mg/kg Cr supplementation groups including tumour necrosis factor-α (TNF-α), interleukin 1ß (IL-1ß) and interleukin 8 (IL-8) (P < 0.05), and 0.4mg/kg-12.0 mg/kg Cr supplementation significantly improved the relative expressions of hepatic heat shock protein 70 (HSP70) and heat shock protein 90 (HSP90) (P < 0.05). The present study indicated that dietary Cr supplementation might have no significant effect on immune capacity and antioxidant capacity under normal physiological conditions, whereas it played an important role in relieving HS.

Methionine played a positive role in improving the intestinal digestion capacity, anti-inflammatory reaction and oxidation resistance of grass carp, Ctenopharyngodon idella, fry.

A study was carried out to appraisal the function of methionine on intestinal digestion and the health of grass carp (Ctenopharyngodon idella) fry (initial weight 0.36 ± 0.01 g). The fry were fed graded dietary methionine levels (0.33%-1.20% dry matter) in 18 recirculatory tanks (180 L). After an 8-week breeding experiment, the results revealed that 0.71%-1.20% dietary methionine levels markedly upregulated the mRNA levels of intestinal digestion including trypsin, amylase, chymotrypsin and AKP, and 0.71%-0.87% dietary methionine level significantly increased intestinal trypsin activities compared with the 0.33% dietary methionine level. For inflammation, 0.71%-1.20% dietary methionine levels downregulated the mRNA levels of NF-κBp65, IL-1ß, IL-6, IL-8, IL-15 and IL-17D, whereas upregulated the mRNA levels of anti-inflammatory cytokines, including IL-4/13B, IL-10 and IL-11. In terms of antioxidants, although dietary methionine levels had no significant effect on the expression of most core genes of the Nrf2/ARE signaling pathway, such as Nrf2, Keap 1, GPx4, CAT, Cu/Zn-SOD. Furthermore, dietary methionine levels had no significant effect on the expression of p38MAPK, IL-12p35, TGF-ß2 and IL-4/13A. 0.71%-1.20% dietary methionine levels still increased the mRNA levels of GPx1α, GSTR and GSTP1. Furthermore, higher intestinal catalase activity and glutathione contents were also observed in fry fed 0.71%-1.20% diets. In summary, 0.71%-1.20% dietary methionine levels played a positive role in improving the intestinal digestion capacity of digestion, anti-inflammatory reaction and oxidation resistance of grass carp fry. This study provided a theoretical basis for improving the survival rate and growth of grass carp fry.

Mulberry leaf meal: A potential feed supplement for juvenile Megalobrama amblycephala "Huahai No. 1".

This study was performed to evaluate the potential application of mulberry leaf meal (ML) and fermented mulberry leaf meal (FML) as feed supplements in aquatic animals for developing varieties of practical and economical feed ingredients. Juveniles Megalobrama amblycephala were fed a basal diet (35.7% crude protein, 10.4% crude lipid; control group) supplemented with 2.22% and 4.44% mulberry leaf meals (ML2, ML4) and fermented mulberry leaf meals (FML2, FML4) for 8 weeks. Generally, the two-way ANOVA showed the supplementation level exhibited a prominent effect on the growth performance and physiological status of fish. Furthermore, the two-way ANOVA showed the supplementary fermented mulberry leaf meal increased plasma complement 4 (C4) content (P < 0.05). The weight gain rate (WGR, 145.87%) and the specific growth rate (SGR, 1.63%) were significantly increased in FML2 group compared with the control group (P < 0.05). The muscle crude lipid content and hepatosomatic index (HSI) were higher in FML2 group than that in ML2 group (P < 0.05). The hepatic GSH content in ML4 group and CAT, T-SOD activities in FML4 group were significantly increased compared with the control group (P < 0.05). The hepatic MDA content in FML4 group was significantly decreased compared with the FML2 group (P < 0.05). Total cholesterol (TC) contents showed a significant decrease in ML4 and FML4 groups compared with the control group (P < 0.05). Regarding the gene expression, sirtiun 1 (Sirt1) gene expression was elevated in FML2 group compared with the ML2 group (P < 0.05). Compare to the control group, FML2 diet significantly increased the expression of i-kappa-B alpha (IKBα) gene in liver, and decreased the expression of forkhead box O1 α (FoxO1α), toll-like receptors 4 (TLR4) and nuclear factor-kappa B (NF-κB) genes (P < 0.05). In conclusion, 2.22% FML promoted the growth performance of M. amblycephala and enhanced the anti-inflammatory responses by inhibiting TLR4/NF-κB signaling pathway. On the other hand, 4.44% FML reduced plasma lipid content (hypolipedemic effect) and improved the hepatic antioxidant capacity of M. amblycephala.

