Berberine attenuates oxidative stress and hepatocytes apoptosis via protecting mitochondria in blunt snout bream <Emphasis Type="Italic">Megalobrama amblycephala</Emphasis> fed high-fat diets

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.     

Effects of dietary linolenic acid on growth,fatty acid composition,immune function and antioxidant status of juvenile blunt snout bream,Megalobrama amblycephala

A nine‐week feeding trial was performed to determine the dietary linolenic acid (LNA; 18:3n–3) requirements of juvenile blunt snout bream. Six iso‐nitrogenous, semi‐purified diets were prepared with different concentrations of LNA (0–25 g/kg). Dietary LNA had no significant effects on survival rate. However, final fish weight, weight gain (WG), specific growth rate (SGR) and feed efficiency ratio (FER) increased with increasing dietary LNA concentrations up to 20 g/kg. Dietary LNA increased muscle LNA and total n‐3 polyunsaturated fatty acid (PUFA) contents, but decreased total saturated fatty acid content. Fish fed 20 g/kg LNA had the highest plasma alkaline phosphatase activity, total protein, albumin and white blood cell count levels. Additionally, fish fed 20 g/kg LNA had higher triglyceride levels than control fish. Plasma glucose increased with increasing dietary LNA concentrations. Superoxide dismutase and glutathione peroxidase activities significantly increased with increasing dietary LNA concentrations up to 15 g/kg. Based on SGR and FER, the optimal dietary LNA requirements of juvenile blunt snout bream were 17.5 and 15.6 g/kg respectively.     

Effects of dietary choline supplementation on growth performance,lipid deposition and intestinal enzyme activities of blunt snout bream Megalobrama amblycephal fed high‐lipid diet

This study aimed to investigate the effects of dietary choline supplementation on growth, lipid deposition and intestinal enzyme activities of Megalobrama amblycephala. Fish were fed four diets with two lipid levels (50 and 150 g kg^?1) and two choline supplementations (600 and 1600 mg kg^?1) for 8 weeks. Feed conversion ratio (FCR), viscerosomatic index (VSI), hepatosomatic index (HSI), intraperitoneal fat (IPF) ratio, whole‐body and muscle lipid contents, intestinal lipase activities and lipoprotein lipase (LPL) activities all increased significantly (P < 0.05) as lipid levels increased, whereas the opposite was true for whole‐body and muscle moisture contents and intestinal amylase activities. VSI, IPF ratio and whole‐body lipid contents all decreased significantly (P < 0.05) with increasing dietary choline supplementations. Weight gain, muscle moisture content all increased significantly (P < 0.05) with increasing dietary choline supplementations when dietary lipid levels reached 150 g kg^?1, whereas the opposite was true for FCR, IPF ratio, IPF and liver LPL activities. In addition, abnormal hepatocytes were found in the liver of fish fed 150 g kg^?1 lipid with 600 mg kg^?1 choline supplementation. The result of this study indicated that extra choline supplementation can improve growth performance, intestinal enzymes activities and reduce excessive lipid deposition of M. amblycephala fed high lipid.     

Effects of dietary carbohydrate/lipid ratios on growth performance,body composition and glucose metabolism of fingerling blunt snout bream Megalobrama amblycephala

This study aimed to investigate the optimum dietary carbohydrate/lipid (CHO/L) ratio for fingerling blunt snout bream Megalobrama amblycephala (average initial weight: 6.61 ± 0.03 g). Six isonitrogenous and isoenergetic diets were formulated to contain various CHO/L ratios ranging from 1.62 to 24.20. Each diet was tested in four replicates for 10 weeks. Specific growth rate (SGR), feed conversion ratio and nitrogen and energy retention all improved significantly (P < 0.05) as dietary CHO/L ratios decreased from 24.20 to 5.64 but showed little difference (P > 0.05) as dietary CHO/L ratios ranged from 2.45 to 5.64. Hepatosomatic index increased significantly (P < 0.05) as dietary CHO/L ratios increased, whereas intraperitoneal fat ratio showed an opposite trend (P < 0.05). Opposite to moisture content, lipid content of whole body and carcass all increased significantly (P < 0.05) as dietary CHO/L ratios decreased. Liver lipid content showed little difference (P > 0.05) among all the treatments, while liver glycogen content increased significantly (P < 0.05) with increasing CHO/L ratios. High dietary carbohydrate enhanced the activities of liver hexokinase, pyruvate kinase and glucose‐6‐phosphate dehydrogenase but did not induce hyperglycaemia. Based on the second‐order polynomial regression analysis of SGR, the optimal dietary carbohydrate and lipid contents for fingerling blunt snout bream were 291.7 and 81.4 g kg⁻¹, respectively, with a corresponding dietary CHO/L ratio of 3.58.     

