Grass carp feed, method and application

Abstract

The invention relates to the technical field of grass carp feeding, in particular to grass carp feed, a method and application. The grass carp feed provided by the embodiment of the invention can promote the growth of grass carp, improve the T-SOD level of grass carp serum, improve the antioxidant defense capability of grass carp, promote the claudin c gene expression of grass carp intestinal tract, promote the cludin gene expression of grass carp intestinal tract, promote the ZO-2 gene expression of grass carp intestinal tract, enhance the barrier of grass carp intestinal tract, promote the Mucin-2 gene expression of grass carp intestinal tract and reduce the LPS level in grass carp serum; the IL-22 level in serum of the grass carp is improved, and the intestinal immune function of the grass carp is enhanced. The grass carp feed can be used as a raw material or preparation for preventing or treating enteritis of grass carp, can reduce abuse of antibiotics and drugs, and promotes green, healthy and sustainable development of aquaculture industry in China.

Description

Technical Field

[0001] The present application relates to the technical field of grass carp feeding, and in particular to grass carp feed, methods and applications. Background Art

[0002] Grass carp (Ctenopharyngodon idella) is a herbivorous fish with the important economic characteristics of fast growth and high yield. However, the incidence of enteritis in grass carp is high. Enteritis is one of the most common diseases in grass carp farming and has a high mortality rate. Enteritis is the most common and serious disease in aquaculture, which causes considerable losses to my country's aquaculture industry every year. Pathogenic infection, nutritional imbalance, dietary trace element deficiency, environmental pollution, animal genetic defects, etc. can all lead to the occurrence of fish enteritis. Diseased fish often show similar intestinal symptoms: severe damage to the intestinal barrier, especially the destruction of tight junction structure, cell necrosis, inflammatory cell infiltration, and significant increase in intestinal permeability. Antibiotics are commonly used drugs for the treatment of enteritis, but the use of antibiotics often causes drug residues, the production of drug-resistant bacteria, impaired intestinal homeostasis, disruption of flora balance and barrier dysfunction. Finding alternatives to antibiotics to prevent the occurrence of enteritis has become an urgent problem that needs to be solved in the aquaculture industry. Summary of the invention

[0003] In view of this, the embodiments of the present application provide a grass carp feed, method and application.

[0004] Based on this, the embodiments of the present application disclose at least the following technical solutions:

[0005] In a first aspect, an embodiment discloses a grass carp feed, which comprises, by weight: 800-1200 parts of soybean meal containing 43% protein, 1000-1500 parts of rapeseed meal, 1000-1500 parts of rapeseed cake, 1200-2000 parts of fish meal, 500-1000 parts of corn germ meal, 1500-2000 parts of flour, 300-400 parts of soybean oil, 20-40 parts of L-lysine hydrochloride, 10-30 parts of DL-methionine, 1-5 parts of ethoxyquin, 20-40 parts of choline chloride, 200-300 parts of monocalcium phosphate, 20-40 parts of mineral premix, 20-30 parts of vitamin premix, 1000-1500 parts of bentonite, and 9-27 parts of the antimicrobial peptide shown in SEQ ID NO: 1.

[0006] In the second aspect, an embodiment discloses a method for improving the antioxidant defense ability of grass carp, comprising feeding the grass carp feed described in the first aspect at 9:00 a.m. and 17:00 p.m. every day until the young fish are full; and regularly replacing 1 / 3 of the aerated water in the breeding barrel at 20:00 every night, and cleaning the excrement at the bottom of the tank with a sewage suction device, and continuing feeding for 60 days.

[0007] In the third aspect, an embodiment discloses a method for enhancing the intestinal barrier of grass carp, comprising feeding the grass carp feed described in the first aspect at 9:00 a.m. and 17:00 p.m. every day until the young fish are full; and regularly replacing 1 / 3 of the aerated water in the breeding barrel at 20:00 every night, and cleaning the excrement at the bottom of the tank with a sewage suction device, and continuing feeding for 60 days.

[0008] In a fourth aspect, an embodiment discloses a method for increasing the level of IL-22 in grass carp serum, comprising feeding the grass carp feed described in the first aspect at 9:00 a.m. and 17:00 p.m. every day until the young fish are full; and regularly replacing 1 / 3 of the aerated water in the breeding barrel at 20:00 p.m. every night, and cleaning the excrement at the bottom of the tank with a sewage suction device, and continuing feeding for 60 days.

[0009] In a fifth aspect, the embodiment discloses an application of the grass carp feed described in the first aspect, wherein the application is selected from at least one of the following:

[0010] Promote the growth of grass carp;

[0011] Improve the disease resistance of grass carp;

[0012] Improve the intestinal morphology of grass carp;

[0013] Enhance the integrity and function of the intestinal barrier of grass carp;

[0014] Improve the serum T-SOD level of grass carp;

[0015] Improve the antioxidant defense capacity of grass carp;

[0016] Promote the expression of claudin c gene in the intestine of grass carp;

[0017] Promote the expression of occludin gene in the intestine of grass carp;

[0018] Promote the expression of ZO-2 gene in the intestine of grass carp;

[0019] Promote the expression of Mucin-2 gene in the intestine of grass carp;

[0020] Increase the level of IL-22 in grass carp serum;

[0021] Reduce LPS levels in grass carp serum;

[0022] Reduce the intestinal permeability of grass carp;

[0023] Preparation of preparations for preventing enteritis;

[0024] Preparation of preparations for treating enteritis;

[0025] Improve the intestinal microbial composition of grass carp;

[0026] Improve the intestinal microbial diversity of grass carp;

[0027] Regulate metabolic pathways in grass carp;

[0028] Regulating interactions between intestinal microorganisms in grass carp.