Dietary chlorella (Chlorella vulgaris) supplementation effectively improves body color, alleviates muscle inflammation and inhibits apoptosis in largemouth bass (Micropterus salmoides).

Muscle quality, antioxidant status, and inflammatory and apoptotic molecule expression were investigated in juvenile largemouth bass fed five levels of Chlorella for 60 days. The results showed that muscle quality can be improved by increasing the muscle crude protein content, muscle and skin brightness value (L*), redness value (a*) and yellowness value (b*) in Chlorella-supplemented diets without affecting the growth and muscle fiber development of fish. Chlorella supplementation did not cause oxidative stress in muscle, but optimal Chlorella administration alleviated the muscle inflammatory response by downregulating the nuclear factor κB (NF-κB)-mediated proinflammatory factors such as interleukin 1ß (IL-1ß) and interleukin 8 (IL-8). Moreover, anti-apoptotic effects were induced by upregulation of anti-apoptotic genes, such as b cell lymphoma-2 (bcl-2) and myeloid cell leukemia-1 (mcl-1), and downregulation of pro-apoptotic genes, including bcl2-associated x (bax) and caspase3. In conclusion, Chlorella improved muscle quality, alleviated muscle inflammation and resisted muscle apoptosis.

Effects of replacing fishmeal with methanotroph (Methylococcus capsulatus, Bath) bacteria meal (FeedKind®) on growth and intestinal health status of juvenile largemouth bass (Micropterus salmoides).

A ten-week feeding trial evaluated the feasibility of methanotroph (Methylococcus capsulatus) bacteria meal (FeedKind®, FK) as a fishmeal substitute in largemouth bass (Micropterus salmoides) diets. Six isonitrogenous and isoenergetic diets with different inclusion levels of FK (0 (fishmeal group), 43, 86, 129, 172 and 215 g/kg) were formulated to replace 0, 50, 100, 150, 200 and 250 g/kg fishmeal, respectively. The results showed that FK inclusion level could reach 129 g/kg without significantly affecting growth or feed coefficient rate (P > 0.05), while growth performance was decreased and feed coefficient rate increased when FK inclusion levels exceeded 129 g/kg (P < 0.05). Increase in FK inclusion levels tended to reduce plasma total cholesterol and total triglyceride whilst plasma total protein, albumin, alanine aminotransferase and aspartate aminotransferase in FK treatment groups were unchanged compared with fishmeal group (P > 0.05). FK inclusion levels at 43 g/kg and 86 g/kg were not detrimental to intestinal morphology whilst it was unfavourable when FK inclusion levels exceeded 86 g/kg as the total length of intestinal wall thickness and villus height, villus height were obviously decreased compared with fishmeal group (P < 0.05). As regards to inflammatory cytokine genes, FK instead of fishmeal increased the expression levels of TLR2, RelA, TNF-α, IL-1ß, IL-10 and TGF-ß, 43 g/kg and 86 g/kg FK decreased the expression level of Caspase-3 (P < 0.05). In conclusion, 129 g/kg FK can replace 150 g/kg fishmeal without negative effects on the growth performance, and replacing 100 g/kg fishmeal with 86 g/kg FK is more beneficial to intestinal health.

A study of the potential effect of yellow mealworm (Tenebrio molitor) substitution for fish meal on growth, immune and antioxidant capacity in juvenile largemouth bass (Micropterus salmoides).