Complete replacement of fish meal by plant protein ingredients with dietary essential amino acids supplementation for juvenile blunt snout bream (Megalobrama amblycephala)

Six extruded diets were formulated with a graded level of fish meal (FM); diet 1 and diet 2 were formulated with 80 g/kg FM; diet 3 and diet 4 were formulated with 40 g/kg FM; and diet 5 and diet 6 were devoid of FM. Hence, diet 2, diet 4 and diet 6 were supplemented with essential amino acids (EAAs). The diets were fed throughout an eight‐week feeding trial. The results revealed that specific growth rate (SGR), feed conversion ratio (FCR), body weight gain (BWG) and protein efficiency ratio (PER) were influenced by FM reduction and improved by the addition of EAA (p < 0.05). A survival rate (SR), whole body content of protein, lipid, moisture and ash, and plasma total protein (TP), albumin (ALB), triglyceride (TG) and urea (UN) did not influence by dietary treatments (p > 0.05). Additionally, target of rapamycin (TOR) pathway did not influence by dietary treatments (p > 0.05). Expression levels of hepatic peptide transporter 1 (Pept1) and peptide transporter 2 (Pept2) decreased against FM reduction and improved significantly in the groups fed diet 4 and diet 6. In summary, the findings revealed that diet containing plant proteins mixture supplemented with EAA could totally replace FM in the practical diet of blunt snout bream.     

饲料锌对团头鲂幼鱼生长性能、血清生化指标和抗氧化功能的影响

以酪蛋白和明胶为蛋白源,七水硫酸锌(Zn SO4·7H2O)为Zn源,分别配制成7种Zn含量(7.4 mg/kg、20.3 mg/kg、32.1 mg/kg、51.0 mg/kg、84.4 mg/kg、169.7 mg/kg、332.4 mg/kg)的半纯化饲料,投喂初始体重为(3.6±0.1)g团头鲂(Megalobrama amblycephala)12周,考察Zn对团头鲂幼鱼生长性能、血清生化指标和抗氧化功能的影响,确定团头鲂幼鱼对饲料Zn的需要量。结果表明,随着饲料Zn含量增加,团头鲂增重率、特定生长率和全鱼Zn含量呈先增加后稳定的趋势;全鱼水分含量显著降低(P0.05),粗蛋白含量显著增加。饲料Zn含量对团头鲂饲料系数无显著影响。饲料中添加Zn显著影响血清总蛋白、尿素氮、高密度脂蛋白胆固醇、总胆固醇以及甘油三酯含量,而对血清白蛋白含量和碱性磷酸酶活性无显著影响。随着饲料中Zn含量的增加,团头鲂肝丙二醛含量显著降低(P0.05),而肝过氧化氢酶和超氧化物歧化酶活性在各处理间均无显著差异。折线回归分析表明,团头鲂幼鱼(体重3.6~26.7 g)获得最佳生长时对饲料Zn需要量为32.6 mg/kg,获得最大鱼体Zn含量时Zn的需要量为47.6 mg/kg。本研究旨在确定团头鲂幼鱼对饲料中Zn的需要量,为配制团头鲂高效环保饲料提供科学依据。     

Effects of dietary supplementation of fructooligosaccharide on growth performance,body composition,intestinal enzymes activities and histology of blunt snout bream (Megalobrama amblycephala) fingerlings

