A method for raising Pengze crucian carp seedlings with high stress resistance and disease resistance

Abstract

The invention discloses a method for raising seedlings of Pengze crucian carp with high stress resistance and disease resistance, and belongs to the field of aquaculture technology. The method includes the step of adding a composite additive to fish feed to feed Pengze crucian carp; the composite additive is made of kaempferol and hesperidin in a mass ratio of (1-4): (1-2). The present invention screens a variety of plant natural extract components and finds for the first time that adding a composite additive prepared by kaempferol and hesperidin to fish feed is beneficial to the growth of Pengze crucian carp, and can effectively improve the nonspecific immunity of Pengze crucian carp and fight fish diseases caused by bacteria such as fish-harming myxococci, significantly enhance the stress resistance and disease resistance of Pengze crucian carp, and confirms through the results of the toxicity test that the additive compounded by kaempferol and hesperidin can completely replace antibiotics, improve the disease symptoms of Pengze crucian carp infected with bacteria, significantly reduce the infection mortality rate, and lay an important foundation for the development of "green fishery" of Pengze crucian carp.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, in particular to a method for breeding Pengze crucian carp with high stress resistance and disease resistance. Background Art

[0002] Pengze Crucian Carp is a premium crucian carp variety in my country. Beginning in 1983, the Jiangxi Provincial Fisheries Science Research Institute and the Jiujiang Municipal Fisheries Science Research Institute collaborated to select large, fast-growing, and uniformly colored wild crucian carp from Taibai Lake and Dingjia Lake in Pengze County, Jiangxi Province. After seven years and six generations of breeding, the resulting species boasts exceptional traits such as large individuals, rapid growth, easy reproduction, wide adaptability, tolerance to hypoxia, strong disease resistance, a well-formed body, compact scales, high protein content, a high meat consumption rate, and a pleasant taste. Pengze Crucian Carp has become a key freshwater crucian carp variety due to its high protein, low fat content, high edible rate, and nutritional value, meeting modern dietary needs.

[0003] Today, Pengze crucian carp, with its excellent characteristics and adaptability to aquaculture areas, has been well-received by farmers and the market, and the aquaculture area is constantly expanding. However, farmers often implement high-density aquaculture in pursuit of higher economic returns. However, in such a high-density aquaculture environment, the rapid growth of Pengze crucian carp can easily induce infections such as spore parasites and myxococci, as well as hemorrhagic diseases, which affect the growth efficiency of Pengze crucian carp. The use of antibiotics in aquaculture can reduce the losses caused by disease in Pengze crucian carp, but the use of antibiotics does not meet the development requirements of "green fisheries". Therefore, it is necessary to select a natural, non-toxic active ingredient to completely replace antibiotics to improve the high-strength stress resistance and disease resistance of Pengze crucian carp seedlings.

[0004] Plant extracts are made from plants as raw materials. According to the needs of the purposes of the final product to be extracted, through a physical and chemical extraction and separation process, a certain one or more active ingredients in the plant are obtained and concentrated, without changing the product formed by its active ingredient structure. It has been found that various plant extract components have special physiological activity and medical effects, such as ephedrine for treating asthma in Ephedra, hyoscyamine for antispasmodic and analgesic effects in Hyoscyamine, etc. There are also many studies that adopt various plant extracts to mix as additives for breeding feed to promote animal growth, and there are few reports about whether plant extracts can replace antibiotics and be applied to Pengze crucian carp seedlings breeding. Therefore, the present invention intends to provide a plant extract component to completely replace antibiotics, in order to improve the stress resistance and disease resistance of Pengze crucian carp. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for raising Pengze crucian carp seedlings with high stress resistance and disease resistance to solve the problems existing in the above-mentioned prior art. The present invention discovers for the first time that an additive compounded with kaempferol and hesperidin can completely replace antibiotics. At an effective dose, it can effectively improve the nonspecific immunity of Pengze crucian carp and fight fish diseases caused by bacteria such as fish-harming myxococci, thereby enhancing the stress resistance and disease resistance of Pengze crucian carp, and laying an important foundation for the development of "green fishery" of Pengze crucian carp.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a method for raising Pengze crucian carp seedlings with high stress resistance and disease resistance. The method comprises the steps of adding a composite additive into fish feed and feeding the Pengze crucian carp; the composite additive comprises kaempferol and hesperidin.

[0008] Furthermore, based on fish feed, the added amount of the composite additive is 50-400 mg / kg, and the composite additive is prepared from kaempferol and hesperidin in a mass ratio of (1-4): (1-2).

[0009] Furthermore, the added amount of the composite additive is 200 mg / kg, and the composite additive is prepared from kaempferol and hesperidin in a mass ratio of 3:1.

[0010] Furthermore, the composite additive enhances the stress resistance and disease resistance of the Pengze crucian carp by increasing the growth rate, nonspecific immunity and survival rate of the Pengze crucian carp after being infected with bacteria.