This study aimed to evaluate the effects of partial replacement of fish meal (FM) with yellow mealworm (Tenebrio molitor, TM) on the growth performance, food utilization and intestinal immune response of juvenile largemouth bass (Micropterus salmoides). Seven diets containing increasing levels of TM (FM substitution) were designed (approximately 0% (0%), 4% (11.1%), 8.1% (22.2%), 12.2% (33.3%), 16.3% (44.4%), 20.4% (55.5%), and 24.5% (66.6%), designated TM0, TM11, TM22, TM33, TM44, TM55, and TM66, respectively). 420 fish were randomly selected and placed in 21 cages (1 m*1 m*1 m, 7 treatments for triplicate, 20 fish per cage). Fish (initial weight 6.25 ± 0.03 g) were fed seven isonitrogenous (47%) and isocaloric (19 MJ kg-1) diets to satiety twice daily for 8 weeks. Compared to the control group (TM0), TM11 showed no significant difference in the weight gain rate (WGR), specific growth rate (SGR) or feed conversion ratio (FCR), while all other TM inclusion groups presented different degrees of decline. There was no significant difference in the whole-body composition among all groups (P > 0.05). Plasma total protein (TP), triglyceride (TG) and albumin (ALB) contents were significantly decreased in TM55 and TM66 (P < 0.05). The highest plasma aspartate transaminase (AST) activity was observed in TM66 (P < 0.05). TM33, TM44 and TM55 showed the lowest activities of plasma alanine amiotransferase (ALT) and alkaline phosphatase (ALP) (P < 0.05). Moreover, increased mRNA levels of superoxide dismutase (SOD) and catalase (CAT) were measured in the TM11 to TM55 groups, while intestinal SOD activity peaked in TM11 (P < 0.05). With the exception of TM11, the other TM inclusion groups showed significant inhibition of the relative expression of RelA, C3 and TNF-α (P < 0.05). All experimental groups exhibited lower expression of IL-10 than TM0 (P < 0.05). The TM11 group showed significantly upregulated expression of IL-1ß and TGF-ß (P < 0.05). In addition, TLR2 expression was increased in TM11 and TM22 (P < 0.05). Considering enzyme activities and immune-related gene expression, TM supplementation levels should not exceed 4% (TM11).

Culture salinity modulates Nrf2 antioxidant signaling pathway and immune response of juvenile Genetically Improved Farmed Tilapia (GIFT) (Oreochromis niloticus) under different dietary protein levels.

This study aimed to evaluate that dietary protein levels and culture salinity levels affect the health status of juvenile genetically improved farmed tilapia (GIFT, Oreochromis niloticus). Graded protein levels of six diets were prepared, ranging from 18.20% to 49.49% (dry basis), and were used in cultured GIFT at two salinity levels (0‰ and 8‰) for 8 weeks. The results suggested that appropriate protein levels reduced pro-inflammatory gene expressions in the intestine including interleukin 1ß (IL-1ß), interleukin 8 (IL-8) and tumour necrosis factor-α (TNF-α) mRNA levels at two salinity levels (P < 0.05). 8‰ salinity significantly decreased the expression levels of IL-1ß, TNF-α and nuclear factor-kappa B (NF-κB) (P < 0.05). The anti-inflammatory factor interleukin 10 (IL-10) was significantly increased by 36.42% protein level (P < 0.05). Regarding antioxidant capacity, appropriate protein levels and 8‰ salinity significantly improved the antioxidant capacity of fish by regulating the activities of intestinal total superoxide dismutase (T-SOD), glutathione peroxidase (GPx), and the levels of glutathione (GSH) and malondialdehyde (MDA). Furthermore, appropriate protein levels and 8‰ salinity also significantly enhanced the antioxidant gene expressions associated with the Nrf2/keap1 signaling pathway by regulating the expression levels of heme oxygenase-1 (HO-1), GPx, catalase (CAT) and superoxide dismutase (SOD). According to GPx activities and the mRNA levels of IL-10, the optimum dietary protein levels for GIFT juveniles were 31.12%-32.18% (0‰) and 34.25-35.38% (8‰) based on second-degree polynomial regression analysis. The present study found that appropriate protein levels and 8‰ culture salinity are critical in maintaining the health of GIFT juveniles by improving antioxidant and immune capacity.