A feeding trial was conducted to investigate the effects of dietary fructooligosaccharide (FOS) on growth performance, body composition, intestinal enzymes activities and histology of fingerling Megalobrama amblycephala. A total of 1200 fish (1.42 ± 0.01 g) were fed diets containing graded levels of FOS (0, 0.5, 1, 2, 4 and 8 g kg⁻¹ diet) for 8 weeks in a recirculating system indoor. The weight gain, specific growth rate (SGR) and survival rate were all improved in dietary supplementation of FOS fed fish. Increasing FOS levels resulted in both higher whole‐body lipid and lower moisture contents, whereas ash and protein contents showed no significant differences among all the treatments. Intestinal amylase, protease, Na⁺, K⁺‐ATPase, alkaline phosphatase, γ‐glutamyl transpeptidase and creatine kinase activities all increased with dietary FOS levels up to 4 g kg⁻¹ (P < 0.05). Transmission electron microscopy analysis indicated that microvilli length in the mid‐intestine was significantly increased with increased dietary FOS levels (P < 0.05). In conclusion, dietary supplementation of FOS could confer benefits on growth performance, intestinal digestive and absorptive ability, histology of fingerling Megalobrama amblycephala.     

Effects of dietary tryptophan levels on growth performance,whole body composition and gene expression levels related to glycometabolism for juvenile blunt snout bream,Megalobrama amblycephala

To investigate the effects of dietary tryptophan on growth and glycometabolism in juvenile blunt snout bream, 450 fish (initial weight 23.33 ± 0.03 g) were fed six practical diets with graded levels of tryptophan (from 0.79 g/kg to 5.96 g/kg dry matter) for 8 weeks. Results showed that final weight, per cent weight gain (PWG), protein efficiency rate, feed intake and feed conversion ratio (FCR) were significantly improved by 2.80 g/kg diet. The maximum values of protein and ash were observed in 2.80 g/kg diet, while moisture was minimum. Lipid content of fish fed 3.95 g/kg diet was significantly higher than other diets. The highest plasma insulin‐like growth factor‐1 (IGF‐1) content was observed in 0.79 g/kg diet. In the liver, IGF‐1 mRNA levels were significantly downregulated by 2.80 g/kg dietary tryptophan, while glucokinase levels were by 3.95 g/kg, while glucose‐6‐phosphatase and phosphoenolpyruvate carboxykinase mRNA levels showed a converse trend compared with IGF‐1. Based on PWG and FCR, the optimal dietary tryptophan level was determined to be 1.99 g/kg (6.20 g/kg of dietary protein) and 1.96 g/kg (6.11 g/kg of dietary protein), respectively, using broken‐line regression analysis.     

Dietary histidine affects intestinal antioxidant enzyme activities,antioxidant gene expressions and inflammatory factors in juvenile blunt snout bream (Megalobrama amblycephala)

Six semipurified diets of graded histidine levels (from 4.1 to 14.2 g/kg) were fed to fish for 8 weeks. The results showed that the intestinal content of malondialdehyde (MDA), activities of total superoxide dismutase (T‐SOD) and glutathione peroxidase (GPx) were noticeably lower in 8.0 g/kg diet compared to control group. While the lowest activities of copper–zinc superoxide dismutase (Cu/Zn‐SOD) and manganese superoxide dismutase (Mn‐SOD) were observed in 9.9 g/kg diet, the intestinal activities of total antioxidant capacity (T‐AOC) and catalase (CAT) in 14.2 g/kg diet were noticeably higher than those in 8.0 and 9.9 g/kg diets. Plasma biochemical indexes were not significantly affected by dietary histidine levels. In the intestine, the Kelch‐like ECH‐associated protein 1 (Keap1) mRNA levels were increased in 8.0 g/kg diet, which suppressed the nuclear translocation of nuclear factor E2‐related factor 2 (Nrf2), and subsequently decreased CAT, GPx1, Cu/Zn‐SOD and Mn‐SOD expression levels. The lowest mRNA levels of interleukin 8 (IL‐8) and tumour necrosis factor‐α (TNF‐α) were observed in 8.0 g/kg diet, whereas the highest mRNA levels of transforming growth factor‐β (TGF‐β) and interleukin 10 (IL‐10) were observed in 8.0 g/kg diet. These results indicated that dietary histidine plays a major role in maintaining intestinal health in juvenile blunt snout bream.     

