| Ctenopharyngodon idella is the largest economic fish in freshwater culture in China,which is very susceptible to disease under high temperature.The purpose of this study is to study the key genes of the effect of high temperature on the immune response of grass carp in the early,middle and late stages of immune response by transcriptome analysis.Then,the effects of synbiotic of yupingfeng polysaccharide(YPF-P)and synbiotic of Angelica polysaccharide(ASP)on the immune function of grass carp under high temperature stress are explored,which laid a research basis for the application of Chinese medicine polysaccharide synbiotic in additives.The specific research contents and results are as follows:1.Transcriptome analysis of effects of high temperature stress on grass carp immune responseThe grass carp(20±2 g)was divided into normal temperature group(28℃)and high temperature group(34℃).After 24 h of temperature stress,the Aeromonas hydrophila LPS was injected and the spleen tissues of grass carp were collected at 0,6,24 and 72 h after that for transcriptome sequencing.In this study,1497,126,383,4841 differentially expressed mRNAs,28,28,20,and 141 differentially expressed miRNAs,62,3,15,and 1133 pairs of miRNA-mRNA were screened at four time points in high-temperature group and the normal temperature group,respectively.This study have found that genes of Glutathione metabolism,Proteasome,Protein processing in endoplasmic reticulum,and Cytokine-cytokine receptor interaction were involved in the immune response of grass carp under high temperature stress.This provides a theoretical basis for scientifically explaining that grass carp is extremely susceptible to diseases under high temperature conditions,and also provides a research target for subsequent control research.2.Effect of YPF-P synbiotics on the immunoregulation of grass carp under heat stressGrass carp(20±2 g)was divided into four groups for 21 d feeding experiment.The control group was fed with basic diet.The experimental group was fed with0.08%,0.12% and 0.20% YPF-P respectively in the basic diet.Before feeding,1%compound probiotics(Lactobacillus,Streptococcus thermophilus and Bacillus subtilis)was added.The serum biochemical indexes,liver biochemical indexes and liver related gene expression of grass carp were measured at 0,6,24 and 48 h after high temperature(32～34℃)stress.The results showed that the activities of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in each concentration group were lower than that in the control group before and after stress,while the activities of ALT and AST of 0.12% YPF-P group was significantly lower before and after stress(P<0.05).The total superoxide dismutase(SOD)activity of 0.12% and0.20% YPF-P groups was higher than that of the control group before and after stress,while the SOD activity of 0.12% YPF-P group was significantly increased at 0 h and24 h(P<0.05).The activities of total antioxidant capacity(T-AOC),glutathione peroxidase(GSH-PX),and glutathione reductase(GR)in each concentration group were higher than that in the control group before and after stress.The GSH-PX activity of 0.12% and 0.2% YPF-P group was significantly increased at 6 h(P<0.05),and the GR activity of 0.12% YPF-P group was significantly increased at 0 h and 48 h(P<0.05).The content of malondialdehyde(MDA)in each concentration group was lower than that in the control group after stress,while the MDA content of 0.12%YPF-P group was significantly reduced at 24 h and 48 h(P<0.05).Compared with the control group,in 0.12% and 0.20% YPF-P groups,the expression of Nrf2 and Cu/Zn-SOD were significantly increased at 0 h and 6 h(P<0.05),while the expression of Keap1 was significantly reduced at 0 h,24 h and 48 h(P<0.05).In 0.12% YPF-P group,the expression of HSP70 was significantly increased at 24 h and 48 h(P<0.05),and the expression of GSH-Px was significantly increased at 6 h and 24 h(P<0.05).The expression of GR of 0.12% YPF-P group was significantly increased before and after stress(P<0.05).The expression of MHC II in each concentration groups was significantly increased before and after stress(P<0.05).The expression of IL-8 in0.08% and 0.12% YPF-P groups was slightly lower than that in the control group at24 h and 48 h,but there was no significant difference(P>0.05).The above results showed that the addition of 0.12% YPF-P synbiotic had certain immunoregulatory activity,which could improve the anti-oxidative stress ability of grass carp and have a good immune regulation effect.3.Effect of ASP synbiotics on the immunoregulation of grass carp under heat stressGrass carp(20±2 g)was divided into four groups for 21 d feeding experiment.The control group was fed with basic diet.The experimental group was fed with0.08%,0.12% and 0.20% ASP respectively in the basic diet.Before feeding,1%compound probiotics was added.The serum biochemical indexes,liver biochemical indexes and liver related gene expression of grass carp were measured at 0,6,24 and48 h after high temperature(32～34℃)stress.Compared with the control group,in0.12% and 0.20% ASP groups,the ALT activity of was significantly reduced at 0 and6 h(P<0.05),and the AST activity was significantly reduced before and after stress(P<0.05).The SOD activity of 0.12% and 0.20% ASP groups was significantly increased at 0,24 and 48 h(P<0.05).In 0.20% ASP group,the alkaline phosphatase(AKP)activity was significantly increased at 0 h(P<0.05)and the MDA content was significantly reduced at 48 h(P<0.05).The activities of lysozyme(LZM)and T-AOC in each concentration group were slightly higher than that in the control group before and after stress(P>0.05).The CAT activity in each concentration group was higher than that in the control group before and after stress,and the CAT activity of 0.2%ASP group was significantly increased at 6 h(P<0.05).Compared with the control group,in 0.20% ASP group,the expression of Nrf2 was significantly increased at 0-24h(P<0.05),while the expression of Keap1 was significantly reduced at 6 h and 24 h of stress(P<0.05).In the 0.12% and 0.20% ASP groups,the expression of Cu/Zn-SOD was significantly increasedat at 0 h and 6 h(P<0.05),and the expression of CAT was significantly increased at 6 h and 48 h respectively(P<0.05).The expression of IL-8 of 0.12% and 0.20% ASP groups was slightly lower than that of the control group after stress(P>0.05).The expression of HSP70 and HSP90 of 0.08%and 0.20% ASP groups were significantly increased at all time points after stress(P<0.05).The expression of MHC II in each concentration groups was significantly increased before and after stress(P<0.05).The results showed that 0.2% ASP synbiotic could improve the immunity and anti-oxidative stress of grass carp. |
PDF Full Text Request