Molecular cloning and expression mechanism of Mnp65 in Megalobrama amblycephala response to Aeromonas hydrophilia challenge.

p65 is one of the important subunits of the inflammation-related transcription factor NF-κB. In the present study, we cloned and identified the p65 from Megalobrama amblycephala (Mnp65) by homologous cloning and RACE technique. The full-length Mnp65 cDNA consisted of 2331 bp, and included one open reading frame encoding a 604-amino acid putative protein. The protein sequence included a DNA binding motif, a well conserved N-terminal Rel-homology domain (RHD), and a C-terminal IG-like plexins transcription (IPT). Mnp65 was closely related with the other p65 proteins of Cypriniformes and clearly distinct from that of Perciformes and Salmoniformes in terms of sequence homology. Mnp65 homodimer may interact with IκBα in the IPT domain based on the predicted 3D structure of IκBα/Mnp65 complex. Mnp65 was ubiquitously expressed in M. amblycephala tissues, and the highest levels were detected in muscle and liver. Intragastric infection with Aeromonas hydrophila caused respiratory burst and cytokine storm from 8 h to 48 h, showing significantly higher level of respiratory burst activities and significantly high cytokines levels, such as TNF-α, IL-1ß, IL-6, IL-8 etc., compared to 0 h. In addition, the bacterial challenge downregulated the IkBα, and upregulated Mnp65 and TNF-α in the liver. IkBα-Mnp65 was regulated by the negative feedback of cytokine storm, to increase IkBα and decrease Mnp65. Then cytokine storm was relieved at 96 h. Finally, severe intestinal inflammation was observed from 24 h to 48 h after infection, characterized by extensive villous necrosis, epithelial hyperplasia and lymphocyte infiltration, all of which were relieved at 96 h. Taken together, Mnp65 plays a crucial role in the physiological response of teleost fish to bacterial infection.

Molecular cloning and functional characterization of the hypoxia-inducible factor-1α in bighead carp (Aristichthys nobilis).

HIF-l is the earliest documented and most widely studied hypoxia-inducible factor (HIF) and plays a key role in the cell hypoxia signal transduction pathway. Particularly, the HIF-1α protein is sensitive to oxygen and plays a critical role in hypoxia regulation. This study is the first to report on the molecular cloning and characterization of HIF-1α in bighead carp (Aristichthys nobilis; anHIF-1α). The full-length cDNA of anHIF-1α was 2361 bp, and encodes an estimated 674 amino acids with a predicted molecular mass of 76.10 kDa and a theoretical isoelectric point of 7.72. Moreover, the conserved basic Helix-Loop-Helix domain along with two Per-ARNT-Sim domains (A/B), and C-TAD were identified in this protein. Interestingly, the tertiary structure of the anHIF-1α protein was found to be extremely similar to that of mice. Multiple comparison and phylogenetic tree results demonstrated that anHIF-1α was highly conserved. Under normoxic conditions, anHIF-1α mRNA transcripts could be detected in all tissues examined with the highest expression level in the heart. With gradually decreasing oxygen concentrations, anHIF-1α mRNA level was upregulated significantly in the gill, liver, kidney, spleen, intestine, brain, and muscle tissues (P < 0.05). Similarly, anHIF-1α was expressed in all examined bighead carp tissues, and the results suggested that the upregulation of anHIF-1α at the transcriptional level may be an important stress response adaptation to hypoxia in bighead carp. Finally, based on the tertiary structure comparative analyses between anHIF-1α with mouse HIF-1α, we think the physiological function, and protein structure of HIF-1α could be compared between fish and mammal in the future.

The immunoreaction and antioxidant capacity of juvenile blunt snout bream (Megalobrama amblycephala) involves the PI3K/Akt/Nrf2 and NF-κB signal pathways in response to dietary methionine levels.