[0029] The grass carp feed provided in the embodiment of the present application can promote the growth of grass carp, increase the level of T-SOD in grass carp serum, improve the antioxidant defense ability of grass carp, promote the expression of claudin c gene in the intestine of grass carp, promote the expression of occludin gene in the intestine of grass carp, promote the expression of ZO-2 gene in the intestine of grass carp, enhance the intestinal barrier of grass carp, promote the expression of Mucin-2 gene in the intestine of grass carp, enhance the intestinal immune function of grass carp, and increase the level of IL-22 in the serum of grass carp. The grass carp feed has the function of being a raw material or preparation for preventing or treating enteritis of grass carp, and provides a reference for its application in aquaculture, thereby reducing the abuse of antibiotics and drugs, and promoting the green, healthy and sustainable development of my country's aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The images are of grass carp intestinal epithelial cells of the control group (A), low-dose treatment group (B), medium-dose treatment group (C) and high-dose treatment group (D) provided in the examples, with a magnification of 10000×, and "TJ" stands for tight junction.

[0031] Figure 2 The MDA content (A), T-SOD enzyme activity (B) and oxidative stress-related gene mRNA levels (C) of grass carp in the Control, LD, MD and HD groups provided in the examples; different letters represent significant differences among different groups (P < 0.05).

[0032] Figure 3 The mRNA levels of claudin c, occludin, ZO-2 and Mucin-2 genes in the intestine of grass carp in the Control, LD, MD and HD groups provided in the example; different letters on the bar graph represent significant differences among different groups (P < 0.05).

[0033] Figure 4 The IL-22 content (A) and LPS content (B) of grass carp in the Control, LD, MD and HD groups provided in the examples. Different letters on the bar graph represent significant differences among different groups (P<0.05).

[0034] Figure 5 This is an analysis diagram of the intestinal microbial composition of grass carp in the Control, LD, MD and HD groups provided in the example.

[0035] Figure 6This is a cross-analysis diagram of the intestinal microorganisms of grass carp in the Control, LD, MD and HD groups provided in the example.

[0036] Figure 7 This is an analysis chart of the dominant genus composition of grass carp intestinal microorganisms in the Control, LD, MD and HD groups provided in the example.

[0037] Figure 8 This is a graph showing the α-diversity analysis of the intestinal microorganisms of grass carp in the Control, LD, MD and HD groups provided in the examples.

[0038] Fig. 9 This is a Beta diversity analysis diagram of the grass carp intestinal microorganisms in the Control, LD, MD and HD groups provided in the example.

[0039] Fig.10 This is a prediction analysis diagram of the intestinal microbial functions of grass carp in the Control, LD, MD and HD groups provided in the example.

[0040] Fig.11 This is a diagram showing the interaction analysis between the intestinal microorganisms of grass carp in the Control, LD, MD and HD groups provided in the examples. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0042] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers used in this application to express the amount, percentage, and other numerical values ​​should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods.

[0043] The term "part" refers to parts by weight only. Specifically, any weight can be used as 1 part, such as 0.001g, 0.01g, 0.05g, 0.1g, 0.5g, 1g, 10g, 100g, 1000g, 5000g, 10000g, etc. There is no specific limitation on the absolute weight of 1 part.

[0044] Embodiment A grass carp feed is prepared using 43% protein soybean meal, rapeseed meal, rapeseed cake, fish meal, corn germ meal, flour, soybean oil, L-lysine hydrochloride, DL-methionine, ethoxyquinoline, choline chloride, calcium dihydrogen phosphate, mineral premix, vitamin premix, bentonite and the antimicrobial peptide shown in SEQ ID NO: 1 as raw materials. The grass carp feed can promote the growth of grass carp, increase the level of T-SOD in grass carp serum, improve the antioxidant defense ability of grass carp, promote the expression of claudin c gene in the intestinal tract of grass carp, promote the expression of occludin gene in the intestinal tract of grass carp, promote the expression of ZO-2 gene in the intestinal tract of grass carp, promote the expression of Mucin-2 gene in the intestinal tract of grass carp, enhance the intestinal barrier of grass carp, and increase the level of IL-22 in the serum of grass carp. The grass carp feed has the function of being a raw material or preparation for preventing or treating enteritis of grass carp, and provides a reference for its application in aquaculture, thereby reducing the abuse of antibiotics and drugs, and promoting the green, healthy and sustainable development of my country's aquaculture industry.

[0045] Based on this, the embodiment provides a grass carp feed, which includes, by weight: 800-1200 parts of soybean meal containing 43% protein, 1000-1500 parts of rapeseed meal, 1000-1500 parts of rapeseed cake, 1200-2000 parts of fish meal, 500-1000 parts of corn germ meal, 1500-2000 parts of flour, 300-400 parts of soybean oil, 20-40 parts of L-lysine hydrochloride, 10-30 parts of DL-methionine, 1-5 parts of ethoxyquinoline, 20-40 parts of choline chloride, 200-300 parts of monocalcium phosphate, 20-40 parts of mineral premix, 20-30 parts of vitamin premix, 1000-1500 parts of bentonite, and 9-27 parts of antimicrobial peptides as shown in SEQ ID NO: 1.

