阅读说明：本技术 池塘种植鱼腥草修复无乳链球菌感染引起罗非鱼肝脏损伤的方法 (Method for repairing tilapia liver injury caused by streptococcus agalactiae infection by planting houttuynia cordata in pond ) 是由 祝璟琳 杨弘 邹芝英 李大宇 肖炜 于 2019-11-08 设计创作，主要内容包括：本发明涉及一种池塘种植鱼腥草修复无乳链球菌感染引起罗非鱼肝脏损伤的方法,它包括以下步骤：在池塘水面上制作浮床并种植鱼腥草；对罗非鱼感染前以及在罗非鱼感染后的多个时间点对罗非鱼进行肝脏采集并进行肝脏生化、抗氧化性能、组织病理和<I>HSP</I>70指标检测；对数据进行比较。本发明能改善链球菌感染所造成罗非鱼肝AST、ALT上升,减缓链球菌感染引起T-AOC下降,增加肝脏中SOD、GSH-PX、CAT和NOS等抗氧化酶的活性,减少脂质过氧化MDA的产生、促进受损蛋白的降解与清除,显著减轻无乳链球菌感染引起罗非鱼的病理损伤。(The invention relates to a method for repairing tilapia liver injury caused by streptococcus agalactiae infection by planting houttuynia cordata in a pond, which comprises the following steps: manufacturing a floating bed on the water surface of the pond and planting houttuynia cordata; liver collection of tilapia mossambica before and after tilapia mossambica infection and liver biochemistry, antioxidation performance, histopathology and liver identification HSP 70, detecting the index; the data are compared. The invention can improve AST and ALT rise of tilapia liver caused by streptococcus infection, slow down T-AOC reduction caused by streptococcus infection, increase activities of antioxidase such as SOD, GSH-PX, CAT and NOS in liver, reduce generation of lipid peroxidation MDA, promote degradation and removal of damaged protein, and remarkably reduce pathological damage of tilapia caused by streptococcus agalactiae infection.)

1. A method for repairing tilapia liver injury caused by streptococcus agalactiae infection by planting houttuynia cordata in a pond is characterized by comprising the following steps:

a. manufacturing a floating bed on the water surface of a tilapia culture pond, planting houttuynia cordata on the floating bed, wherein the root system of the houttuynia cordata extends into the water of the culture pond, and controlling the planting area of the houttuynia cordata to be 5 ~ 15% of the water surface area of the culture pond;

b. after tilapia is cultured for 90 days, liver of tilapia is collected, and liver biochemistry, oxidation resistance, histopathology and liver biochemical analysis are carried out on tilapiaHSP70, detecting indexes, and immediately performing streptococcus agalactiae artificial infection on tilapia after collection;

c. liver collection of tilapia at multiple time points after tilapia infection and liver biochemistry, antioxidant performance, histopathology and liver identificationHSP70, detecting the index;

d. subjecting the liver obtained in step b to biochemical treatment, antioxidant activity, histopathological treatment andHSP70 data and Biochemical, antioxidant, histopathological and Biochemical Properties of the liver obtained in step cHSP70, to show the effect of the houttuynia cordata planted in the pond on repairing liver injury of tilapia caused by streptococcus agalactiae infection.

2. The method for repairing tilapia liver injury caused by streptococcus agalactiae infection by planting houttuynia cordata in the pond according to claim 1, wherein the height of the houttuynia cordata is 10 ~ 25 cm, and the planting distance of the houttuynia cordata is 10 ~ 30 cm.

3. The method for repairing tilapia liver injury caused by streptococcus agalactiae infection by planting houttuynia cordata in the pond according to claim 1, wherein the floating bed is made of polyvinyl chloride tubes, and the size of the floating bed is 1 ~ 4m x 1 ~ 4 m.

4. The method for repairing tilapia liver injury caused by streptococcus agalactiae infection by planting houttuynia cordata in the pond according to claim 1, which is characterized in that: the liver biochemistry in the step b and the step c at least comprise AST index and ALT index.

5. The method for repairing tilapia liver injury caused by streptococcus agalactiae infection by planting houttuynia cordata in the pond according to claim 1, which is characterized in that: the oxidation resistance in the step b and the step c at least comprises a T-AOC index, an SOD index, a GSH-PX index, a CAT index, an NOS index and an MDA index.