A 75-day rearing trail was designed to evaluate the immunoreaction and antioxidant capacity of juvenile blunt snout bream in response to dietary methionine levels. Three practical diets were extruded to feed juveniles with graded methionine levels (0.40%, 0.84% and 1.28% dry matter). The data indicated that the plasma concentrations of immunoglobulin M (IgM), complement component 3 (C3) and glutathione (GSH) in the 0.84% methionine diet were markedly upper than those in the 0.40% group (P < 0.05). The activities of plasma antioxidant parameters involving catalase (CAT), total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC) and glutathione peroxidase (GPx) were significantly increased by the 0.84% diet compared with the 0.40% diet, whereas plasma alanine aminotransferase (ALT) and malondialdehyde (MDA) levels were significantly induced by 0.40% methionine (P < 0.05). Compared with the 0.40% group, 0.84% dietary methionine dramatically upregulated the mRNA expression levels of protein kinase B (Akt), phosphoinositide 3-kinase (PI3K) and nuclear factor erythroid 2-related factor 2 (Nrf2) pathway related genes including CAT, manganese superoxide dismutase (Mn-SOD), heme oxygenase 1 (HO-1) and glutathione peroxidase-1 (GPx-1) in the kidney and liver, and downregulated Kelch-like ECH-associated protein 1 (Keap1) mRNA levels (P < 0.05). Compared with the 0.40% group, the 0.84% dietary methionine strikingly suppressed the mRNA levels of renal and hepatic nuclear factor-kappa B (NF-κB) and pro-inflammatory cytokines (interleukin 1ß (IL-1ß), tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6)), however, improved the mRNA expression levels of anti-inflammatory cytokines involved renal and hepatic transforming growth factor-ß (TGF-ß) and hepatic interleukin 10 (IL-10) (P < 0.05). Renal IL-10 and interleukin 8 (IL-8) mRNA expression levels were not markedly influenced by experimental diets (P > 0.05). Dietary methionine (0.84%) significantly upregulated renal and hepatic heat stress protein 70 (Hsp70), renal B-cell lymphoma-2 (Bcl-2) gene expression levels compared with the 0.40% diet (P < 0.05). In a word, the data represented that 0.84% dietary methionine could enhance the immune and antioxidant capacity of this fish species by inducing PI3K/Akt/Nrf2 pathway and inhibiting NF-κB pathway.

Transcriptome profiling and histology changes in juvenile blunt snout bream (Megalobrama amblycephala) liver tissue in response to acute thermal stress.

To understand the precise mechanism and the pathways activated by thermal stress in fish, we sampled livers from juvenile Megalobrama amblycephala exposed to control (25 °C) and test (35 °C) conditions, and performed short read (100 bp) next-generation RNA sequencing (RNA-seq). Using reads from different temperature, expression analysis identified a total of 440 differentially-expressed genes. These genes were related to oxidative stress, apoptosis, immune responses and so on. We used quantitative real-time reverse transcriptase PCR to assess the differential mRNA expression of selected genes that encode antioxidant enzymes and heat shock proteins in response to thermal stress. Fish exposed to thermal stress also showed liver damage associated with serum biochemical parameter changes. The set of genes identified showed regulatory modulation at different temperatures, and therefore could be further studied to determine how thermal stress damages M. amblycephala livers and the possible roles of reactive oxygen species in this process.

Emodin ameliorates metabolic and antioxidant capacity inhibited by dietary oxidized fish oil through PPARs and Nrf2-Keap1 signaling in Wuchang bream (Megalobrama amblycephala).