[0011] Furthermore, improving the nonspecific immunity includes improving the activities of superoxide dismutase (SOD), alkaline phosphatase (AKP) and peroxidase (POD).

[0012] Furthermore, the bacteria include Myxococcus piscicola.

[0013] The present invention also provides a feed for improving the stress resistance and disease resistance of Pengze crucian carp. The feed is prepared by adding a composite additive prepared from kaempferol and hesperidin into fish feed in an amount of 50-400 mg / kg.

[0014] Furthermore, the mass ratio of kaempferol to hesperidin in the composite additive is 3:1.

[0015] The present invention discloses the following technical effects:

[0016] By screening a variety of natural plant extract components, the present invention discovered for the first time that adding 50-400 mg / kg of a composite additive prepared with kaempferol and hesperidin to fish feed is beneficial to the growth of Pengze crucian carp, and can effectively improve the nonspecific immunity of Pengze crucian carp and fight fish diseases caused by bacteria such as fish-harming myxococci, significantly enhance the stress resistance and disease resistance of Pengze crucian carp, and the results of toxicity test confirm that the additive compounded with kaempferol and hesperidin can completely replace antibiotics, improve the disease symptoms of bacterial infection in Pengze crucian carp, and significantly reduce the infection mortality rate, laying an important foundation for the development of "green fishery" of Pengze crucian carp. DETAILED DESCRIPTION

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0019] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0020] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0021] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0022] Example 1 Screening of active ingredients from different plant extracts

[0023] 1. Experimental design and breeding management

[0024] Six aquaculture ponds of similar specifications (6m×5m×2m (depth)) were randomly selected from the Fisheries Research Institute of Jiujiang Academy of Agricultural Sciences, and 6,000 Pengze crucian carp fry were stocked in each aquaculture pond.

[0025] Five plant extracts (hesperidin, chlorogenic acid, quercetin, luteolin, and kaempferol) (purchased from Shaanxi Yongyuan Biotechnology Co., Ltd.) were added to conventional Pengze Crucian Carp feed (provided by the Pengze Crucian Carp Breeding Farm in Jiujiang City) at a dose of 100 mg / kg. These five experimental groups served as the control group, which received no plant extracts. Following conventional pond management, the Pengze Crucian Carp feed was fed two to three times daily at a designated location for a three-week breeding trial.

[0026] 2. Index detection

[0027] 2.1 Body length

[0028] At the end of the breeding experiment, 10 Pengze crucian carp were sampled from each group and their body lengths were measured. The results are as follows:

[0029] Table 1 Body length of each group of Pengze crucian carp

[0030]

[0031]

[0032] According to Table 1, compared with the control group, adding hesperidin, chlorogenic acid, quercetin, luteolin and kaempferol to the breeding feed can significantly increase the growth rate of Pengze crucian carp, among which hesperidin and kaempferol have better effects.

[0033] 2.2 Nonspecific immune indicators

[0034] At the end of the breeding experiment, 20 Pengze crucian carp were randomly selected from each group, and blood was collected from their tail veins. After being placed at 37℃ for 1 hour, it was kept at 4℃ overnight; it was centrifuged at 10000r / min at 4℃ for 15 minutes, and the upper serum was collected and stored at -80℃ for later use.

[0035] The activity of nonspecific immune enzymes in serum, including superoxide dismutase (SOD), alkaline phosphatase (AKP), and peroxidase (POD), was determined using kits from the Nanjing Jiancheng Biochemical Engineering Research Institute. The corresponding procedures were performed according to the instructions. The test results are as follows:

[0036] Table 2 The activity of nonspecific immune enzymes in the serum of Pengze crucian carp in each group

[0037] Group SOD activity (U / mL) AKP activity (IU / L) POD activity (mU / L) control group 92 45 39 Hesperidin group 133 74 68 Chlorogenic acid group 108 62 51 Quercetin group 121 57 44 Luteolin group 116 66 52 Kaempferol group 138 75 67

[0038] As shown in Table 2, compared with the control group, adding hesperidin, chlorogenic acid, quercetin, luteolin and kaempferol to the breeding feed can effectively improve the activity of three nonspecific immune enzymes of Pengze crucian carp, thereby enhancing the immunity and stress resistance of Pengze crucian carp, among which hesperidin and kaempferol enhanced the nonspecific immunity most significantly.

[0039] 3. Virus infection test

[0040] 3.1 Preparation of live bacterial solution

[0041] The fish-harming Myxococcus bacteria used in the experiment were provided by the Fish Disease Laboratory of the Institute of Hydrobiology, Chinese Academy of Sciences. A liquid culture medium containing 0.05% tryptone, 0.05% yeast extract, 0.02% beef extract, and 0.05% sodium acetate, with a pH of 7.2-7.4, was used. The culture medium was sterilized by autoclaving at 0.1 MPa, 121.5°C, for 20 minutes before inoculation. The culture medium was then shaken in an incubator at (25±1)°C for 48 hours, washed three times with sterile saline, and diluted to 10 9 The live bacterial solution with a concentration of cells / mL is ready for use.

