| The hemorrhage maked grass carp suffering from haemorrhages in gills and, ulceration, abscesses, exophthalmia and abdominal distension. This disease has strong contagious, fast morbidity and high mortality. Nowadays, antibiotics are widely used to get rid of the infections, their constant application leads to bacterial resistance, consequently cause adverse. Vaccination is an effective precaution for controlling fish diseases. Vaccines are inconvenient which is the major limiting factor to apply in aquaculture. There is an increasing interest in using herbs in aquaculture. Herbs are easy to prepare, no-toxic side effects, no drug residue and no drug resistance. Therefore, screening drugs from traditional Chinese medicine to effectively control hemorrhage is of great significance for aquaculture.Rhizoma Coptidis, the rhizome of Coptis chinensis Franch, has been widely used in traditional Chinese medicine for thousand of years. It is known to harbor a diversity of active ingredients, including berberine, coptisine, palmatine, jatrorrhizine and epiberberine. Rhizoma Coptidis alkaloid have a common quinolizidine ring system, which were reported to exhibit similar pharmacological effects. The purpose of this study was to evaluate the effects of Fibrous root of Coptis chinensis Franch (FRC) and berberine (BBR) on non-specific immunity in grass carp and antibacterial activity in vitro against Aeromonas hydrophila, enhancing the ability of grass carp on resistance against hemorrhage, and preliminary explore the effects of Rhizoma Coptidis alkaloid on the of immune related factors.The following is the main contents and results of this project:1. The in vitro antibacterial activity of FRC and BBR were investigated with the double dilution method. The minimum inhibitory concentration (MIC) method showed strong inhibitory effects. Their MICs were 3.125 mg/mL and 39.062 mg/L, respectively.2. In the experiment of the effects of FRC and BBR on the survival rate (SR), healthy fish with approximately the same weight were randomly assigned to seven groups, containing normal control group (NC), model group (MD),1.00%,0.50%, 0.25%,0.10% of FRC and 0.05% of BBR. Experiments were divided into therapeutic experiment and prophylactic experiment. During the two experiments, NC and MD were fed with basal diet. In prophylactic experiment, the experimental groups were fed with corresponding diets twice a day for 14 days. After that, the fish of all groups except NC were injected with 0.2 mL of the suspension of A. hydrophila, the NC were injected with 0.2 mL of sterile saline. The SR were noted daily for 14 days. The experiment was repeated three times, the final three times taking the average. The results showed that compared with the MD, SR of grass carp after challenge with A. hydrophila in different experimental groups were improved. The 0.50% FRC treatment conferred the best protection against A. hydrophila infection,74.29%. The NC registered no mortality. At the beginning of the therapeutic experiment, the fish of all groups except NC were injected with 0.2 mL of the suspension of A. hydrophila, the NC were injected with 0.2 mL of sterile saline. After that, fish were fed with corresponding diets at the twice a day for 14 days. The SR was noted daily for 14 days. The experiment was repeated three times, finally taking the average of 3 experiments. The results showed that, compared with the MD, all experimental groups led to an increase in SR. The highest survival rate was 0.05% BBR group,67.14%. The NC registered no mortality. Two results indicate that FRC and BBR can enhance the disease resistance of grass carp against A.hydrophila.3. In the experiment of the effects of FRC and BBR on the non-specific immunity, healthy fish with approximately the same weight were randomly assigned to six groups, containing NC,1.00%,0.50%,0.25%,0.10% of FRC and 0.05% of BBR. During the experiment, the NC is always fed with basal diet, five experimental groups were fed with corresponding diets twice a day for 28 days. Six fish from all groups were sampled and blood was collected from the caudal vein on the 14th and 28th days for serum lysozyme activity, NBT activity and complement C3 level assays. The results showed that the immune parameters of experiment groups were enhanced compared with NC during the experiment. On the 14th day, the lysozyme activity in 1.00% FRC showed a significantly (P<0.05) higher lysozyme activity than the NC. The highest NBT activity was observed in 0.50% FRC group on the 14th day. However, significant differences (P<0.05) in complement C3 levels were observed only in 1.00% FRC groups on the 28th day as compared to the MD. The results indicated that dietary FRC and BBR could enhance the non-specific immunity of grass carp.4. In the experiment of the effects of Rhizoma Coptidis alkaloid on the of immune related factors, healthy fish were randomly assigned to five groups, containing NC, 0.05% of BBR,0.05% of Coptisine,0.05% of Epiberberine and 0.05% of Palmatine. During the experiment, the NC is always fed with basal diet, experimental groups were fed with corresponding diets twice a day for 14 days. The level of three immune related factors in liver, intestine and spleen of interleukin-1 (IL-1β), intefferon (IFN) and tumor necrosis factor α (TNFα) were examined on the 14th day. The results showed that palmatine can enhance the level of IL-1β in liver, intestine and spleen; Coptisine can enhance the level of IL-1β in liver and intestine; BBR can only enhance the level of IL-1β in intestine. The results showed that all experimental groups can inhibit the level of IFN in liver; However, all experimental groups can enhance the level of IFN in spleen; Only palmatine can enhance the level of IFN in intestine. The results showed that the palmatine and coptisine can enhance the level of TNFα in liver; However, all experimental groups can inhibit the level of TNFα in spleen; Palmatine and coptisine can enhance level of TNFαin intestine. |
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