Technical Field

The invention relates to a method for repairing tilapia liver injury caused by streptococcus agalactiae infection by planting houttuynia cordata in a pond, and belongs to the technical field of aquaculture.

Background

China is tilapia mossambica (Oreochromisspp.) yield and export, fish are affected by environmental stresses such as hypoxia and high temperature with the increase of the degree of intensification, which can cause the suppression of immune response of fish and the susceptibility to infection by pathogenic bacteria. It has been shown that the stress caused by the fluctuation of the environmental factors of pond water is closely related to the outbreak of streptococcicosis. Therefore, it is very important to improve the pond culture environment. At present, tilapia farmers mixedly culture litopenaeus vannamei (a) and (b) in ponds by planting water spinach, medicinal plants and the like on water surfacesLitopenaeus vannamei) Grass carp (A)Ctenopharyngodon idella) Bighead atractylodes rhizome (A)Aristichys nobilis ) And the like, the self-cleaning capacity of the water body is improved, the stress of high-concentration ammonia nitrogen, nitrite and pathogenic bacteria is reduced, and the morbidity and mortality of the fish body are reduced. Chinese herbal medicines have the potential to replace antibiotics and immunoprophylaxis agents in aquaculture, and they can be used in whole or in parts (leaves or roots) or in the form of extract compounds or feed additives, alone or in combination with probiotics or other immunostimulants. Herba Houttuyniae (houttuynia cordata Thunb.)Houttuynia cordata) Is a plant of houttuynia of Saururaceae, contains bioactive substances such as alkaloid, flavonoid, essential oil and polysaccharide, and has antiviral, antitumor, antibacterial, antiinflammatory, and antioxidant effects. Studies such as Serina and the like show that the houttuynia cordata floating bed can better promote the self-purification function of the water body of a tilapia pond and improve the nonspecific immunity and survival rate of the tilapia. Zhengyao and the like discover that the culture of the gift tilapia can be obviously enhanced by planting the houttuynia cordata on the water surface of the culture pondO. niloticusGIFT strain) bile, and reduces the probability of infecting the streptococcus by GIFT tilapia.

The fish liver is an important metabolic organ in the fish body and is involved in digestion, metabolism, excretion, detoxification, immunity and the like in the bodyThe function is very important for the growth and health of the fish body. The antioxidant defense system in fish body comprises enzymatic system and non-enzymatic system, wherein the enzymatic system comprises antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and Catalase (CAT), and protects cells and cell membranes from oxidative damage. Oxidative stress can cause host inflammatory reaction, cause tissue damage and diseases, and the tissue section technology is an important method for observing pathological changes of fish tissues. The research finds that the heat shock protein 70 (HSP70) As a nonspecific endogenous protective protein for cells, it plays an important role in preventing cell damage caused by stress, recovering damaged cells, and the like. Earlier studies have shown that heartleaf houttuynia herb planted in an aquaculture pond can significantly reduce the artificial infection of tilapia with streptococcus agalactiae (S. agalactiae) ((R))Streptococcus agalactiae) The mortality rate after the cultivation is 10% of the optimum planting rate of houttuynia cordata, but the mechanism is not completely clear.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a method which has a certain repairing effect on tilapia liver injury caused by streptococcus agalactiae infection and can improve the capability of tilapia in resisting streptococcus infection.

According to the technical scheme provided by the invention, the method for repairing tilapia liver injury caused by streptococcus agalactiae infection by planting houttuynia cordata in the pond comprises the following steps:

a. manufacturing a floating bed on the water surface of a tilapia culture pond, planting houttuynia cordata on the floating bed, wherein the root system of the houttuynia cordata extends into the water of the culture pond, and controlling the planting area of the houttuynia cordata to be 5 ~ 15% of the water surface area of the culture pond;

b. after tilapia is cultured for 90 days, liver of tilapia is collected, and liver biochemistry, oxidation resistance, histopathology and liver biochemical analysis are carried out on tilapiaHSP70, detecting indexes, and immediately performing streptococcus agalactiae artificial infection on tilapia after collection;

c. liver collection of tilapia at multiple time points after tilapia infection and liver biochemistry, antioxidant performance, histopathology and liver identificationHSP70, detecting the index;

d. to pairThe biochemical and antioxidant properties and histopathology of the liver obtained in step bHSP70 data and Biochemical, antioxidant, histopathological and Biochemical Properties of the liver obtained in step cHSP70, to show the effect of the houttuynia cordata planted in the pond on repairing liver injury of tilapia caused by streptococcus agalactiae infection.

