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This research investigated the effects of dietary cholesterol (CHO) on growth, molting performance, CHO deposition, and lipid metabolism of Chinese mitten crab (Eriocheir sinensis). Six diets were formulated to contain 0% (control), 0.1%, 0.2%, 0.4%, 0.8%, and 1.6% CHO and were administered to E. sinensis 43.35 ± 0.05 g , for 16 weeks. From our results, crabs fed with 0.2% and 0.4% CHO diet, the final body weight (FBW), and weight gain ratio (WGR) increased significantly and the feed conversion ratio (FCR) decreased significantly compared with that of the control group. Furthermore, the molting performance of E. sinensis was significantly improved in crabs fed 0.2% CHO. Meanwhile, 0.2% CHO tends to upregulate molt-promoting genes and downregulate the expression of molt-inhibiting genes. It indicated that CHO might improve the molting performance of crabs by regulating ecdysteroid signal pathway transduction. In addition, dietary CHO can significantly increase the content of CHO in the hepatopancreas, muscle, and hemolymph. Moreover, dietary CHO increases the content of triglyceride (TG) in the hepatopancreas and hemolymph. The gene-related lipid metabolism shows that CHO may increase the lipid content by promoting lipid synthesis and inhibiting lipolysis. According to the results of this study, the dietary CHO requirement of E. sinensis is 0.27% for growth requirement and 0.45% for tissue CHO deposition based on the broken-line model.

Blunt snout bream (Megalobrama amblycephala) were randomly assigned into three diets: normal-carbohydrate diet (NCD, 30% carbohydrate, w/w), high-carbohydrate diet (HCD, 43% carbohydrate), and HCB (HCD supplemented with 50 mg/kg berberine (BBR)). After 10 weeks' feeding trial, the results showed that higher levels of plasma glucose, triglyceride, and total cholesterol were observed in HCD-fed fish than in NCD-fed fish, while HCB feeding significantly ameliorated this effect. Moreover, HCB feeding remarkably reversed HCD-induced hepatic glycogen and lipid contents. In insulin signaling, BBR inclusion restored HCD-induced suppression of insulin receptor substrate mRNA expression and elevation of forkhead transcription factor 1 mRNA expression. In glucose metabolism, upregulated glucose transporter 2 and glycogen synthase mRNA expressions in the HCD group were observed compared to the NCD group. However, BBR adding reduced the mRNA expressions of glycogen synthase, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase and increased the transcriptional levels of glucose transporter 2 and pyruvate kinase. In lipid metabolism, BBR supplementation could reverse downregulated hepatic carnitine palmitoyl transferase I mRNA expression and upregulated hepatic acetyl-CoA carboxylase and fatty acid synthetase mRNA expressions in the HCD group. Taken together, it demonstrates that BBR could improve glucose metabolism of this species via enhancing liver's glycolysis and insulin signaling, while inhibiting liver's glycogen synthesis and gluconeogenesis. It also indicates that BBR could reduce the metabolic burden of the liver by inhibiting fat synthesis and promoting lipid decomposition, and then enhance fat uptake in peripheral tissues.

An 8-week feeding trial was performed to test the effects of glycyrrhetinic acid (GA) on growth and some gene expression of hepatic lipid metabolism in channel catfish (initial body weight, 3.5 ± 0.02 g) fed high-fat diets. Fish were fed the control diet, high-fat diet (HFD), and HFD supplemented with 0.4, 0.8, and 1.2 mg/kg GA in 15 tanks at a stocking density of 21 fish/tank. Fish fed HFD were significantly lower in body weight gain and specific growth rate but higher in feed intake and feed conversion ratio in comparison to the control. Supplement of GA at 1.2 mg/kg remarkably improved these parameters as compared to the control diet. High levels of cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), and low levels of high-density lipoprotein (HDL) in plasma were observed in fish fed HFD; the opposite was observed for fish fed HFD supplemented with GA. The transcription of fatty acid synthase (FAS), sterol regulatory element-binding protein-1 (SREBP1), liver X receptor alpha (LXRα), and hormone-sensitive lipase (HSL) was upregulated, while that of carnitine palmitoyltransferase I (CPT1), peroxisome proliferator-activated receptors alpha (PPARα), acyl-CoA oxidase (ACO), and microsomal triglyceride transfer protein (MTTP) mRNA expression were downregulated in fish fed HFD. The opposite was observed in fish fed HFD supplemented with GA as well as the control group. In conclusion, supplementing the HFD with GA at 1.2 mg/kg could improve the growth performance and lipid metabolism of channel catfish consuming HFD.

One hundred and sixty crabs (average initial weight: 51.32±0.08 g) were fed with four experimental diets containing cottonseed meal protein hydrolysate (CPH) at 0% (CPH0, control), 0.3% (CPH0.3), 0.6% (CPH0.6), and 1.2% CPH (CPH1.2). The experiment results show that no difference was observed in specific growth rate and survival rate of crabs fed with CPH diet. Moisture content of crabs fed with CPH0.6 diet was significantly reduced than that of the CPH0 group. Superoxide dismutase and catalase activities of crabs fed with CPH0.6 diet were significantly increased and the difference was not significant between the CPH0.3 and CPH0.6 groups. Malondialdehyde content of CPH0.3 group was significantly lower than that of the CPH0 group. Lysozyme, alkaline phosphatase, and acid phosphatase activities of CPH0.3 diet crabs were significantly higher than that of the CPH0 group. Glutamic-pyruvic transaminase activity of crabs fed with CPH0.3 diet was significantly decreased compared to the CPH0 group. The relative expression levels of Toll1, Toll2, MyD88, LITAF, and ILF-2 of crabs fed with CPH0.3 diet were significantly higher than that of the CPH0 group. The expression level of SOCS2 showed an opposite pattern. After CPH perfusion, the expression levels of SOCS2 and Toll1 in intestine at time 3 h and SOCS2 in hepatopancreas at time 18 h increased significantly to the highest value. The expression level of Toll2, MyD88, LITAF decreased at times 6 h, 6 h, 12 h, respectively, then increased gradually. Therefore, supplementation of dietary CPH could improve antioxidant capacity and immune function; the appropriate supplement dosage of CPH for crab could be 0.3%–0.6%. Furthermore, the short-term CPH stimulation could significantly increase or decline the expression levels of immune-related genes at different times after CPH perfusion.