[0042] 3.2 Artificial infection test

[0043] At the end of the breeding experiment, 20 Pengze crucian carp were randomly selected from each experimental group, and 40 Pengze crucian carp were randomly selected from the control group and divided equally into a control challenge group and a control antibiotic group. The above groups were placed in a pre-prepared 60cm×50cm×20cm foam box for the challenge test. Pengze crucian carp were injected intramuscularly with an injection dose of 0.5mL of live bacterial solution per fish. They were observed for 1 week and the incidence and mortality of the fish were recorded. During the challenge period, the control antibiotic group was fed fish feed supplemented with 10mg / kg ampicillin, the experimental groups were fed fish feed supplemented with the respective plant extracts, and the control challenge group was fed conventional fish feed. All groups were raised normally, fed fish feed according to the daily water volume, and the water temperature was controlled at (25±1)℃.

[0044] 3.3 Infection and Death

[0045] Table 3 Morbidity and mortality of Pengze crucian carp in each group

[0046]

[0047]

[0048] Note: “-” means no obvious symptoms, “+” means mild symptoms, “++” means obvious symptoms, and “+++” means severe symptoms.

[0049] Table 3 shows that compared with the control challenge group, adding antibiotics, hesperidin, chlorogenic acid, quercetin, luteolin and kaempferol to the breeding feed can effectively improve the symptoms of Pengze crucian carp infected with fish pest myxococcus, significantly reduce the infection mortality rate, and improve the disease resistance of Pengze crucian carp. Among them, hesperidin and kaempferol have the best effect in resisting diseases, and can completely replace antibiotics to enhance the ability of Pengze crucian carp to resist diseases.

[0050] Example 2 Compounding of Kaempferol and Hesperidin

[0051] To clarify the combined effect of kaempferol and hesperidin, the experimental design method of Example 1 was used. Kaempferol and hesperidin were added alone or in combination according to the ratio shown in Table 4, and then added at a rate of 100 mg / kg to the conventional feed for Pengze crucian carp seedlings to determine the combined ratio of kaempferol and hesperidin.

[0052] According to the method of Example 1, the body length, nonspecific immune index and challenge infection test of Pengze crucian carp with different ratios were tested, and the experimental results are as follows:

[0053] Table 4 Effects of different ratios on Pengze crucian carp seedlings

[0054]

[0055] Table 4 shows that the combination of kaempferol and hesperidin has a significant synergistic effect on the growth, immunity, and disease resistance of Pengze crucian carp. The combined effect is most pronounced when the mass ratio of kaempferol to hesperidin is 3:1. Therefore, a 3:1 mass ratio of kaempferol to hesperidin is preferred as a composite additive.

[0056] Example 3 Screening the dosage of composite additives

[0057] In order to clarify the dosage of the composite additive, the experimental design method of Example 1 was referred to, and the composite additive was added to the conventional breeding feed of Pengze crucian carp at an addition amount of 50, 100, 150, 200, 300, and 400 mg / kg, respectively, to determine the optimal dosage.

[0058] According to the method of Example 1, the body length, nonspecific immune index and challenge infection test of different groups of Pengze crucian carp were tested, and the experimental results are as follows:

[0059] Table 5 Effects of different compound additive dosages on Pengze crucian carp

[0060]

[0061] According to Table 5, the addition amount of the compound additive in the range of 50-400 mg / kg can effectively improve the growth rate and nonspecific immunity of Pengze crucian carp and enhance the disease resistance of Pengze crucian carp. When the addition amount is 200 mg / kg, the growth rate and nonspecific immunity of Pengze crucian carp are the strongest, and the disease mortality rate is the lowest.

[0062] In summary, the present invention, through screening of various natural plant extract components, discovered for the first time that adding kaempferol and hesperidin in a 3:1 mass ratio to conventional fish feed can promote the growth of Pengze crucian carp and enhance its immunity and disease resistance. Furthermore, challenge tests confirmed that at effective doses (50-400 mg / kg), kaempferol and hesperidin can completely replace antibiotics to prevent and treat fish diseases, providing new ideas for the development of "green fisheries."

[0063] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for raising seedlings of crucian carp for improving the survival rate of crucian carp infected with Myxococcus spp., characterized in that: The method comprises the steps of adding a composite additive into fish feed and feeding the Pengze crucian carp; based on the fish feed, the added amount of the composite additive is 200 mg / kg, and the composite additive is prepared from kaempferol and hesperidin in a mass ratio of 3:1.

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