Preferably, the height of the houttuynia cordata is 10 ~ 25 cm, and the planting distance of the houttuynia cordata is 10 ~ 30 cm.

Preferably, the floating bed is made of polyvinyl chloride pipes, and the size of the floating bed is 1 ~ 4m multiplied by 1 ~ 4 m.

Preferably, the liver biochemistry of step b and step c at least comprises AST index and ALT index.

Preferably, the oxidation resistance in the step b and the step c at least comprises a T-AOC index, an SOD index, a GSH-PX index, a CAT index, an NOS index and an MDA index.

The invention has the following advantages:

1. the invention can obviously improve AST and ALT rise of tilapia liver caused by streptococcus infection, protect tilapia liver against acute liver injury;

2. the invention can improve the oxidation resistance stress capability of tilapia, slow down the reduction of T-AOC caused by streptococcus infection, obviously increase the activities of antioxidant enzymes such as SOD, GSH-PX, CAT, NOS and the like in liver, increase the scavenging capability of free radicals, reduce the generation of lipid peroxidation MDA, and has the protection effect on the acute oxidation stress injury induced by streptococcus agalactiae;

3. the invention can obviously reduce the pathological damage of tilapia caused by streptococcus infection;

4. after the invention is implemented, the tilapia passes through the liverHSP70, the high expression is involved in the refolding of the damaged polypeptide, and the degradation and the elimination of the damaged protein are promoted.

Drawings

FIG. 1 is a graph showing the variation of liver alanine Aminotransferase (ALT) after tilapia in different houttuynia cordata groups of the present invention is infected with Streptococcus agalactiae.

FIG. 2 is a graph showing the change of aspartate Aminotransferase (AST) in liver after tilapia in different houttuynia cordata groups of the present invention is infected with Streptococcus agalactiae.

FIG. 3 is a graph showing the change of total antioxidant capacity (T-AOC) of liver after infecting Streptococcus agalactiae with GIFT tilapia from different houttuynia cordata groups of the present invention.

FIG. 4 is a graph showing the change of liver superoxide dismutase (SOD) after the tilapia in different houttuynia cordata groups is infected with streptococcus agalactiae.

FIG. 5 is a graph showing the change of the activity of glutathione peroxidase (GSH-PX) in liver after the tilapia in different houttuynia cordata groups is infected with Streptococcus agalactiae.

FIG. 6 is a graph showing the change of the activity of the Catalase (CAT) in the liver of the tilapia in different houttuynia cordata groups infected with Streptococcus agalactiae.

FIG. 7 is a graph showing the change of the content of Malondialdehyde (MDA) in liver of genetically improved farmed tilapia in different houttuynia cordata groups after infecting Streptococcus agalactiae.

FIG. 8 is a graph showing the change of liver Nitric Oxide Synthase (NOS) activity after infecting Streptococcus agalactiae with GIFT tilapia from different houttuynia cordata groups of the present invention.

FIG. 9 shows the liver of tilapia gilvus infected with Streptococcus agalactiae after houttuynia cordata is planted in the pond of the inventionHSP70 mRNA expression level.

Fig. 10 is a pathological map of tilapia liver 0h after infection of 0% area group.

Fig. 11 is a pathological map of tilapia liver 48h after infection in 0% area group.

Fig. 12 is a pathological picture of tilapia liver 0h after infection in 5% area group.

Fig. 13 is a pathological map of tilapia liver 48h after infection in 5% area group.

Fig. 14 is a pathological picture of tilapia liver 0h after infection in 10% area group.

Fig. 15 is a pathological map of tilapia liver 48h after infection in 10% area group.

FIG. 16 is a pathological picture of tilapia liver 0h after infection in 15% area group.

Fig. 17 is a pathological map of tilapia liver 48h after infection in 15% area group.

Detailed Description

The present invention will be further described with reference to the following specific examples.