Glucagon has emerged as the main regulator of extracellular amino acid homeostasis. Insufficient glucagon signaling results in hyperaminoacidemia, which drives adaptive proliferation of glucagon-producing α cells. Aside from mammalian target of rapamycin complex 1 (mTORC1), the role of other amino acid sensors in the α cell proliferation has not been described. Here, using gcgr-deficient zebrafish and cultured mouse islets, we show that α cell proliferation requires the calcium sensing receptor (CaSR) and downstream extracellular signalregulated protein kinase (ERK1/2). Inactivation of casr dampened α cell proliferation, which can be rescued by re-expression of CaSR or activation of the downstream Gq, but not Gi, signaling in α cells. CaSR was also unexpectedly necessary for mTORC1 activation in α cells. Furthermore, co-activation of Gq and mTORC1 induced α cell proliferation independent of hyperaminoacidemia. These results reveal another amino acid sensitive mediator, and identify major pathways necessary and sufficient for hyperaminoacidemia-induced α cell proliferation.

To investigate the effects of dietary icariin (ICA) supplementation on acute oxidative stress and hepatopancreatic injury induced by lipopolysaccharide (LPS) injection in Eriocheir sinensis, an 8-week feeding trial of crabs was conducted using 4 diets with different supplementation levels of ICA (0, 50, 100, and 200 mg/kg diet weight, respectively), and then challenged with LPS of 400 μg/kg body weight for 6 h. Results showed that 100 mg/kg ICA supplementation increased the antioxidant capacity, reduced the stress-related indicators in haemolymph, strengthen the mitochondrial membrane potential, and reduce apoptosis compared to the single LPS-treated crabs. The expressions of apoptosis-related genes and proteins were also evaluated to further understand the effects of dietary ICA pretreatment on LPS-induced cell apoptosis. As a result, dietary 100 mg/kg diet weight ICA pre-addition significantly down-regulated the expression of HSP60, HSP70, Caspase 3c, Caspase 8, Caspase 3, Caspase 9, P38, and Bax (P 0.05), and alleviated the suppressed expression of PI3K, AKT, MEK, and Bcl-2 (P 0.05) in crabs challenged with LPS. Overall, this research reveals that ICA supplementation of 100 mg/kg diet weight could enhance the resistance to oxidative damage and apoptosis in E. sinensis facing LPS challenge.

Juvenile blunt snout bream Megalobrama amblycephala were reared under three photoperiods, 8 L: 16D, 12 L: 12D and 16 L: 8D (L: light; D: dark) for over 8 weeks. The results showed that growth performance was significantly enhanced by long photoperiod. Contrary to feed conversion ratio, final length, final weight, weight gain and feed intake increased significantly as illumination time increased from 8 h daily to 16 h daily. Low size heterogeneity and whole-body lipid content in fish exposed to long photoperiod were also observed. Both relative mRNA expression level of GH and IGF-I increased as illumination time increased from 8 h to 16 h daily. The lowest value of plasma cortisol was observed at the middle the photophase while the highest value was observed at the transition between the day and night span. Hepatic MDA content significantly increased as illumination time increased from 8 h daily to 16 h daily. The activities of hepatic catalase and glutathione peroxidase were lowest in fish exposed to 16 L: 8D photoperiod and significantly lower than that in fish exposed to 8 L: 16D. These results indicate that photoperiod manipulation may not only improve growth performance but also reduce size heterogeneity. However, prolonged photoperiod could cause chronic stress since plasma cortisol level was higher in the long photoperiod group, leading to an increasing oxidative stress.

One hundred and twenty crabs (initial weight: 51.32 ± 0.06 g) were fed three experimental diets with cottonseed meal protein hydrolysate (CPH) at 0, 6 and 12 g/kg for 60 days. The results showed that CPH0.6 diet significantly improved the 1‐hr feed intake, protease activity and weight gain of crabs. In thoracic ganglia, 4E‐binding protein (4E‐BP) expression level of crab fed the diets containing CPH was significantly higher than CPH0 group. In cerebral ganglia, protein kinase B and 4E‐BP expression levels of crab fed CPH0.6 diets were significantly lower than CPH0 group. In mid‐intestine, target of rapamycin (TOR) relative expression level of crabs fed the diets containing CPH was significantly lower than CPH0 group. 4E‐BP expression level showed an opposite pattern. In thoracic ganglia, cerebral ganglia and mid‐intestine, leptin receptor expression level of crabs fed CPH0.6 diet was significantly lower than CPH0 group. Neuropeptide Y and neuropeptide Y receptor expression level of crab fed CPH0.6 diet was significantly higher than CPH0 group. In conclusion, our results indicated that dietary supplementation of 6 g/kg CPH could stimulate appetite and quicken feeding rate via the TOR signalling pathway.