Dietary lipids and fatty acids are involved in cell metabolism and animal physiological regulation. However, oxidized lipids could induce oxidative stress and disorder normal growth and physiological health in fish. A 12-week rearing experiment with 6% fish oil (6F), 6% oxidized fish oil (6OF) and emodin supplemented diets (6F + E, 6OF + E) was conducted to evaluate the protective mechanism of emodin on oxidized fish oil stress in Megalobrama amblycephala. Results indicate that, under oxidized fish oil stress, emodin rescued the growth performance inhibition, improved special growth ratio (SGR), and reduced feed conversion ratio (FCR) and hepatosomatic index (HSI); rescued intestine histological impairment, ameliorated the structural expansion and membrane damage of mitochondria in intestine cells, and increased the length and intensity of intestinal villus. Moreover, emodin enhanced serum immune and antioxidant enzyme activity, increased metabolic activity through PPARs signaling, increased antioxidant capacity through PPARs and Nrf2-Keap1 signaling based on the transcriptional expression of specific genes. These results indicate emodin could be used as an effective immunostimulant to protect organism form oxidative stress induced by dietary oxidized lipid. This may provide insights for oxidized lipid prevention in aquaculture production.

Serum biochemistry, liver histology and transcriptome profiling of bighead carp Aristichthys nobilis following different dietary protein levels.

Dietary protein plays a major role in determining the rate of fish growth and overall health. Given that the liver is an important organ for metabolism and detoxification, we hypothesized that optimal dietary protein levels may benefit liver function. Herein, we investigated the effects of dietary protein level on serum biochemistry, liver histology and transcriptome profiling of juvenile bighead carp Aristichthys nobilis fed for 8 weeks on a diet supplemented with high protein (HP, 40%), low protein (LP, 24%) or optimal protein (OP, 32%; controls). The results revealed a significant change in liver morphology in LP and HP groups compared with the OP group, coupled with increased serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity. RNA sequencing (RNA-Seq) analysis of the liver transcriptome yielded 47 million high-quality reads using an Illumina platform, which were de novo assembled into 80,777 unique transcript fragments (unigenes) with an average length of 1021 bp. Subsequent bioinformatics analysis identified 878 and 733 differentially expressed unigenes (DEGs) in liver in response to LP and HP diets, respectively. KEGG enrichment analysis of DEGs identified immune and metabolism-related pathways, including Toll-like receptor signaling, PI3K-Akt signaling, NF-κB signaling, complement and coagulation, peroxisome, nitrogen metabolism, PPAR signaling, and glycolysis and gluconeogenesis pathways. Transcriptome profiling results were validated by quantitative real-time PCR for 16 selected DEGs. The findings expand our understanding of the molecular mechanisms underlying the effects of dietary protein level on liver function in bighead carp.

Effects of dietary fenugreek seed extracts on growth performance, plasma biochemical parameters, lipid metabolism, Nrf2 antioxidant capacity and immune response of juvenile blunt snout bream (Megalobrama amblycephala).

Dietary administration of some plant-derived substances have been proved of great economic value in aquaculture. In order to investigate the effects of dietary fenugreek seed extracts (FSE) on juvenile blunt snout bream (Megalobrama amblycephala), a feeding trial was conducted for 8 weeks. The results showed that final weight (FW), weight gain (WG), feed conversion ratio (FCR) and specific growth rate (SGR) were not significantly affected by dietary FSE levels. The whole body lipid contents of fish fed with 0.04%, 0.08% and 0.16% FSE diets were significantly lowered compared to the control group. Dietary FSE diets significantly affected plasma complement component 3 (C3), immunoglobulin M (IgM), albumin (ALB) and total protein (TP). The relative expressions of acetyl CoA carboxylase (ACC), fatty acid synthase (FAS) and sterol regulatory element binding protein-1 (SREBP1) mRNA in the liver of fish decreased significantly with increasing dietary FSE levels from 0% up to 0.04%. FSE supplementation diets lowered the liver pro-inflammatory genes expressions by regulating tumor necrosis factor-α (TNF-α) and interleukin 8 (IL-8) mRNA levels and increased anti-inflammatory genes expression by regulating transforming growth factor (TGF-ß) and interleukin 10 (IL-10). FSE diets increased growth factor-1 (IGF-1) and target of rapamycin (TOR) mRNA levels from 0% up to 0.04%, 0.04% FSE diets significantly increased growth factor-1 (IGF-1) mRNA levels and S6 kinase-polypeptide 1 (S6K1) mRNA levels compared to the control group. 0.04% FSE diets significantly increased superoxide dismutase (SOD) activities and 0.08% FSE diets significantly increased catalase (CAT) and glutathione peroxidase (GPx) activities, 0.16% FSE diets significantly increased total antioxidant capacity (T-AOC) activities compared to the control group. Additionally, compared to the control group, 0.04% dietary FSE significantly up-regulated nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA levels and glutathione peroxidase-1 (GPx1) mRNA levels, at the same time, 0.02%, 0.04%, 0.08%, 0.16% FSE diets significantly down-regulated kelch-like ECH-associated protein 1 (Keap1) mRNA levels. However, no significant effects were observed on copper zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD). Our study indicated that dietary FSE could improve plasma biochemical parameters, regulate lipid metabolism related genes, promote Nrf2 antioxidant capacity and enhance immune response of juvenile blunt snout bream.