Effects of dietary docosahexaenoic acid on growth performance,fatty acid profile and lipogenesis of blunt snout bream (Megalobrama amblycephala)

Six diets were designed to investigate the effects of dietary docosahexaenoic acid (22:6n‐3; DHA) levels (0.5, 1.3, 2.3, 4.2, 8.1 and 15.9 g/kg diets) on growth performance, fatty acid profile and expression of some lipogenesis‐related genes of blunt snout bream (Megalobrama amblycephala). Fish (average weight: 26.40 ± 0.11 g) were randomly fed one of six diets for 8 weeks. Results indicated that the final body weight (FBW) and specific growth rate (SGR) of fish fed 1.3 g/kg DHA were significantly higher than other groups except for the 2.3 g/kg DHA (p < .05). Compared with other groups, the number of lipid droplet clusters of the liver stained with oil red O in the 2.3 g/kg DHA group was the highest, which was consistent with the lipid contents of whole body and liver. The DHA proportion in liver and muscle significantly increased with the increasing dietary DHA levels (p < .05), which reflected fatty acid profiles of diets. The highest mRNA expressions of acetyl‐CoA carboxylase α (ACCα), fatty acid synthase (FAS) and sterol regulatory element‐binding protein‐1 (SREBP‐1) occurred in the 1.3 g/kg DHA group, followed by 2.3 g/kg DHA. In summary, the supplementation of 1.3–2.3 g/kg DHA could improve growth performance and lipogenesis, and the dietary DHA could improve DHA and PUFA proportion in liver and muscle.     

Molecular characterization and identification of facilitative glucose transporter 2 (GLUT2) and its expression and of the related glycometabolism enzymes in response to different starch levels in blunt snout bream (<Emphasis Type="Italic">Megalobrama amblycephala</Emphasis>)

Facilitative glucose transporters (GLUT) are transmembrane transporters involved in glucose transport across the plasma membrane. In this study, blunt snout bream GLUT2 gene was cloned, and its expression in various tissues and in liver in response to diets with different carbohydrate levels (17.1; 21.8; 26.4; 32.0; 36.3; and 41.9% of dry matter). Blunt snout bream GLUT2 was also characterized. A full-length cDNA fragment of 2577 bp was cloned, which contains a 5′-untranslated region (UTR) of 73 bp, a 3′-UTR of 992 bp, and an open reading frame of 1512 bp that encodes a polypeptide of 503 amino acids with predicted molecular mass of 55.046 kDa and theoretical isoelectric point was 7.52. The predicted GLUT2 protein has 12 transmembrane domains between amino acid residues at 7–29; 71–93; 106–123; 133–155; 168–190; 195–217; 282–301; 316–338; 345–367; 377–399; 412–434; and 438–460. Besides, the conservative structure domains located at 12–477 amino acids belong to the sugar porter family which is the major facilitator superfamily (MFS) of transporters. Blunt snout bream GLUT2 had the high degree of sequence identity to four GLUT2s from zebrafish, chicken, human, and mouse, with 91, 63, 57, and 54% identity, respectively. Quantitative real-time (qRT) PCR assays revealed that GLUT2 expression was high in the liver, intestine, and kidney; highest in the liver and was regulated by carbohydrate intake. Compared with the control group (17.1%), fed by 3 h with higher starch levels (32.0; 36.3; and 41.9%), increased plasma glucose levels and glycemic level went back to basal by 24 h after treatment. Furthermore, higher dietary starch levels significantly increase GLUT2, glucokinase (GK), and pyruvate kinase (PK) expression and concurrently decrease phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6P) mRNA levels (P?<?0.05), and these changes were also back to basal levels after 24 h of any dietary treatment. These results indicate that the blunt snout bream is able to regulate their ability to metabolize glucose by improving GLUT2, GK, and PK expression levels and decreasing PEPCK and G6P expression levels.     