High-fat diets are associated with fatty liver and aberrant hepatic lipid metabolism, and glycyrrhetinic acid (GA) has been shown to exert a beneficial effect on lipolysis and fat deposition in fish. In the present study, we evaluated the effect of GA on the growth performance and expression of hepatic lipid transport related genes in blunt snout bream ( Megalobrama amblycephala) fed a high fat diet. Two hundred and sixteen fish (average body weight: 45.57 g ± 0.98 g) were fed three experimental diets (6% fat/L6 group, control, 11% fat/L11 group, and 11% fat with 0.3 mg kg −1 GA/L11GA group) for 8 weeks. Compared to the control group, the weight gain and specific growth rate of high-fat fed group at the end of the trialwere significantly improved ( P P > .05). Dietary supplementation with 0.3 mg kg −1 GA significantly decreased the hepatosomatic index, viscera/body ratio, and intraperitoneal fat ratio ( P P P > .05). In conclusion, GA significantly rescued high-fat diet induced hepatic lipid accumulation and metabolism dysfunction in M. amblycephala by stimulating hepatic fatty acid transport and β-oxidation. Dietary GA may be used as a promising supplement to alleviate high-fat diet induced side effects on M. amblycephala.

The Chinese mitten crab is an important economic species in the Chinese aquaculture industry due to its rich nutritional value and distinct flavor. The hepatopancreas is a popular edible part of the Chinese mitten crab, and therefore, hepatopancreatic health directly determines its quality. However, a large-scale outbreak of hepatopancreatic necrosis syndrome ("Shuibiezi" disease in Chinese), which is caused by abiotic agents correlated with cyanobacteria bloom outbreaks, adversely affects the Chinese mitten crab breeding industry. Cyanobacterial blooms that occur in high-density farming ponds can produce microcystin-LR (MC-LR), which is hepatotoxic in fish and mammals. Hepatopancreas toxicity of MC-LR (0, 25, 50 and 75 μg/kg) was investigated after 48 h of exposure. The MC-LR can cause hepatopancreatic injury by inducing hepatopancreatic structural damage, subcellular structural changes, and cell apoptosis, followed by enhanced lipid peroxidase, reactive oxygen species, and apoptosis-related enzyme (Caspase 3, 8, and 9) activities. These in turn promote gene and protein expression of apoptosis-associated proteases (Caspase 3, 7, and 8, Bcl-2, and Bax), and alter antioxidant system responses (superoxide dismutase, glutathione S-transferase, glutathione peroxidase, glutathione reductase activities, and glutathione content). The present study is the first report on MC-LR hepatotoxicity in the Chinese mitten crab and confirms hepatopancreas toxicity, providing a theoretical basis for enhancing MCs resistance and developing preventive and curative measures against hepatopancreatic disease in the Chinese mitten crab breeding industry.

Global warming is favouring the incidence, intensity and duration of harmful cyanobacterial blooms. Microcystin-LR (MC-LR), a hepatotoxic agent, is produced during cyanobacterial blooms. To understand the molecular mechanisms of acute hepatotoxic effect of low doses of MC-LR in crab, we examined differentially expressed genes in samples of the hepatopancreas of Chinese mitten crab (Eriocheir sinensis) collected in 48 h after injections of MC-LR at doses of 0, 25, 50, and 75 µg/kg. The results revealed that MC-LR induced changes in corresponding gene led to the accumulation of triglycerides. MC-LR exposure affected sterol metabolism. Apoptosis-related genes such as Fas-L, Bcl-XL, Cytc, AiF, p53, PERK, calpain, CASP2, CASP7, α-tubulin, PARP, GF, G12, and PKC were upregulated. Conversely, expression levels of CASP10 and ASK1 were downregulated. Genes related to the regulation of actin cytoskeleton (Rho, ROCK, MLCP, MLC, PAK, and PFN) were upregulated. Further, expression levels of genes encoding fatty acid elongation-related enzymes were upregulated, but the expression of genes related to fatty acid synthesis was slightly down regulated. Taken together, these results demonstrated the hepatic toxicity and molecular mechanisms of changes in lipid metabolism, immune and apoptosis in Chinese mitten crab under the MC-LR-induced stress, which is the first report on crabs and performs a comprehensive analysis and a new insight of the molecular toxicological responses in crabs.

In Eriocheir sinensis aquaculture, crabs feeding on an artificial diet without steroid hormones available in ice-fresh fish or snails show delayed ovarian maturation and late listing. Icariin (ICA), a safe plant-derived monomer compound, can effectively promote estradiol (E2) synthesis and ovarian development in mammals, but its effect in crustaceans has not been reported. We investigated the effects of ICA on estrogen synthesis, vitellogenesis, and oocyte development in Chinese mitten crabs fed with one of four diets containing 0, 50, 100, and 200 mg/kg ICA, respectively, for 8 weeks. Results showed that vitellogenin (VTG) contents, gonadosomatic indexes (GSIs), and oocyte volumes in ICA50 and ICA 100 crabs were significantly higher than those in the control. The highest upregulated expression of VTG in hepatopancreases and ovaries was detected in ICA100 crabs. Hemolymph E2 contents were also significantly increased in ICA100 crabs. Enzyme-linked immunosorbent assay (ELISA) and western blot analysis indicated that the increase in E2 concentrations was attributable to an elevation in aromatase protein levels in ovaries through the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP response element binding (CREB) protein pathway. These findings suggest that ICA could serve as a natural additive to resolve the problem of delayed ovarian maturation of crabs raised in ponds.

