| This study was conducted to determine the effects of vitamin C on the growth,muscle composition,immunity and high pH coercion in crayfish,Procambarus clarkia by means of undertaking growth trials,physiology and biochemistry as well as immunoenzymatic activity detection,and transcriptome analysis.This study will provide further proofs for improving nutrient requirement parameters and guiding the production of compound feed in crayfish.The research results are as follows.1.The effects of high PH stress on antioxidant capacity and transcriptome analysis in crayfish.Two experiments were set up:pH 7.6(control group)and pH 10.2(pH stress group).Each group was respectively sampled for measuring superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and malondialdehyde(MDA)levels in hepatopancreas at 0 h,3 h,6 h,12 h,24 h,5 d,10 d and 15 d.Transcriptome sequencing was used to analyze the differentially expressed genes in hepatopancreas of the two groups under 5 d stress.The results showed that the activity of SOD presented the ascending trend at first and then descending with the extension of stress time and MDA was increased significantly(P<0.05)after 5 d pH stress in pH stress group compared with control group.In the early stages of stress(3-24 h),the activity of GSH-Px in experimental group was apparently lower than control group.A total of 79,814 Unigenes were obtained by transcriptome sequencing,which could be annotated to all 7 databases.Comparative transcriptome analysis identified 4,596 up-regulated genes and 4,604 down-regulated genes.Most of the genes related to immunity,energy metabolism and apoptosis.The function of all genes were clustered into 2772 GO categories and 34 KEGG metabolic pathways.Glutathione metabolism,drug metabolism cytochrome P450,citrate cycle,antigen processing and presentation,metabolism of xenobiotics by cytochrome P450,PPAR signaling pathway and estrogen signaling pathway were significantly enriched.In conclusion,high pH treatment of crayfish could reduce antioxidant capacity of hepatopancreas,and it significantly affects gene expression in metabolic pathways such as immunity,energy,and apoptosis2.A single factor gradient method was used to prepared the six isonitrogenous and isoenergy diets with the vitamin C content were 0(control group),30,60,120,240 and 480 mg/kg(experimental group),and the growth test lasted for 60 days.The results showed that the weight gain rate(WGR)and specific growth rate(SGR)were firstly increased and then decreased,the inflection point was dietary vitamin C level 120 mg/kg.The meat content in 480 Vc mg/kg group was the highest(P<0.05);Compared with the control group,plasma lysozyme(LZM),acid phosphatase(ACP)and alkaline phosphatase(AKP)activities in 120 Vc mg/kg group were significantly increased(P<0.05).Conclusion:Appropriate vitamin C(120 mg/kg)can promote the growth and improve the activity of plasma immunoenzyme of crayfish,but excessive amount of vitamin C can inhibit the growth and immunity.3.After 60 days feeding,10 cayfish were exposed from each repeat of each test group to pH stress(pH 10.2)test for 5 days.The indexes of plasma stress,hepatopancreas antioxidant indexes,gill metabolic enzyme activities,and PPARy,Keap-1 and Nrf2 gene expressions were investigated at 0 d,1 d and 5 d,respectively.The results showed that the plasma glucose(GLU)and hepatopancreas catalase(CAT)expression levels in 120 Vc mg/kg group were significantly lower than control group before and after stress(P<0.05).Compared with the control group,the content of cortisol(CORTISOL)in plasma decreasing after stress in 120 Vc mg/kg group(P<0.05).The activity of SOD in hepatopancreas activity was significantly increased after stress when dietary vitamin C content was 60 to 120 Vc mg/kg(P<0.05).Under pH stress,the activity of lactate dehydrogenase(LDH)in gills of crayfish in each group increased,Compared with the control group,the hepatopancreas malondialdehyde level in experimental groups was significantly lower than that in the control group on day 5 after stress except 30 mg/kg group(P<0.05)while the gill Na+K+-ATPase activity was significantly higher than that of the control group(P<0.05);The relative expression level of Keap-1 gene in all groups except 60 mg/kg Vc group was significantly lower than that in control group(P<0.05),and the relative expression of PPARy and Nrf2 gene was significantly higher than that of control group(P<0.05)in 120 mg/k g Vc group under pH stress at 1 d.In conclusion,appropriate vitamin C supplementation(120 mg/kg)can improve the non-specific immunity and immune gene expression of crayfish and effectively enhance the ability to resist stress. |
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