[0046] In some embodiments, the grass carp feed includes, by weight, 1000 parts of soybean meal containing 43% protein, 1462 parts of rapeseed meal, 1200 parts of rapeseed cake, 1800 parts of fish meal, 800 parts of corn germ meal, 1800 parts of flour, 350 parts of soybean oil, 30 parts of L-lysine hydrochloride, 20 parts of DL-methionine, 3 parts of ethoxyquin, 30 parts of choline chloride, 250 parts of monocalcium phosphate, 30 parts of mineral premix, 25 parts of vitamin premix, 1200 parts of bentonite, and 9 parts of antimicrobial peptides.

[0047] In some embodiments, the grass carp feed includes, by weight, 1000 parts of soybean meal containing 43% protein, 1462 parts of rapeseed meal, 1200 parts of rapeseed cake, 1800 parts of fish meal, 800 parts of corn germ meal, 1800 parts of flour, 350 parts of soybean oil, 30 parts of L-lysine hydrochloride, 20 parts of DL-methionine, 3 parts of ethoxyquin, 30 parts of choline chloride, 250 parts of monocalcium phosphate, 30 parts of mineral premix, 25 parts of vitamin premix, 1200 parts of bentonite, and 18 parts of antimicrobial peptides.

[0048] In some embodiments, the grass carp feed includes, by weight, 1000 parts of soybean meal containing 43% protein, 1462 parts of rapeseed meal, 1200 parts of rapeseed cake, 1800 parts of fish meal, 800 parts of corn germ meal, 1800 parts of flour, 350 parts of soybean oil, 30 parts of L-lysine hydrochloride, 20 parts of DL-methionine, 3 parts of ethoxyquin, 30 parts of choline chloride, 250 parts of monocalcium phosphate, 30 parts of mineral premix, 25 parts of vitamin premix, 1200 parts of bentonite, and 27 parts of antimicrobial peptides.

[0049] In some embodiments, the vitamin premix includes, by weight, 50-100 parts of vitamin B1, 100-150 parts of vitamin B2, 80-150 parts of vitamin B6, 5-15 parts of vitamin B12, 300-400 parts of vitamin D, 2000-3000 parts of vitamin E, 3000-4000 parts of vitamin K; 300-400 parts of niacinamide, 300-500 parts of calcium pantothenate, 2000-3000 parts of VC phosphate, 500-600 parts of inositol, 10-20 parts of folic acid, 50-100 parts of biotin, 10-15 parts of ethoxyquin, 2000-3000 parts of rice husk powder, and 5-10 parts of zeolite powder.

[0050] In some embodiments, the vitamin premix includes, by weight, 82 parts of vitamin B1, 128 parts of vitamin B2, 93 parts of vitamin B6, 10 parts of vitamin B12, 329 parts of vitamin D, 2480 parts of vitamin E, 3280 parts of vitamin K; 350 parts of niacinamide, 400 parts of calcium pantothenate, 2600 parts of VC phosphate, 530 parts of inositol, 14 parts of folic acid, 86 parts of biotin, 15 parts of ethoxyquin, 2798 parts of rice husk powder, and 5 parts of zeolite powder.

[0051] In some embodiments, the mineral premix includes, by weight, 30-50 parts of calcium iodate, 200-300 parts of sulfuric acid pentahydrate, 1300-1800 parts of ferrous sulfate monohydrate, 700-800 parts of zinc sulfate monohydrate, 100-200 parts of manganese sulfate monohydrate, 3000-4000 parts of magnesium sulfate monohydrate, 10-20 parts of sodium selenite, 30-50 parts of cobalt chloride, 2000-3000 parts of zeolite powder and 1000-1100 parts of rice husk powder.

[0052] In some embodiments, the mineral premix comprises, by weight, 50 parts of calcium iodate, 240 parts of sulfuric acid pentahydrate, 1500 parts of ferrous sulfate monohydrate, 740 parts of zinc sulfate monohydrate, 135 parts of manganese sulfate monohydrate, 3500 parts of magnesium sulfate monohydrate, 15 parts of sodium selenite, 40 parts of cobalt chloride, 2700 parts of zeolite powder and 1080 parts of rice husk powder.

[0053] Based on this, the embodiment also discloses a method for improving the antioxidant defense ability of grass carp, comprising feeding the grass carp feed provided in the above embodiment at 9:00 a.m. and 17:00 p.m. every day until the young fish are full; and regularly replacing 1 / 3 of the aerated water in the breeding barrel at 20:00 every night, and cleaning the excrement at the bottom of the tank with a sewage suction device, and continuing feeding for 60 days.

[0054] Based on this, the embodiment also discloses a method for enhancing the intestinal barrier of grass carp, comprising feeding the grass carp feed provided in the above embodiment at 9:00 a.m. and 17:00 p.m. every day until the young fish are full; and regularly replacing 1 / 3 of the aerated water in the breeding barrel at 20:00 every night, and cleaning the excrement at the bottom of the tank with a sewage suction device, and continuing feeding for 60 days.