T-2 toxin is a secondary metabolite produced by Fusarium spp. that is a major cereal and animal feed contaminant. T-2 toxin has numerous adverse effects on animals, including hepatotoxicity. Arginine (Arg) is closely associated with the regulation of immune responses and antioxidant activity in tissues. The objective of the present study was to evaluate the protective effects of dietary Arg against oxidative damage and immune responses of the hepatopancreas induced by T-2 toxin in Chinese mitten crab. According to the results, 3.17% Arg in the diet decreased alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activity in the haemolymph significantly, when compared with the levels of activity in the T-2 toxin group. Arg supplementation also increased superoxide dismutase and glutathione peroxidase activity, while decreasing malondialdehyde concentrations in the hepatopancreas, when compared with the levels in the T-2 toxin group. In addition, 3.17% Arg in the diet increased acid phosphatase and alkaline phosphatase activity in the hepatopancreas, as well as albumin concentrations in the haemolymph, when compared with the T-2 toxin group. Dietary Arg also regulated the expression of antioxidant enzyme-related genes (mitochondrial manganese superoxide dismutase, cytosolic manganese superoxide dismutase, and catalase) and immune related genes (prophenoloxidase, NF-κB-like transcription factor Relish, and lipopolysaccharide-induced TNF-α factor) to alleviate the damage associated with the T-2 toxin. Furthermore, Arg ameliorated damage to the hepatopancreas microstructure in the crabs. The results of the present study indicate that dietary Arg could enhance the antioxidant and immune capacity of Chinese mitten crab against oxidative damage and immune injury to the hepatopancreas induced by T-2 toxin.

The present study was conducted to characterize the full-length cDNA of c-Jun N-terminal kinase (JNK) in Procambarus clarkii (Pcjnk) and evaluate its potential function under different molt cycle. The full-length cDNA of Pcjnk covered 2937 bp with an open reading frame of 1320 bp, encoding 439 amino acids. A typical conserved TPY motif (118Thr-Pro-120Tyr) was found in Pcjnk. Quantitative real-time PCR (qRT-PCR) analysis revealed a constitutive expression of Pcjnk in the tested tissue, with the highest expression occurring in the hepatopancreas. Additionally, the present study initially revealed that relative mRNA expression of Pcjnk and apoptosis level were significantly higher in the premolt stage (D1/D2 and D3/D4 stage) as compared to other molt stages. In contrast to the levels of superoxide dismutase (SOD) and malondialdehyde (MDA), catalase (CAT) and glutathione peroxidase (GPX) level decreased significantly from the intermolt stage (C stage) to the premolt stage (D1/D2 and D3/D4 stage), then increased from the premolt stage to the postmolt stage (A and B stage). The results obtained in the present study indicated that molt could cause apoptosis induced by oxidative stress through the activation of JNK in Procambarus clarkii.

Eriocheir sinensis is an important aquaculture species in China, and its yield and quality are threatened by oxidative stress caused by deteriorating water conditions. Reduced glutathione (GSH) is an endogenous antioxidant, but whether dietary GSH can increase the resistance of E. sinensis to environmental stress remains unclear. Therefore, in this study, crabs were fed with dietary GSH (0, 300, 600, 900, and 1200 mg/kg diet weight) for up to 10 weeks to determine the effects of different dietary GSH concentrations on growth, antioxidant capacity, and immunity of E. sinensis. The results showed that the weight gain rate and survival rate increased significantly as dietary GSH levels increased from 0 to 900 mg/kg, but decreased at 1200 mg/kg. Compared with the control group, the diet supplemented with 900 mg/kg GSH not only increased the concentration of GSH in the haemolymph and hepatopancreas, but also enhanced the activity of glutathione peroxidase (GSH-Px) (p

A 10-week feeding trial was performed to evaluate the effects of different types and levels of carbohydrates in growth performance, apparent digestibility coefficients and skin-associated mucosal non-specific immune parameters in blunt snout bream (Megalobrama amblycephala). Fish were randomly fed four diets containing two carbohydrates (glucose and starch) diets and two carbohydrates levels (330 and 440 g kg−1). High carbohydrate levels remarkably increased the weight gain rate (WGR), apparent digestibility of dry matters, protein and carbohydrates, body crud protein content, plasma levels of aspartate transaminase (AST), and skin-associated mucosal levels of immunoglobulin M (IgM), HDL cholesterol, lysozyme (LZM), advanced the transcriptions of mucin 2 (Muc2), mucin 5b (Muc5b) and apolipoprotein A-I (apoA-I), whereas the opposite was true for feed conversion ratio (FCR), plasma levels of IgM, skin-associated mucosal levels of major histocompatibility complex (MHC) and β-Defensins, and the transcriptions of heat shock protein 60 (Hsp60). In addition, carbohydrate types of glucose remarkably increased the survival rate, apparent digestibility of dry matters, protein and carbohydartes, body crud ash, plasma levels of total protein (TP), globulin (GLB), immunoglobulin M (IgM), complement C3 and complement C4 and the transcriptions of Muc5b. Whereas the carbohydrate types of starch remarkably increased viscerosomatic index (VSI), hepatosomatic index (HSI), condition factor (CF), abdominal fat percentage (AFP), apparent digestibility of liquid, advanced the transcriptions of Muc2, apoA-I and heat shock protein 70 (Hsp70). Significant interactions between different types and levels of dietary carbohydrates were also observed in WGR, apparent digestibility of dry matters, protein and liquid, body crud ash, plasma levels of TP, albumin (ALB) and AST, skin-associated mucosal levels of major histocompatibility complex (MHC) and β-Defensins, and the transcriptions of Muc2 and Muc5b. Our results indicate that inclusion of high level of glucose in the diet of blunt snout bream could improve growth performance, nonspecific immunity, and increase the efficiency of protein, which is suggesting that high level of glucose could be used in feed production. However, the proportion of the specific formula of glucose using in feed needs further study.