Assessment of reference dietary amino acid pattern for juvenile red sea bream, <Emphasis Type="Italic">Pagrus major</Emphasis>

Four semi-purified diets, containing crystalline amino acids (CAAs), were fed to juvenile red sea bream, Pagrus major in order to ascertain the ideal dietary amino acid pattern for this species. A control diet containing 50% casein–gelatin as protein sources, but no CAAs were fed to the fish. The other diets contained 30% casein–gelatin and 20% CAAs. CAAs were added to diets to simulate with amino acid pattern of the red sea bream eggs protein (REP), red sea bream larvae whole body protein (RLP), red sea bream juvenile whole body protein (RJP), and brown fishmeal protein (BFP). The juveniles (average initial body weight, 1.58 ± 0.01 g) were maintained in triplicate tanks and fed twice daily for 30 days. The highest weight gain was observed in juveniles fed the RJP diet. No significant difference was observed in juveniles fed the RLP and BFP diet. Feed efficiency ratio, protein efficiency ratio and amino acid retention in the whole body were significantly (p < 0.05) affected by the simulated dietary amino acid patterns. The essential amino acid profile and A/E ratios of the whole body after the growth trial showed little difference among the dietary treatments. The results suggest that red sea bream juveniles are able to utilize high amounts of CAA in coated form. The amino acid pattern of RJP could be used as an appropriate of reference dietary amino acid for this species.     

Involvement of the miR-462/731 cluster in hypoxia response in <Emphasis Type="Italic">Megalobrama amblycephala</Emphasis>

MicroRNAs (miRNAs) are non-coding small RNAs showing both evolutionarily conserved and unique features and are involved in nearly all biological processes. In the present study, the role played by miR-462/731 cluster miRNAs in hypoxia response in Megalobrama amblycephala, an important freshwater fish, was investigated. The M. amblycephala miR-462/731 cluster locus and their 5′ flanking sequences were sequenced and analyzed. In M. amblycephala and other teleost fish species, the mature sequences of miR-462 and miR-731 were identical and hypoxia-responsive elements (HREs) were identified upstream of the miR-462/731 loci. The two miRNAs were significantly induced in the liver, spleen, gill, muscle, and brain after hypoxia treatment. The expression of both miRNAs was also upregulated in cells that received treatment which mimicked hypoxia. Furthermore, reporter assay revealed that M. amblycephala HREs can be activated by hypoxia. Taken together, the 462/731 cluster may play a role in the regulation of the hypoxia response in M. amblycephala.     

Molecular cloning and expression analysis of <Emphasis Type="Italic">Megalobrama amblycephala</Emphasis> transferrin gene and effects of exposure to iron and infection with <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

Transferrin (Tf) plays an important function in iron homeostasis and metabolism of organisms. In this study, we identified and characterized the Tf gene in Megalobrama amblycephala and evaluated its expression in basal conditions as well as after iron overload and experimental infection with Aeromonas hydrophila. Furthermore, we studied the iron binding properties of recombinant Tf. The full-length M. amblycephala Tf complementary DNA (cDNA) (GenBank accession no.: KX698308) of 2245 bp was cloned and contained a 1953 bp open reading frame (ORF) encoding 650 amino acid residues and flanked by a 68 bp 5′ and a 204 bp 3′ untranslated regions (UTR). Predicted conservative structure illustrated that M. amblycephala Tf consisted of two conservative Tf domains. Amino acid sequence alignment revealed that M. amblycephala Tf had high similarity with that of cyprinids deposited in Genbank, and phylogenetic analysis showed that M. amblycephala Tf clustered with Ctenopharyngodon idella and Hypophthalmichthys molitrix. Tissue expression pattern analyses demonstrated that the liver was the main Tf mRNA expressing organ, being significantly higher than other tissues (p < 0.05). In the liver, Tf mRNA expression in fish artificially injected with the pathogenic bacteria A. hydrophila was significantly upregulated, reaching a peak at 12 h post injection (hpi) and then decreasing afterward. The expression in FeCl₃-injected fish showed a similar tendency, but reached a peak at 8 hpi. Meanwhile, fish serum iron significantly decreased following A. hydrophila injection, but increased to peak at 4 hpi and then decreased in FeCl₃-injected fish. The recombinant M. amblycephala Tf showed iron binding capacity using CAS analysis. These results are helpful to understand the structure and regulation of expression of Tf, as well as the specific function of Tf for both immune responses and iron homeostasis.     