Effects of dietary tryptophan levels on antioxidant status and immunity for juvenile blunt snout bream (Megalobrama amblycephala) involved in Nrf2 and TOR signaling pathway.

Dietary administration of tryptophan has been proved improving growth performance of fish. An 8-week feeding trial was conducted to investigate the effects of dietary tryptophan level on antioxidant capacity and immune response through Nrf2 and TOR signaling pathway. The results showed that, 0.08% tryptophan level significantly increased plasma aspartate aminotransferase (AST), while immunoglobulin M (IgM) and alkaline phosphatase (ALP) were strikingly increased by 0.40% level. The level of plasma complement component 3 (C3), alanine aminotransferase (ALT) and albumin (ALB) were independent of tryptophan supplementation. Total superoxide dismutase (T-SOD), catalase (CAT), total antioxidant capacity (T-AOC) and glutathione (GSH) activity were increased with increasing dietary tryptophan level until 0.40% and then decreased, while the level of malondialdehyde (MDA) showed a reverse trend. 0.19% and 0.28% tryptophan level significantly improved the glutathione peroxidase 1 (GPx-1) activity. Compared with 0.08% dietary tryptophan level, 0.40% level significantly improved nuclear factor erythroid 2-related factor 2 (Nrf2), GPx, manganese superoxide dismutase (Mn-SOD), CAT and transforming growth factor-ß (TGF-ß) mRNA level, while Kelch-like ECH-associated protein 1 (Keap1) and interleukin 1ß (IL-1ß) mRNA level were significantly decreased. The relative expression of copper zinc superoxide dismutase (Cu/Zn-SOD), heme oxygenase-1 (HO-1), target of rapamycin (TOR), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), protein kinase B (Akt) and interleukin 10 (IL-10) were significantly improved by 0.28% diet, while the mRNA level of tumor necrosis factor-α (TNF-α) and nuclear factor-kappa B (NF-κB) were increased by 0.08% diet. Interleukin 8 (IL-8) mRNA level was not significantly affected by dietary tryptophan. Based on MDA and T-SOD value, the optimal dietary tryptophan level of juvenile blunt snout bream was determined to be 0.33% (1.03% of dietary protein) and 0.36% (1.13% of dietary protein), respectively, using quadratic regression analysis.

The effects of crowding stress on the growth, physiological response, and gene expression of the Nrf2-Keap1 signaling pathway in blunt snout bream (Megalobrama amblycephala) reared under in-pond raceway conditions.

We evaluated the effects of high density stress on growth performance, antioxidant parameters, and Nrf2 pathway signaling molecules after different lengths of exposure (30, 60, or 90 days) of Megalobrama amblycephala to in-pond raceway aquaculture systems (IPRS). M. amblycephala (average initial weight 2.33 ±â€¯0.15 g) were reared at two different initial densities (low density group [LD] had 534 fish/m3 and high density group [HD] had 1073 fish/m3) for 90 days. The growth performance was adversely influenced by the high stocking density. The HD group had elevated white blood cell counts, hemoglobin content, and hematocrit on days 60 and 90. The mRNA levels of NOX2 on days 60 and 90, Nrf2 on days 30, 60, and 90, Keap1 on day 30, Bach1 on days 30 and 60, SOD on day 30, and CAT on day 30 were significantly higher in the HD group than in the LD group. Similarly, higher trends were observed in the enzymatic activities of SOD on day 60, CAT on days 60 and 90, and GPx on day 60 in the HD group, compared to the LD group. Furthermore, HD bream showed an increased MDA content on days 60 and 90 compared to that of the LD group. This study demonstrates that high density-induced antioxidant defenses were involved in modifications to the enzymatic and transcriptional regulation of Nrf2-Keap1 signaling molecules and that M. amblycephala growth was reduced in a crowded IPRS.