An 8-week feeding trial was conducted to evaluate growth performance, whole-body composition, metabolism and digestion of Chinese soft-shelled turtle Pelodiscus sinensis fed with fish meal replacement by rice protein concentrate and squid paste supplementation diets. Three isonitrogenous and isoenergetic practical diets were formulated with the inclusion of 60% fishmeal (CT), 42% fish meal +18% rice protein concentrate (RP) and 42% fish meal +18% rice protein concentrate +1% squid paste (feeding attractant) (RPS), respectively. Microcapsule lysine was supplemented in RP and RPS diets to balance the amino acid profile. Turtles (initial weight 30.65 g) were fed thrice (6:00, 12:00 and 18:00) daily to apparent satiation. The results showed that growth performance in RP group showed no significant difference with that in CT group ( P > .05). In addition, daily squid paste supplementation significantly improved growth performance, nutrients retention and relative feed intake ( P P Pelodiscus sinensis with microcapsule lysine supplementation. Besides, diet with squid paste inclusion could improve feed intake and growth performance of Pelodiscus sinensis .

A 12-week feeding trial was performed to evaluate the effects of metformin on growth performance, energy sensing, insulin signaling pathway, glycolipid metabolism and glucose tolerance of blunt snout bream fed high-carbohydrate diets. Fish were randomly fed four diets containing two dietary carbohydrate levels (30 and 43%) and two metformin levels (0 and 0.25%). High carbohydrate levels remarkably increased tissue glycogen and lipid contents, hepatic adenosine triphosphate (ATP) and adenosine monophosphate (AMP) contents and the ATP/AMP ratio, plasma levels of glucose, insulin, triglyceride, glycated serum protein (GSP), advanced glycation end products (AGES) and lactic acid, hepatic t-AMPK protein levels and the transcriptions of AMPKα1, AMPKα2, target of rapamycin (TOR), insulin receptor (IR), ribosomal protein S6 kinase-polypeptide 1 (S6K1), glucokinase (GK), pyruvate kinase (PK), glucose transporter 2 (GLUT 2), glycogen synthase (GS), acetyl-CoA carboxylase α (ACCα), fatty acid synthase (FAS) and sterol regulatory element-binding protein-1 (SREBP1), whereas the opposite was true for weight gain, relative feed intake (RFI), feed conversion ratio (FCR), pyruvate levels, p-AMPK level, p-AMPK/t-AMPK ratio and the transcriptions of insulin receptor substrate 1 (IRS1), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), fructose-1,6-biphosphatase (FBPase), carnitine palmitoyltransferase IA (CPT IA), peroxisome proliferator activated receptor α (PPARα) and acyl-CoA oxidase (ACO). In addition, metformin supplementation remarkably increased tissue glycogen contents, plasma lactic acid level, hepatic AMP content, t-AMPK and p-AMPK level, p-AMPK/t-AMPK ratio and the transcriptions of AMPKα1, AMPKα2, IR, IRS1, GK, PK, GLUT 2, GS, CPT IA, PPARα and ACO, while the opposite was true for the remaining indicators. Significant interactions between dietary carbohydrate and metformin were also observed in RFI, FCR, tissue glycogen and lipid contents, hepatic ATP/AMP ratio, plasma metabolites, the phosphorylation of AMPK and the transcriptions of TOR, IRS1, S6K1, PK, G6Pase, GLUT 2, GS, ACCα, FAS, SREBP1, CPT IA, PPARα and ACO. Furthermore, metformin improved the glucose tolerance of fish fed high-carbohydrate diets after glucose loading. Overall, our findings indicated that metformin should be benefits the intermediary metabolism of Megalobrama amblycephala fed high-carbohydrate diets through the enhancement of energy sensing, insulin sensitivity, glycolysis, glycogenesis and fatty acid oxidation coupled with the suppression of gluconeogenesis and lipogenesis.

The blunt snout bream (Megalobrama amblycephala) is prone to a glucose metabolic disorder when it is exposed to a high-carbohydrate diet over a long period of time. The purpose of this study was to determine whether dietary L-tryptophan (L-trp) improves glucose regulation in blunt snout bream via effects on long non-coding RNAs (lncRNAs). Fish were fed a normal-carbohydrate diet (CN), a high-carbohydrate diet (HC), or a high-carbohydrate diet supplemented with L-trp (CL) for 9 weeks, then high-throughput sequencing was used to analyze hepatic RNA libraries. Totals of 4951 lncRNAs and 8357 mRNAs were identified. Of these, 189 differentially expressed lncRNAs and 634 differentially expressed mRNAs were identified, some of which were validated using qPCR. Enrichment analysis of the mRNAs that were co-expressed with lncRNAs identified 32 potential cis-regulatory and 892 potential trans-regulatory relationships. Functional analysis showed that these co-expressed genes were represented in 151 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including 8 cis-KEGG enrichment pathways and 143 trans-KEGG enrichment pathways. Of the top 20 significant trans-enriched pathways, most were related to glucose metabolism, L-trp metabolism, and lipid metabolism, which suggested that L-trp might regulate glycolipid metabolism. The significantly differentially expressed mRNAs and lncRNAs that were upregulated in the HC group and downregulated in the CL group were used to construct a co-expression network. In total, 15 lncRNAs and 13 mRNA genes were included in this network, and most of the target genes were involved in the amelioration of insulin resistance and the maintenance of glucose homeostasis. Thus, we have generated a catalog of hepatic lncRNAs for the blunt snout bream and laid the foundation for further research into the regulation of glucose metabolism by dietary L-trp-induced lncRNAs.