Effects of dietary selenium on the growth,selenium status,antioxidant activities,muscle composition and meat quality of blunt snout bream,Megalobrama amblycephala

An 8‐week feeding trial was performed to investigate effects of dietary selenium (sodium selenite, Na₂SeO₃; selenium nanoparticle, Nano‐Se; selenium yeast, Se‐yeast) on the growth, selenium status, antioxidant activities, muscle composition and meat quality of blunt snout bream. Na₂SeO₃ and Nano‐Se were supplemented at 0.2 mg Se kg^?1, and Se‐yeast was supplemented at 0.1, 0.2, 0.4 and 0.8 mg Se kg^?1 in basal diet; no Se was added as a control. The results indicated that groups of 0.2 and 0.4 mg kg^?1 Se‐yeast had significantly higher weight gain and Nano‐Se, 0.2, 0.4 mg kg^?1 Se‐yeast had significantly lower feed conversation ratio compared with the control group. The Se concentrations of whole body, muscle and liver linearly increased with increasing dietary Se‐yeast levels. Group of 0.4 mg kg^?1 Se‐yeast significantly increased activities of catalase and glutathione peroxidase. Muscle colour of Nano‐Se, 0.2, 0.4 mg kg^?1 Se‐yeast groups, the water‐holding capacity of 0.4, 0.8 mg kg^?1 Se‐yeast groups showed significant differences compared with the control group. The results suggest that Se‐yeast and Nano‐Se had a better growth performance than Na₂SeO₃ at 0.2 mg Se kg^?1, and supplementing appropriate Se‐yeast in diet can increase antioxidant activities and enhance meat quality of blunt snout bream.     

The effect of dietary folic acid on biochemical parameters and gene expression of three heat shock proteins (HSPs) of blunt snout bream (<Emphasis Type="Italic">Megalobrama amblycephala)</Emphasis> fingerling under acute high temperature stress

The effects of dietary folic acid on biochemical parameters and gene expression of three heat shock proteins (HSPs) of blunt snout bream (Megalobrama amblycephala) fingerling under acute high temperature stress. Six dietary folic acid groups (0.0, 0.5, 1.0, 2.0, 5.0, and 10.0) mg/kg diets were designed and assigned into 18 tanks in three replicates each (300 l/tank) and were administered for 10 weeks in a re-circulated water system. The fingerlings with an initial weight of 27.0 ± 0.03 g were fed with their respective diets four times daily. At the end of the experiment, samples were collected before challenge, 0, 24, 72 h, and 7 days. Serum total protein (TP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), cortisol, glucose, complement C3 (C3), complement C4 (C4, immunoglobulin M (IgM) hepatic superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and the expression of heat shock protein 60 (HSP60), 70 (HSP70), and 90 (HSP90) were studied. The results showed that fish fed with dietary folic acid between 1.0, 2.0, and 5.0 mg/kg significantly (P < 0.05) increased serum TP, C3, C4 hepatic SOD, CAT, and the expression of HSP60, HSP70, and HSP90 before and after temperature challenge of 32 °C. Also, serum ALP, cortisol, glucose, and hepatic MDA were significantly (P < 0.05) reduced by supplementation of dietary folic acid level 1.0, 2.0, and 5.0 mg/kg before and after the same temperature challenge of 32 °C. Before stress, 0, 24, 72 h, and 7 days significantly (P < 0.05) affects serum biochemical parameters, immune and antioxidant capacities, and expression level of three HSPs. Furthermore, there was no statistical evidence to show that dietary folic acid inclusion level and temperature duration have significant interactive effect on serum biochemical parameters, antioxidant parameters, and gene expression level (P > 0.05) of the three HSPs. However, there were statistical significant interactive effect between dietary folic acid inclusion level and temperature duration on serum C3 and C4 (P < 0.05) except IgM (P > 0.05). The present results indicate that supplementation of basal diet from 1.0 mg/kg; 2.0 and 5.0 mg/kg can enhance acute high temperature resistance ability in M. amblycephala fingerling to some degree and improve physiological response, immune and antioxidant capacities, and expression level of three HSPs.