[0055] Based on this, the embodiment also discloses a method for increasing the level of IL-22 in grass carp serum, comprising feeding the grass carp feed provided in the above embodiment at 9:00 a.m. and 17:00 p.m. every day until the young fish are full; and regularly replacing 1 / 3 of the aerated water in the breeding barrel at 20:00 p.m. every night, and cleaning the excrement at the bottom of the tank with a sewage suction device, and continuing feeding for 60 days.

[0056] Based on this, the embodiment further discloses the application of the grass carp feed provided in the above embodiment, and the application is selected from at least one of the following:

[0057] Promote the growth of grass carp;

[0058] Improve the disease resistance of grass carp;

[0059] Improve the intestinal morphology of grass carp;

[0060] Enhance the integrity and function of the intestinal barrier of grass carp;

[0061] Improve the serum T-SOD level of grass carp;

[0062] Improve the antioxidant defense capacity of grass carp;

[0063] Promote the expression of claudin c gene in the intestine of grass carp;

[0064] Promote the expression of occludin gene in the intestine of grass carp;

[0065] Promote the expression of ZO-2 gene in the intestine of grass carp;

[0066] Promote the expression of Mucin-2 gene in the intestine of grass carp;

[0067] Increase the level of IL-22 in grass carp serum;

[0068] Reduce LPS levels in grass carp serum;

[0069] Reduce the intestinal permeability of grass carp;

[0070] Preparation of preparations for preventing enteritis;

[0071] Preparation of preparations for treating enteritis;

[0072] Improve the intestinal microbial composition of grass carp;

[0073] Improve the intestinal microbial diversity of grass carp;

[0074] Regulate metabolic pathways in grass carp;

[0075] Regulating interactions between intestinal microorganisms in grass carp.

[0076] The present invention is further described below in conjunction with more specific embodiments. Of course, the following embodiments should not be construed as limiting the present application.

[0077] 1. Preparation of grass carp feed

[0078] According to the composition of grass carp feed shown in Table 1, various raw materials were crushed, sieved, fully mixed and expanded to make grass carp feed. In Table 1, mineral premix was provided by Beijing Da Bei Nong Technology Group Co., Ltd. (Beijing, China), and vitamin premix was provided by Beijing Da Bei Nong Technology Group Co., Ltd. (Beijing, China). Note: Mineral premix (g / kg): calcium iodate, 5g; copper sulfate pentahydrate, 24g; ferrous sulfate monohydrate, 150g; zinc sulfate monohydrate, 74g; manganese sulfate monohydrate, 13.5g; magnesium sulfate monohydrate, 350g; sodium selenite, 1.5g; cobalt chloride, 4g; zeolite powder, 270g; rice husk powder, 108g.

[0079] Vitamin premix (g / kg): Vitamin B1, 8.2g; Vitamin B2, 12.8g; Vitamin B6, 9.3g; Vitamin B12, 1g; Vitamin D, 32.9g; Vitamin E, 248g; Vitamin K, 328g; Nicotinamide, 35g; Calcium pantothenate, 40g; VC phosphate, 260g; Inositol, 53g; Folic acid, 1.4g; Biotin, 8.6g; Ethoxyquin, 1.5g; Rice husk powder, 279.8g; Zeolite powder, 0.5g.

[0080] 2. Feeding in groups

[0081] Grass carp were purchased from Honghu Fishery in Wuhan City, Hubei Province. Before the start of the breeding experiment, they were temporarily kept in the breeding pond for 3 weeks for acclimation. A total of 252 juvenile fish with consistent specifications and healthy physique (average weight 46.85g, average body length 14.06cm) were selected and randomly divided into 12 breeding barrels (200L / barrel), with 21 fish in each barrel. The breeding barrels were randomly divided into 4 groups: control group (Control), low-dose group (LD), medium-dose group (MD) and high-dose group (HD), with 3 replicates in each group.

[0082] Spray feed once every 3 days, and store the dried feed in a refrigerator at 4℃ for feeding grass carp. After feeding, feed the young fish at 9:00 am and 17:00 pm every day until they are full. After 30 minutes of feeding, observe whether there is any leftover feed. If there is, remove it, dry it, weigh it and record it. At 20:00 pm every night, regularly replace 1 / 3 of the aerated water in the breeding tank.

[0083] Use a suction device to clean the excrement at the bottom of the tank, continue feeding for 60 days, and pay attention to the health of the fish at any time during the experiment. During the breeding period, the water quality was kept good, the water temperature was 28.5±0.5℃, the light cycle was 12 / 12 (light 12h / dark 12h), the pH was kept at 7.5±0.2, the total ammonia nitrogen content was less than 0.2mg / L, and the nitrite content was less than 0.01mg / L.