This study aimed to investigate the effects of dietary fructooligosaccharide (FOS) on the growth, antioxidant capability, immune response and disease resistance of Chinese mitten crab Eriocheir sinensis . Groups of crab (17.74 ± 0.39 g) were distributed into 18 cement pools (33 crabs per pool) with replicates and fed experimental diets supplemented with FOS at 0, 0.5, 1.0, 1.5, 2.0 and 2.5 g/kg (designated as control, TF1, TF2, TF3, TF4 and TF5, respectively). After 60 days of feeding trial, crabs were challenged by Aeromonas hydrophila and the survival was recorded for the next 96 h. The results indicated that crabs fed TF4 had significantly higher weight gain (WG) than crabs fed control diet, whereas WG decreased with FOS level increasing to 2.5 g/kg. There was no significant difference ( P > 0.05) in SGR among all experimental groups. Catalase (CAT) and superoxide dismutase (SOD) activities in crabs fed TF4 diet were significantly higher than those in control group, while malondialdehyde (MDA) concentrations in crabs fed TF4 diet were significantly lower than that in control group. Crabs fed TF4 diet showed significantly higher alkaline phosphatase (AKP) activity compared to the control group. There was a trend that acid phosphatase (ACP) activity was elevated with FOS levels increasing up to 2.0 g/kg and decreased subsequently, and crabs fed TF4 diet showed significantly higher ACP activity compared to the control group. Supplementation of 2.0 g/kg FOS up-regulated the mRNA expression of interleukin-2 enhancer binding factor 2 ( ILF2 ) and peroxinectin ( PX ), and down-regulated the mRNA expression of lipopolysaccharide-induced TNF-α factor ( LITAF ). Bifidobacteriales abundance in the digestive tract increased significantly with dietary FOS levels increasing from 1.5 to 2.5 g/kg. Crabs fed TF4 showed significantly higher Bacteroides abundance compare to the other groups. After Aeromonas hydrophila challenge, the highest relative percent survival (25.00%) was observed in crabs fed TF4 diet, which was significantly higher than that in control group. Taken together, 2.0 g/kg FOS supplementation might be effective in improving growth performance, antioxidant capability, immune response and disease resistance of Chinese mitten crab.

This study aimed to characterize the full-length complementary DNA (cDNA) of fructose-1,6-bisphosphatase 1b (FBP1b) from fish Megalobrama amblycephala, and investigate its transcriptional response to glucose administration after the adaptation to high-carbohydrate diets. The cDNA obtained covered 1435 bp with an open reading frame of 1014 bp. Sequence alignment and phylogenetic analysis revealed a high degree of conservation (76-96%) among most fish and other vertebrates, retaining one N-linked glycosylation site, one N-terminal acetylation site, 13 phosphorylation sites, one fructose-1,6-bisphosphatase (FBPase) active site, five metal-binding sites, four substrate-binding sites, and several AMP-binding sites. The highest messenger RNA (mRNA) level of FBP1b was observed in liver followed by intestine, whereas relatively low values were detected in heart, gill, and eye. Then, the mRNA levels of FBP1b and the FBPase activity were both determined in the liver of fish injected intraperitoneally with 1.67 g glucose per kilogram body weight after being fed two dietary carbohydrate levels (30 and 42%) for 11 weeks. After the glucose load, the mRNA levels of FBP1b in both treatments decreased significantly to the basal value at 8 h and showed a slight increase afterward. However, the enzymatic activity showed no statistical difference during the first 4 h, but increased remarkably with further increasing times. In addition, both the mRNA levels and activities decreased significantly with increasing dietary carbohydrate levels. The results indicated that the FBP1b of M. amblycephala shared a high similarity with that of the other vertebrates. Its mRNA expression in liver was downregulated remarkably by a glucose administration, as also held true after the long-term adaptation of a carbohydrate-rich diet.

Five 2-week feeding trials were conducted to investigate five stimulants on P. sinensis. Two isonitrogenous and isoenergetic diets were formulated, one contained 600 g kg–1 fishmeal (FM diet) and the other contained 420 g kg–1 fishmeal and 225 g kg–1 animal protein blend (APB diet), and each tested one stimulant with four levels. The feeding stimulant candidates were betaine, 2-carboxy-ethyl dimethyl sulphonium bromide (DMPT), inosine-5′-monophosphate (IMP), taurine and squid extract. In the experiment on each stimulant, turtles (10.85 ± 0.10 g) were equally divided into FM and APB groups and fed with an equal mixture of corresponding diets containing four levels of stimulants, respectively. Each diet contained a unique rare earth oxide as inert marker. Turtles were fed twice daily (8:00 and 17:00), and faeces were collected. Preference for each diet was estimated based on the relative concentration of each marker in the faeces. In the FM group, only IMP showed the enhanced attraction. But in APB groups, all the stimulants, except IMP, showed higher preference than basal diets for at least one inclusion level, and the optimum level was 10 g kg–1 for betaine, 0.1 g kg–1 for DMPT, 0.1 g kg–1 for IMP, 5 g kg–1 for taurine and 10 g kg–1 for squid extract, and the squid extract had the strongest stimulating effect among the stimulants.

