| In this study, five herbs (Radix astragali, Lentinan, Radix ginseng rubra, Shengmai Yu Ping Feng San and Turmeric, respectively) with strong immune stimulating effects on brown spotted grouper (Epinephelus fuscoguttatus), were selected from a total of eight Chinese herbs and two Thai herbs using superoxide anion production and phagocytic activity detection methods. The eight Chinese herbs include Epimedium herb (Epimedium sagittatum), Eucommia bark (Eucommia ulmoides), Radix astragali (Astragalus membranaceus), Lentinan (Lentinus edodes), Radix ginseng rubra (Panax ginseng), Five leaf Gynostemma herb (Gynostemma pentaphyllum), compound1(Shengmai Yu Ping Feng San), and compound2(Shi Quan Da Bu Tang). The two Thai herbs were Turmeric (Curcuma longa) and Fahtalaijons (Andrographis paniculata). Different proportion (0%,0.5%,1.0%and1.5%) of Radix astragali, were added to brown spotted grouper feed for28d. The immune parameters, RBC, WBC, lysozyme activity, superoxide union production and phagocytic activity, were selected to test the immunity of the fed fish. At the same time, gene expression differences of five immune genes, MHC1-α, IL-2, Mx, IFN and TNF, in five organs (gill, kidney, heart, spleen, liver and intestine) were analyzed with fluorescent quantitative PCR. Furthermore, brown spotted grouper were infected with Vibrio vulnificus after fed with the herb feed for28days, and the immunity parameters and immunity-related gene expression differences were analyzed after24h of infection.1. Selection of natural immunostimulant:Active ingredients were extracted from eight Chinese medicine and two Thai herbs using ethyl aqueous extraction and alcohol extraction methods, and screening of natural immunostimulants for brown spotted grouper was carried out by detecting of superoxide anion production with tetrazolium (NBT) reduction and phagocytic activity of its white blood cell. The results showed that, at aqueous extraction concentrations10mg/mL and1mg/mL, the groups with best effect were group compound1and Epimedium herb, respectively, while at aqueous extraction concentration O.lmg/mL, none of experimental groups showed any significant effect. At alcohol extraction concentration10mg/mL, Lentinan group showed the best effect, with the alcohol extraction concentration of5mg/mL, all experimental groups showed no significant effect, at alcohol extraction concentrations of2.5mg/mL, group Radix astragali, Lentinan, Radix ginseng rubra and compound1got the most prominent effect, at alcohol extraction concentrations of1.25mg/mL, group Radix astragali, Radix ginseng rubra, compound1and Turmeric got the best prominent effect, at alcohol extraction concentration of0.625mg/mL, group Radix astragali, Eucommia bark, and Turmeric showed the optimal effect. White blood cell phagocytic activity test showedthatRadix astragali, Lentinan, Radix ginseng rubra and compound1had significant immune enhancement effect on brown spotted grouper (P<0.01). Based on above experimental results, four herbs (Radix astragali, Lentinan, Radix ginseng rubra, Shengmai Yu Ping Feng San and Turmeric respectively) were seleted as effective natural immunostimulants for brown spotted grouper from above experiments. 2. Effect of Radix astragali on the immune hematology in brown spotted grouper. RBC, WBC, lysozyme activity, STI value and phagocytic activity were tested after the fish were fed with0.5%,1.0%or1.5%Radix astragali for28days. Results indicated that at0.5%Radix astragali concentration, lysozyme activity showed a highly significant effect (p<0.01), while phagocytic activity had a significant effect (p<0.05). At1.0%Radix astragali concentration, only lysozyme activity showed a significant effect (p<0.05). At1.5%Radix astragali concentration, RBC, WBC and phagocytic activity were significant different (p<0.05), and STI were highly significant different between the test and control groups (p<0.01). Samples in the test fish injected by Vibrio vulnificus for24h, WBC, RBC and phagocytic activity in all experimental groups were no significant difference with the control group. However, lysozyme activity showed significant and highly significant differences in1.0%and0.5%Radix astragali concentration groups, respectively (p<0.05, p<0.01). In infection experiment,1.5%Radix astragali concentration group had an immune protection rate of86.7%, which was significantly higher than the control group.3. Gene expression analysis of brown spotted grouper: before and after Radix astragali feeding the test group with Radix astragali for28days, immune genes of spleen and gill in test group at1.5%Radix astragali concentration showed the highest expression. In spleen, genes Mx, IL-2, MHC I-α, TNF and IFN expression were140.40times,39.90times,69.70times,115.89times and36.76times as the control group, respectively. In gill, genes Mx, IL-2, MHC I-α, TNF and IFN expression were5.03times,3674.50times,69.70times,8.38times and270.60times as the control group, respectively. After infecting the test group fish with Vibrio vulnificus for24h, immune genes Mx, IL-2, MHC I-a and TNF in group1.5%Radix astragali concentration got the highest expression in spleen, and immune gene IFN in group0.5%Radix astragali concentration got the highest expression. In gill, all immune genes expression in group1.5%Radix astragali concentration was the highest. In spleen, genes Mx, IL-2, MHC I-α, and TNF expression in group1.5%Radix astragali adding were respectively22.11times,206.50times,5.90times and68.99times as the control group. In spleen, gene IFN expression in group0.5%Radix astragali concentration was19.74times as the control group. In gill, genes Mx, IL-2, MHC I-α, TNF and IFN expression in group1.5%Radix astragali concentration were1.17,12.15,1.11,1.49and1.15as the control group, respectively.In summary, four nature herbs with strong immune enhancing effects were preliminarily screened for brown spotted grouper. The immune enhancing effect of Radix astragali on the brown grouper was mainly studied at hematological and gene expression level. It laid a good foundation for the new and safeimmune enhancer selection in salt water fish. It also has broad theoretical and applicational prospect. |
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