[0084] Table 1 Composition of grass carp feed

[0085]

[0086]

[0087] 3. Sample collection

[0088] After 60 days of continuous feeding, 7 fish were randomly selected from each breeding barrel. After anesthesia with tricaine methylsulfate (MS-222) at a concentration of 100 mg / L, the fish were weighed and their lengths were measured. Blood was collected from the tail vein using a syringe, and the collected blood was left to stand overnight at 4°C. The serum obtained was stored in a -80°C refrigerator for subsequent analysis of serum biochemical indicators. The grass carp was then dissected, the viscera and liver were separated, and weighed and measured separately to facilitate the calculation of the viscera-to-body ratio and liver-to-body ratio. The intestine was separated using sterile tweezers, and the anterior, middle, and posterior intestines were accurately distinguished. About 1 cm of the intestine was cut from the same position of each intestinal segment and placed in 4% paraformaldehyde fixative for HE staining analysis. At the same time, a portion of the intestinal segment was collected and placed in 2.5% glutaraldehyde fixative for observation of the intestinal ultrastructure under transmission electron microscopy (TEM). The remaining intestinal tissue was quickly stored in a -80°C refrigerator after being frozen with liquid nitrogen. All animal handling procedures and experiments in this study were reviewed and approved by the Ethics Committee of the Institute of Hydrobiology, Chinese Academy of Sciences.

[0089] 4. Growth performance analysis

[0090] Before and after the start of the breeding experiment, accurately record the initial weight, initial body length, final weight, final body length, visceral mass weight, liver weight and other growth-related data of grass carp, and accurately calculate growth performance indicators such as fatness, weight gain rate, specific growth rate, viscera-to-body ratio, and liver-to-body ratio according to the following formulas.

[0091] Fullness (CF, g / cm 3 ) = 100 × weight / body length 3

[0092] Weight gain (WG, g) = final weight - initial weight

[0093] Weight gain rate (WGR, %) = 100 × (final weight - initial weight) / initial weight

[0094] Specific growth rate (SGR, % / d) = 100 × (ln final weight - ln initial weight) / number of days

[0095] Visceral to body ratio (VSI) = 100 × visceral mass weight / body weight

[0096] Liver to body weight ratio (HSI) = 100 × liver weight / body weight

[0097] 5. Determination of serum biochemical indicators

[0098] According to the instructions of the scientific research kit produced by Nanjing Jiancheng Bioengineering Institute and Jiangsu Enzyme Immunity Industry Co., Ltd., the collected serum samples were tested for a series of specific biochemical indicators, including total superoxide dismutase (T-SOD), malondialdehyde (MDA), lipopolysaccharide (LPS) and interleukin-22 (IL-22).

[0099] 6. HE staining of intestinal tissue

[0100] The collected grass carp front, middle and hind intestines of about 1 cm in length were placed in 4% paraformaldehyde for 48 hours, and the tissues were subsequently processed (washing, dehydration, transparency, waxing, embedding), and then the embedded paraffin blocks were sliced, flattened in warm water, and picked up with a slide, and then the subsequent HE staining and other steps were completed. The slices were photographed and observed using an upright fluorescence microscope. Five parts were randomly selected for each slice, and the intestinal villus height, villus width, muscle layer thickness and crypt depth were measured using Lightools software, and the calculated average value was finally used as the measurement data for this experiment.

[0101] 7. Transmission electron microscopy (TEM)

[0102] The grass carp hind intestine tissue was fixed in 2.5% glutaraldehyde to maintain the integrity of the cell structure, and heavy metal electron-stained sections were obtained through osmium acid fixation, dehydration, and infiltration steps for observation under a transmission electron microscope. The preparation and observation of transmission electron microscope sections were assisted by the Analysis and Testing Center of the Institute of Hydrobiology, Chinese Academy of Sciences.

[0103] 8. Real-time fluorescence quantitative detection

[0104] Total RNA from grass carp intestinal tissue was extracted according to the instructions of Takara Trizol Reagent. The concentration and purity of the extracted RNA were determined using NanoDrop 2000 photometer. The qualified RNA was reverse transcribed into cDNA according to the instructions of TaKaRa reverse transcription kit. Real-time fluorescence quantitative PCR (RT-qPCR) analysis of cDNA was performed using TB Green Master Mix and CFX96TM real-time PCR detection system. β-actin was used as the internal reference gene. -ΔΔCT The relative expression of each target gene was calculated. The primer sequences are shown in Table 2.

[0105] Table 2 Primers used for RT-qPCR

[0106]

[0107] 9. Data statistics and analysis

[0108] SPSS Statistics 27.0 software was used to perform one-way ANOVA on the data. The results were expressed as mean ± standard error (Mean ± SEM). Duncan's multiple range test was used to compare the significance of differences among different treatment groups. The criterion for judging the significance of differences was a P value less than 0.05 (P < 0.05).

[0109] 10. The grass carp feed provided in the embodiment promotes the growth of grass carp and improves the body shape of the fish

[0110] Table 3 shows the final body weight, final body length, weight gain, weight gain rate and specific growth rate of grass carp in each group after 60 days of feeding. The results are expressed as mean ± standard error. Different letters in the same row represent significant differences among the groups (P<0.05), and the same letters or no letters represent no significant differences (P>0.05).

[0111] As shown in Table 3, adding antimicrobial peptides to feed can improve the growth performance of grass carp. Its final body weight, final body length, weight gain, weight gain rate and specific growth rate showed a trend of first increasing and then decreasing with the increase of antimicrobial peptide addition level. These indicators all reached the maximum value in the medium-dose addition group, and the final body weight, weight gain, weight gain rate and specific growth rate of grass carp in the medium-dose group were significantly higher than those in the control group.