Evaluating expression of autophagy-related genes in oriental river prawn Macrobrachium nipponense as potential biomarkers for hypoxia exposure.

Autophagy, a crucial process for maintaining cellular homeostasis, is under the control of several autophagy-related (ATG) proteins, and is highly conserved in most animals, but its response to adverse environmental conditions is poorly understood in crustaceans. Herein, we hypothesised that autophagy acts as a protective response to hypoxia, and Beclin 1, ATG7 and ATG8 in oriental river prawn (Macrobrachium nipponense) were chosen as potential biomarkers under hypoxia exposure; thus, their full-length cDNA sequences were cloned and characterised. Open reading frames (ORFs) of 1281, 2076 and 360 bp, encoding proteins of 427, 692 and 120 amino acid residues, respectively, were obtained. Phylogenetic analysis demonstrated the three M. nipponense proteins do not form a clade with vertebrate homologs. Protein and mRNA levels were investigated in different tissues and developmental stages, and all three were significantly upregulated in a time-dependent manner in the hepatopancreas following hypoxia stress. Biochemical and morphological analysis of hepatocytes revealed that hypoxia increased the abundance of hepatic autophagic vacuoles and stimulated anaerobic metabolism. RNA interference-mediated silencing of ATG8 significantly increased the death rate of M. nipponense juveniles under hypoxia stress conditions. Together, these results suggest that Beclin 1, ATG7 and ATG8 contribute to autophagy-based responses against hypoxia in M. nipponense. The findings also expand our understanding of the potential role of autophagy as an adaptive response against hypoxia toxicity in crustaceans. The results showed that hepatic ATG8 levels may be directly indicative of acute hypoxia in prawns, and provide insight into the time at which hypoxia exposure occurs. Autophagy-related genes expression pattern seems to be sensitive and good biomarkers of acute hypoxia exposure.

Identification and Characterization of Four Autophagy-Related Genes That Are Expressed in Response to Hypoxia in the Brain of the Oriental River Prawn (Macrobrachium nipponense).

Autophagy is a cytoprotective mechanism triggered in response to adverse environmental conditions. Herein, we investigated the autophagy process in the oriental river prawn (Macrobrachium nipponense) following hypoxia. Full-length cDNAs encoding autophagy-related genes (ATGs) ATG3, ATG4B, ATG5, and ATG9A were cloned, and transcription following hypoxia was explored in different tissues and developmental stages. The ATG3, ATG4B, ATG5, and ATG9A cDNAs include open reading frames encoding proteins of 319, 264, 268, and 828 amino acids, respectively. The four M. nipponense proteins clustered separately from vertebrate homologs in phylogenetic analysis. All four mRNAs were expressed in various tissues, with highest levels in brain and hepatopancreas. Hypoxia up-regulated all four mRNAs in a time-dependent manner. Thus, these genes may contribute to autophagy-based responses against hypoxia in M. nipponense. Biochemical analysis revealed that hypoxia stimulated anaerobic metabolism in the brain tissue. Furthermore, in situ hybridization experiments revealed that ATG4B was mainly expressed in the secretory and astrocyte cells of the brain. Silencing of ATG4B down-regulated ATG8 and decreased cell viability in juvenile prawn brains following hypoxia. Thus, autophagy is an adaptive response protecting against hypoxia in M. nipponense and possibly other crustaceans. Recombinant MnATG4B could interact with recombinant MnATG8, but the GST protein could not bind to MnATG8. These findings provide us with a better understanding of the fundamental mechanisms of autophagy in prawns.