This study aimed to determine the optimal dietary niacin requirement of juvenile Megalobrama amblycephala. Fish, with an average weight of 3.62 ± 0.02 g, were randomly divided into six groups and fed six purified diets with graded levels of niacin (0, 10, 20, 30, 40 and 50 mg/kg) three times daily for 8 weeks. The survival rate, weight gain (WG), feed conversion ratio, protein efficiency and protein retention all significantly improved (p .05) with further increases in niacin levels. The supplementation of 30 mg/kg niacin also led to a significant (p .05) but exerted a significant (p

Eriocheir sinensis (E. sinensis) is an important aquaculture species in China. However, deteriorating water environments lead to oxidative stress in these crabs, which subsequently reduces their quality and yield. Glutathione (GSH) is an endogenous antioxidant that is used to mitigate oxidative stress. However, whether dietary GSH can enhance the resistance of E. sinensis to oxidative stress remains unclear. Herein, crabs were fed dietary GSH (the basal diet was supplemented with 0, 300, 600, 900, and 1200 mg/kg diet weight of GSH) for up to 3 weeks and, then, challenged with lipopolysaccharide (LPS; 400 μg/kg body weight). After 6 h, their hepatopancreas were sampled. Diet supplementation with 600 and 900 mg/kg diet weight GSH not only increased the content of GSH in the hepatopancreas, but also enhanced the activities and mRNA expressions of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione-S-transferase (GST) (P

An 8-week feeding trial was conducted to investigate effects of dietary protein levels (37, 40, and 43%) on the growth performance, feed utilization, digestive enzyme activity, and gene expressions of target of rapamycin (TOR) signaling pathway in fingerling yellow catfish. One hundred and eighty fingerlings (average weight 0.77 ± 0.03 g) were equally distributed across four replicate tanks for each of the three treatments, with 15 fish per tank. No difference (P 0.05) was observed in initial body weight, survival rate (SR), hepatosomatic index (HSI), viscera index (VSI), dressing percentage (DP), and condition factor (CF) among all the treatments. The diet containing 40% protein increased significantly (P 0.05) final body weight, weight gain rate (WGR), specific growth rate (SGR), protein efficiency ratio (PER), nitrogen retention (NRE), and energy retention (ERE) in fish. The highest protease activity in the stomach and intestine was observed in the P40 group (P 0.05), while amylase and lipase were not significantly different (P 0.05). The transcriptional levels of IGF-1, IGF-1R, and Akt were significantly (P 0.05) higher in fish fed P40 or P43 than those of fish fed P37. TOR and S6K1 mRNA expressions were significantly (P 0.05) increased in the P40 groups. Hence, the diet containing 40% protein would be suitable for the optimum growth and effective protein utilization of fingerling Pelteobagrus fulvidraco. In vitro, the transcriptional levels of IGF-1, IGF-1R, Akt, TOR, and S6K1 in hepatocyte supplemented with a 40-μM mixed amino acids were significantly (P 0.05) higher compared to other treatments. No difference (P 0.05) was observed in eukaryotic translation initiation factor 4E-binding protein 1 in vivo and in vitro among all the treatments. Effects of dietary protein level on growth performance likely are involved in the activation of TOR signaling pathway in fingerling Pelteobagrus fulvidraco.

The Chinese mitten crab (Eriocheir sinensis) is an economically important aquaculture species in China, with distinct differences in ovarian maturation status between crabs fed with natural diets and artificial diets during the listing period, thus, leading to selling price differentiation. Our previous study showed that dietary supplementation with 100 mg/kg icariin can effectively promote ovarian development of E. sinensis. However, the internal molecular mechanism has not yet been elucidated because of a lack of comprehensive genome sequence information. We compared the ovary transcriptomes of E. sinensis fed with two diets containing 0 and 100 mg/kg ICA using the BGISEQ-500 platform. This yielded 12.54 Gb clean bases and 54,794 unigenes, 13,832 of which were found to be differentially expressed after icariin exposure. Twenty pathways closely related to gonadal development were selected through KEGG analysis. Seven differentially expressed genes relevant to vitellogenesis and oocyte maturation (serine/threonine-protein kinase mos-like, Eg2, cytoplasmic polyadenylation element-binding protein, cyclin B, vitellogenin 1, cathepsin D, and juvenile hormone esterase-like carboxylesterase 1) were validated by qRT-PCR, and four proteins (MEK1/2, ERK1/2, Cyclin B and Cdc2) associated with the progesterone mediated oocyte maturation pathway (i.e., MAPK/MPF pathway) were analyzed by western-blot. The results showed that icariin could promote the synthesis, processing and deposition of vitellogenin in oocytes, and that it also has the potential to promote oocyte maturation (resumption of Meiosis I) by altering the expression of the relevant genes and proteins.

This study aimed to investigate the optimal dietary vitamin B12 requirement of fingerling blunt snout bream Megalobrama amblycephala based on the results of growth performance, intestinal digestive and absorptive capability and responses of the growth hormone (GH)-insulin-like growth factor-I (IGF-I) axis. Fish (initial body weight: 0.71 ± 0.03 g) were randomly assigned to six purified diets containing graded levels of vitamin B12 (0, 0.028, 0.051, 0.12, 0.23 and 0.41 mg/kg) for 12 weeks. Both weight gain (WG) and specific growth rate increased significantly (P Statement of relevance This study investigated the optimal dietary vitamin B12 requirement of fingerling blunt snout bream based on growth performance. The underlying mechanisms were also investigated with emphasis on intestinal digestive and absorptive capability and modulation of the GH-IGF-I axis. Data obtained here might facilitate the development of low-cost formulated feed for aquatic species.