[0112] Table 3

[0113] Group Control LD MD HD IBW(g) 46.16±1.25 45.87±1.35 47.21±1.45 48.17±1.44 IBL(cm) 14.03±0.12 13.99±0.18 14.04±0.13 14.19±0.16 FBW(g) 125.31±6.02b 144.16±6.38ab 149.63±8.00a 144.45±7.31ab FBL(cm) 18.77±0.35 19.46±0.35 19.69±0.48 19.54±0.33 WG(g) 79.15±6.02b 98.28±6.38ab 102.41±7.96a 96.28±7.31ab WGR (%) 171.5±13.05b 214.30±14.03ab 216.75±16.58a 199.92±15.22ab SGR (% / d) 1.63±0.08b 1.88±0.07a 1.88±0.08a 1.78±0.09ab

[0114] Table 4 shows the fatness, organ-to-body ratio, and liver-to-body ratio of grass carp in each group after 60 days of feeding. The results are expressed as mean ± standard error. Different letters in the same row represent significant differences among the groups (P<0.05), and the same letters or no letters represent no significant differences (P>0.05).

[0115] As shown in Table 4, compared with the control group (Control), the liver-to-body ratio and organ-to-body ratio of grass carp in the antimicrobial peptide group (MD) were significantly reduced, while the fatness was significantly increased. This shows that an appropriate dose of antimicrobial peptides can promote the absorption and utilization of nutrients in feed by grass carp, reduce fat deposition, and benefit the growth of grass carp.

[0116] Table 4

[0117] Group Control LD MD HD HSI 2.29±0.09 2.24±0.08 2.14±0.08 2.25±0.07 VSI 10.69±0.53 10.24±0.56 10.13±0.31 10.62±0.43 <![CDATA[CF(g / cm 3 )]]> 1.88±0.05 1.95±0.06 1.98±0.09 1.91±0.04

[0118] 11. The grass carp feed provided in the embodiment improves the disease resistance of grass carp

[0119] Table 5 shows the statistical results of the death of grass carp after challenge with Aeromonas hydrophila (CCTCC HB 20082627). The challenge test process specifically includes:

[0120] After 60 days of feeding, 30 fish (average weight 140.89 g, average length 19.37 cm) were selected from each group for the Aeromonas hydrophila challenge test. Each grass carp was intraperitoneally injected with 200 μl of 2.85×10 7 cfu / mL of Aeromonas hydrophila live bacterial liquid. 24 hours after injection, the number and time of dead fish were recorded for a total of 7 days, and the mortality rate and relative immune protection rate were calculated. Among them, the mortality rate is the percentage of the number of grass carp deaths in each group to the number of poisoned tails, and the relative immune protection rate is the percentage of the difference in the number of grass carp deaths between the control group and the low-dose group, medium-dose group or high-dose group to the number of grass carp deaths in the control group.

[0121] Table 5

[0122] Group Number of fish attacking poison Death tail number mortality rate Relative immune protection rate Control group 30 23 76.7％ 0％ Low dose group 30 17 56.7％ 26.1％ Medium dose group 30 13 43.3％ 43.5％ High dose group 30 19 63.3％ 17.4％

[0123] As shown in Table 5, the control group had the highest cumulative mortality rate of 76.7%. The mortality rate of grass carp in the antimicrobial peptide group after infection with Aeromonas hydrophila was significantly reduced. The cumulative mortality rates of the low and high dose groups were 56.7% and 63.3%, respectively. The grass carp in the medium dose group had the lowest mortality rate after infection, which was 43.3%. At the same time, the relative immune protection rate of the antimicrobial peptide group was significantly higher than that of the control group, which were 26.1%, 43.5%, and 17.4%, respectively. This shows that adding antimicrobial peptides to feed can enhance the resistance of grass carp to Aeromonas hydrophila and produce a good immune protection effect on the fish body.

[0124] 12. The grass carp feed provided in the embodiment improves the intestinal morphology of grass carp

[0125] Table 6 shows the villus length, villus width, muscle layer thickness, and crypt depth of each group of grass carp after 60 days of feeding. The results are expressed as mean ± standard error. Different letters in the same row represent significant differences among the groups (P<0.05), and the same letters or no letters represent no significant differences (P>0.05).

[0126] As shown in Table 6, in general, the effect of antimicrobial peptides on intestinal morphology showed a certain degree of dose effect: with the increase of antimicrobial peptide content, the length, width and muscle layer thickness of the grass carp intestine first increased and then decreased, while the depth of the crypts showed the opposite trend. Specifically, in terms of villus length, in the foregut, the villus length of the antimicrobial peptide group was significantly higher than that of the control group (P < 0.05); the villus length of the midgut was the highest in the low-dose group, but the difference was not significant with the control group; the villus length of the hindgut reached the maximum in the medium-dose group, which was significantly different from the control group (P < 0.05). In terms of villus width, the villus width of the foregut of grass carp in the low-dose group, the hindgut of grass carp in the low-dose group, and the midgut of grass carp in the medium-dose group was the widest, and was significantly wider than that of the control group (P < 0.05). In terms of muscle layer thickness, the muscle layer thickness of the fore, mid, and hind intestines of grass carp in the medium-dose group was the highest, and was significantly higher than that of the control group (P < 0.05). In terms of crypt depth, the intestinal crypt depth of grass carp in the antimicrobial peptide group was significantly lower than that in the control group (P<0.05).

