| In this paper,the effects of three Chinese herbal compounds on growth,intestinal digestive enzymes,intestinal flora,plasma and liver biochemical enzymes,HSP70 and HSC70 expression of Anguilla marmorata during 30 days of feeding were studied.To provide green and effective new choices to the development and application of fishery drugs in aquaculture industry,the effects of three Chinese herbal compounds on disease resistance of Anguilla inarmorata were evaluated comprehensively in combination with Edwardsiella tarda challenge test.The research is divided into the following six parts:1.Evaluation of the effect of Chinese herbal compounds on activation of leukocytes in vitro:The separation of leukocytes from peripheral blood of Anguilla marmorata was optimized by gradient dilution method.The separation effect was determined by staining observation and flow cytometry.To obtain the leukocytesa with a concentration of 1.0×105 cell/ml,the separation of white blood cells by Percoll solution system with 40%density and 30%density was determined.Five kinds of compound liquids(Polysaccharide compound Dt,Pulsatilla chinensis compound Btw,Radix isatidis compound Blg,Rhubarb compound Dh,Thymol compound Blf)were prepared at a concentration of 1.0 mg/ml.They were co-incubated with 1.0×105 CFU/ml leukocytes at room temperature for 2 hours.The respiratory burst activity was measured by the method of Nitroblue tetrazolium(NBT).The results showed that Dt,Btw and Blg could significantly increase the respiratory burst activity of peripheral blood leukocytes of Anguilla marmorata(P<0.05),which increased by 728.57%,413.00%and 1030.00%.,respectively.Flow cytometry was used to analyze the phagocytic activity of leukocytes.The results showed that Dt and Blg could significantly increase the phagocytic activity of leukocytes(P<0.05).Btw could also increase the phagocytic activity of leukocytes,but the difference was not significant(P>0.05).Comprehensive analysis showed that Dt,Btw and Blg activated the leukocytes of the flower buds significantly higher than Dh and Blf.Therefore,Dt,Btw and Blg were selected as the Chinese herbal compounds for the feeding experiment.2.Effects of three Chinese herbal compounds on the growth and digestive enzymes of Anguilla marmorata:500 eels were cultured in four groups of recirculating water for 30 days.During the period,Dt,Btw and Blg were used for feeding.Samples were taken every 10 days to detect the effect of these compounds on the growth and digestive enzymes of Anguilla marmorata.The results showed that Dt could significantly increase the WGR(P<0.05)and promote growth.Dt also increased the intestinal protease,lipase and amylase activities.The optimum action period of Dt on three intestinal digestive enzymes(protease,lipase and amylase)of Anguilla marmorata was about 20,10 and 30 days,respectively.The growth indexes of Btw were better than those of the control group,but was not significant(P>0.05).Btw significantly enhanced the intestinal protease and lipase activity at 30d and 10d,respectively,and it had no enhancement effect on amylase activity(P>0.05).The survival rate of the Blg was the highest,which reaching to 98.29%,but the growth effect was not prominent,and it was not much different from the control group;Blg only had an enhanced effect on the intestinal amylase.Blg have the best effect on the intestinal amylase activity in about 20 days.3.High-throughput sequencing technology was used to analyze the effects of three Chinese herbal compouds on the intestinal flora of Anguilla marmorata.The results showed that the three Chinese herbal compounds could change the diversity of the intestinal flora to a certain extent.During the 30 days period,the structure of the intestinal flora of Anguilla marmorata experienced a process from drastic change to stable.Blg and Dt significantly increased the abundance of beneficial bacteria such as Cetobacterium and Clostridium(P<0.05),and reduced the proportion of pathogenic bacteria and conditional pathogenesis bacteria.Btw has no significant beneficial effects on intestinal flora,and the proportion of pathogenic bacteria such as Salmonella in the intestine is higher than the other groups.4.The effects of three Chinese herbal compounds on the disease resistance of Anguilla marmorata:Effects of Feeding Three Compounds of Chinese Herbal Medicine on Nonspecific Immunity-related Indicators of Anguilla marmorata:the average levels of GPT and GOT in Dt were significantly higher than those in the control group,Btw and Blg groups(P<0.05).It indicated that Dt had a certain metabolic burden on liver;The activity of SOD in the compound groups was improved.but there was no significant difference among the 4 groups(P>0.05).The MDA content in the compound groups was lower than that in the control group.The lowest MDA content was in the Dt group.It indicating that Dt showed the best performance in antioxidants,and there was a correlation between SOD activity and MDA content.The respiratory burst activity of peripheral blood leukocytes in the three compound groups first increased and then decreased during the 30-day treatment period.The highest NBT%were Dt 96.06%,Btw 146.31%,and Blg 289.66%in 20 days.Effects of three Chinese herbal compounds on the expression changes of HSP70 and HSC70 of Anguilla marmorata was were detected by q-PCR.The results showed that the expression level of HSP70 in Dt was first up-regulated to 2.29 times higher than the control group and then down-regulated during the treatment.The expression level of HSC70 in Dt was not significantly changed(P>0.05)The expression of HSP70 and HSC70 in Btw group was first up-regulated to 4.52 times and 6.69 times higher than that of the control group,respectively,and then down-regulated.The expression of HSP70 in Blg was first up-regulated,then down-regulated to the same level as the control group.The expression level of HSC70 in Blg was first up-regulated to 2.54 times higher than in control group after 10 days.In conclusion,among the three compounds,Dt had the least effect on the HSP70 and HSC70,followed by Blg.Anti-infective ability of Anguilla marmorata after feeding three Chinese herbal compounds:After 30 days of feeding,Anguilla marmorata was intraperitoneally injected by 1.0×107 CFU/ml of G1-2,and the attack period was 14 days.According to the protection rate,the ability of three Chinese herbal compounds to enhance the resistance of Anguilla marmorata was evaluated.The results showed that the total survival rate of the four groups was 14.29%in the control group,25.00%in Dt,19.05%in Btw,and 44.90%in Blg.Detection of non-specific immune-related indicators in samples after challenge.The results showed that the MDA content in the compound group was significantly lower than that in the control group(P<0.05).In conclusion,the order of anti-infectivity ability of the three compounds is as follows:Blg>Dt>Btw. |
PDF Full Text Request