High-fat diets may have favorable effects on growth and cost, but high-fat diets often induce excessive fat deposition, resulting in liver damage. This study aimed to identify the hepatoprotective of a Chinese herb (berberine) for blunt snout bream (Megalobrama amblycephala). Fish were fed with a normal diet (LFD, 5 % fat), high-fat diet (HFD, 15 % fat) or berberine-supplemented diets (BSD, 15 % fat with berberine 50 or 100 mg/kg level) for 8 weeks. After the feeding, histology, oxidative status and mitochondrial function of liver were assessed. The results showed that HFD caused fat accumulation, oxidative stress and apoptosis in hepatocytes of fish. Hepatocytes in HFD group appeared to be hypertrophied, with larger liver cells diameter than these of LFD group. Berberine-supplemented diets could attenuate oxidative stress and hepatocytes apoptosis. HFD induced the decreasing mitochondrial complexes activities and bulk density and surface area density. Berberine improved function of mitochondrial respiratory chain via increasing the complex activities. Moreover, the histological results showed that berberine has the potential to repair mitochondrial ultrastructural damage and elevate the density in cells. In conclusion, our study demonstrated that berberine has attenuated liver damage induced by the high fat mainly via the protection for mitochondria.

A full-length cDNA coding glucokinase (GK) was cloned from Megalobrama amblycephala by RT-PCR and RACE approaches. The cDNA obtained covered 2090 bp with an open reading frame of 1431 bp encoding 476 amino acids. Sequence alignment and phylogenetic analysis revealed a high degree of conservation (84–99%) among most fish and higher vertebrates, retaining one hexokinase signature sequence, one ATP-binding domain, two N-linked glycosylation sites and several glucose-binding sites. The highest GK expression was observed in the liver followed by intestine, whereas relatively low values were detected in the muscle, kidney and brain. Then, plasma glucose levels and liver GK activities and expressions were determined in fish subjected to a glucose load after being fed two dietary carbohydrate levels (30% and 42%) for 11 weeks. Plasma glucose levels decreased significantly after high-carbohydrate adaption. Glucose load induced a remarkable increment of glycemia in both groups with the maximum level being attained at 1 h. Thereafter, it decreased significantly to the basal value at 6 h and kept constant afterward. Hepatic GK expressions in both groups increased significantly with the maximum value being attained at 2 h after the glucose load, whereas no statistical difference was observed in enzymatic activities during the first 4 h. Then, both the activities and expressions decreased significantly with further increasing time. In addition, an inducible liver GK activity and expression by dietary carbohydrate levels were both observed. The results indicated that the GK gene of Megalobrama amblycephala showed a typical structure of the hexokinase family, and shared a high similarity with that of the other vertebrates. Hepatic GK expressions and activities of this species were highly inducible by glucose administration, as favored a short postprandial period of hyperglycemia. Furthermore, high dietary carbohydrate improved its glucose tolerance through the enhanced GK expressions and activities. Statement of relevance :This study investigated the mRNA expressions and activities of GK in herbivorous Megalobrama amblycephala subjected to a glucose load after the adaption to dietary carbohydrate levels. The data obtained here will facilitate the understanding of the molecular events involved in the carbohydrate metabolism of fish. It is also helpful for the development of low-protein and high-energy feed for fish.

This study aimed to investigate the effects of feeding rates on stress response, innate immunity and hypoxia resistance of juvenile blunt snout bream Megalobrama amblycephala. Fish were randomly assigned to one of six feeding rates (2, 3, 4, 5, 6 and 7% of body weight/day) for 60 days. Then, fish were subjected to hypoxic conditions and haemato-immunological parameters were analyzed pre- and post-challenge. Low feed ration resulted in decreased liver superoxide dismutase and catalase activities and reduced glutathione contents. Inadequate feeding also adversely affected the immune functions of fish, as was characterized by the relatively low haemato-immunological parameters (including alternative complement (ACH50), myeloperoxidase (MPO), plasma protein profiles and transferring) and high hypoxia-induced mortality. High feed ration did not lead to the improvement in antioxidant capability, immune responses and survival. In addition, plasma cortisol, glucose and transferrin levels as well as lysozyme activities all increased significantly after hypoxia challenge, whereas the opposite was true for plasma ACH50 and MPO activities as well as protein profiles in terms of hypoxia challenge. An interaction between feeding rate and hypoxia was also observed in plasma cortisol, glucose and protein profiles. In conclusion, a feeding rate of 4-5% of body weight/day is optimal to boost the innate immunity of juvenile blunt snout bream. Low ration resulted in decreased antioxidant capability, compromised immune functions and reduced hypoxia resistance, while over feeding did not benefit the health status.

Fermented soybean meal (FSM) is used as a potential substitute for fishmeal in various aquatic species due to its enriched nutrient profile. This study is aimed to investigate the effect of two different kinds of FSM on growth and protein metabolism in the Chinese mitten crabs (Eriocheir sinensis). One hundred and sixty-eight crabs were randomly assigned to 21 cement pools, each with eight crabs. The crabs in the control group were fed a basic diet, without FSM. Six isonitrogenous and isolipidic diets were prepared by replacing fish meal with two kinds (A and B) of FSM at concentrations of 5 %, 10 %, and 15 %, respectively. The results showed that partial replacement of fish meal with FSM could improve the growth performance of and crude protein content in the Chinese mitten crabs. In addition, increased activities of trypsin, pepsin, Na+/K+-ATPase, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase in the Chinese mitten crabs were observed. Furthermore, the hepatopancreas mRNA levels of protein metabolism-related genes, including protein kinase B (Akt), protein S6 kinase 1 (S6k1), 4E-binding protein 1 (4E-BP1), and oligopeptide transporter 1 (Pept1), were higher in the crabs fed FSM-supplemented diets than in the control crabs. The present study concludes that about 10 and 15 % of fish meal could be substituted by FSM A and B, respectively, without having any deleterious effects on growth performance, protein metabolism-related enzyme activities, and the expression ofand protein synthesis- and metabolism-related genes expressions.