[0127] This shows that the grass carp feed provided in the embodiment can significantly increase the length, width and muscle layer thickness of the intestinal villi, reduce the depth of the crypts, and thus improve the intestinal morphology of the grass carp, thereby achieving the purpose of promoting the ability of the grass carp intestine to digest and absorb nutrients.

[0128] Table 6

[0129]

[0130] 13. The grass carp feed provided in the embodiment enhances the integrity and function of the intestinal barrier of grass carp

[0131] like Figure 1The transmission electron microscope (TEM) scans shown in the figure show that the number of microvilli in the intestine of grass carp increased in the low-dose and medium-dose antimicrobial peptide treatment groups, while they were more sparse in the high-dose group. The microvilli in the medium-dose group became longer and more neatly arranged. The tight junction structure of the intestinal epithelium, namely tight junctions, appears as black dense electron bands under an electron microscope. It is located at the top of the epithelial cells and extends along the roots of the villi to the basal layer, strengthening the barrier function between cells. Compared with the control group, the tight junction structure of the intestinal epithelium in the low-dose and medium-dose groups was more neatly fitted and had a higher electron density, thereby better maintaining the integrity and function of the intestinal barrier.

[0132] 14. The grass carp feed provided in the embodiment improves the antioxidant defense ability of grass carp

[0133] like Figure 2 As shown in A, the MDA content in the low-dose group was the lowest, indicating that low-dose antimicrobial peptides may be more effective in reducing oxidative stress in fish. Figure 2 As shown in B, the activity of total superoxide dismutase (T-SOD) in serum showed a trend of first increasing and then decreasing. The T-SOD activity in the medium-dose group was significantly higher than that in the other groups (P<0.05), while the T-SOD level in the control group was lower than that in the other groups. Figure 2 C shows the relative expression of catalase (CAT) gene in intestinal tissue in each experimental group. The results show that the relative expression of CAT in all experimental groups is higher than that in the control group, among which the expression in the low-dose group is the highest. However, as the dose of antimicrobial peptide increases, its expression level gradually decreases. This shows that the grass carp feed provided in the embodiment has a positive regulatory effect on the antioxidant defense system in grass carp.

[0134] 15. The grass carp feed provided in the embodiment regulates the expression of claudin c, occludin, ZO-2 and Mucin-2 genes in the intestine of grass carp

[0135] like Figure 3 The results showed that the expression of genes related to the intestinal barrier function of grass carp was upregulated after treatment with different concentrations of antimicrobial peptides, but the change in expression level showed a complex relationship with the dose of antimicrobial peptides. The expression level of claudin c gene was the highest in the medium-dose antimicrobial peptide treatment group, and then decreased, with significant differences among the groups (P<0.05). At the same time, the relative expression of occludin gene was significantly increased in the groups except the control group (P<0.05), and with the increase of antimicrobial peptide dose, its expression level showed a gradual upward trend, reaching the highest in the high-dose group. The expression level of ZO-2 gene reached a peak in the low-dose antimicrobial peptide treatment group and then gradually decreased. In particular, the expression level of ZO-2 in the low-dose group was significantly higher than that in the control group (P<0.05).

[0136] This shows that adding an appropriate amount of antimicrobial peptides to grass carp feed can promote the expression of genes related to the intestinal barrier function of the intestinal epithelium and mucin genes in the epithelial tissue, enhance the intestinal mucosal (epithelial) barrier and chemical (mucin) barrier function, and make the intestinal mucosal layer structure of the body more stable. However, adding excessive antimicrobial peptides may inhibit the expression of genes related to the intestinal mucus barrier and reduce the intestinal defense function. Therefore, the amount of antimicrobial peptides used needs to be precisely controlled to ensure that they will not have a negative impact on the body while improving intestinal health.

[0137] 16. The grass carp feed provided in the embodiment regulates the expression of inflammatory factors in grass carp

[0138] like Figure 4 As shown in A, the interleukin-22 (IL-22) content in the serum of the experimental group was higher than that of the control group, and the IL-22 content showed a trend of first increasing and then decreasing with the increase of the level of antimicrobial peptide addition. Especially under the condition of medium-dose antimicrobial peptide treatment, the IL-22 content was significantly higher than that of the control group and reached the highest value (P<0.05). Figure 4 As shown in B, the endotoxin (LPS) content in the serum of the experimental group was significantly lower than that of the control group (P<0.05), but there was no significant difference in LPS content between different treatment groups.

[0139] This shows that the grass carp feed provided in the embodiment can reduce the endotoxin level in the grass carp and reduce the inflammatory response. In addition, adding antimicrobial peptides to the grass carp feed provided in the embodiment can promote the synthesis of IL-22, induce the synthesis of intestinal mucin-2, and then weaken the signal transduction derived from bacteria, reduce the serum LPS level, thereby achieving the purpose of enhancing immune function and intestinal barrier and protecting tissues.

[0140] 17. The grass carp feed provided in the embodiment improves the intestinal microbial composition of grass carp

[0141] The differences in intestinal microbial composition of grass carp in the control, low-dose, medium-dose, and high-dose groups were determined. Figure 5 As shown in the figure, the composition of grass carp intestinal microorganisms at the phylum level is mainly Firmicutes, Fusobacteria, Verrucomicrobia, Proteobacteria, Chloroflexi, Actinobacteria, Chlamydiae, Planctomycetes, Tenericutes and Bacteroidetes. Figure 6As shown, the proportion of Fusobacteria in the medium and low dose groups was significantly higher than that in the control group, while the abundance of Fusobacteria in the high dose group was lower than that in the control group. The proportion of Proteobacteria in the antimicrobial peptide group was lower than that in the control group, and the abundance of Chloroflexi in the medium and low dose groups was significantly lower than that in the control group.

[0142] like Figure 7 As shown, the dominant genera in each group include: Cetobacterium, Akkermansia, Fusibacter, Anaerorhabdus, Acetobacterium, Bacillus, Robinsoniella, Rhodobacter, Proteocatella, Mycobacterium, etc. Cetobacterium is the dominant group with the highest proportion in the medium and low dose groups (37.17% and 31.57%, respectively), and accounts for a large proportion in the Fusobacteria, while it accounts for a lower proportion in the control group and high dose group (17.59% and 11.3%, respectively). The abundance of Fusibacterium in the low and high dose groups was significantly higher than that in the control group. The abundance of Anaerorhabdus in the antimicrobial peptide group was significantly reduced compared with the control group.

[0143] This indicates that the grass carp feed provided in the embodiment improves the intestinal microbial composition of grass carp, reduces the composition of pathogenic bacteria therein, and increases the composition and proportion of probiotic bacteria.

[0144] 18. The grass carp feed provided in the embodiment improves the intestinal microbial diversity of grass carp

[0145] The intestinal microorganisms of grass carp were analyzed by 16S rRNA sequencing, and the Good's Coverage values ​​of the four groups of intestinal samples were all ≥99%. Chao 1 index, Simpson index, Shannon index and Faith-pd were calculated to evaluate the α diversity of intestinal microorganisms. Figure 8 As shown, the Chao 1 index, Simpson index, Shannon index and Faith-pd of the low-dose group decreased significantly, while the Chao 1 index and Shannon index of the medium-dose group increased significantly, and the Simpson index and Faith-pd decreased significantly.

[0146] like Fig. 9 As shown, PCoA principal coordinate analysis (i.e. Beta diversity analysis) based on the Bray-Curtis matrix showed that the samples in the control group were clustered together, while the samples in the antimicrobial peptide group were separated from the control group. Further PERMANOVA analysis showed that there was a significant difference in the microbial community composition between the high-dose group and the control group (P<0.05), but there was no significant difference between the other groups.

[0147] 19. The grass carp feed provided in the embodiment regulates the metabolic pathway of grass carp

[0148] The MetaCyc database was used to perform PICRUSt functional prediction analysis of grass carp intestinal bacteria. Fig.10 As shown in the figure, the intestinal microorganisms of grass carp include the following primary functional pathways: biosynthesis, degradation / utilization / assimilation, generation of precursor metabolite and energy, glycan pathways, macromolecule modification, and metabolic clusters. The difference between each experimental group and the control group was detected by independent T test. It was found that the metabolic pathways of amine and polyamine degradation, amino acid biosynthesis, nucleoside and nucleotide degradation, and aromatic compound degradation were significantly enriched in the antimicrobial peptide group. Nucleic Acid Processing, Carbohydrate Degradation, Cell Structure Biosynthesis, Faly Acid and Lipid Degradation metabolic pathways were significantly enriched only in the medium-dose group. Fermentation metabolic pathway was significantly enriched only in the high-dose group (P<0.05).

[0149] 20. The grass carp feed provided in the embodiment regulates the interaction between grass carp intestinal microorganisms

[0150] like Fig.11As shown in the figure, network analysis showed the interaction relationship between the intestinal microorganisms of grass carp in different groups. In the association network, a total of 100 nodes and 1209 edges were identified, with a modularity coefficient of 0.276, a network diameter of 3, an average path length of 1.79, an average degree of 24.18, an average weighted degree of 13.865, and an average clustering coefficient of 0.424. The relationship between the intestinal microorganisms of grass carp was complex, but positive correlation was dominant. Cetobacterium, Fusibacter, Bacillus, Robinsoniella, Akkermansia, Erysipelotrichaceae, and Lachnospiraceae were the main network nodes with positive correlation.

[0151] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A grass carp feed, comprising, by weight: 800-1200 parts of soybean meal containing 43% protein, 1000-1500 parts of rapeseed meal, 1000-1500 parts of rapeseed cake, 1200-2000 parts of fish meal, 500-1000 parts of corn germ meal, 1500-2000 parts of flour, 300-400 parts of soybean oil, 20-40 parts of L-lysine hydrochloride, 10-30 parts of DL-methionine, 1-5 parts of ethoxyquinoline, 20-40 parts of choline chloride, 200-300 parts of calcium dihydrogen phosphate, 20-40 parts of mineral premix, 20-30 parts of vitamin premix, 1000-1500 parts of bentonite, and 9-27 parts of antimicrobial peptide as shown in SEQ ID NO:

1.

2. The grass carp feed according to claim 1 comprises, by weight, 1000 parts of soybean meal containing 43% protein, 1462 parts of rapeseed meal, 1200 parts of rapeseed cake, 1800 parts of fish meal, 800 parts of corn germ meal, 1800 parts of flour, 350 parts of soybean oil, 30 parts of L-lysine hydrochloride, 20 parts of DL-methionine, 3 parts of ethoxyquin, 30 parts of choline chloride, 250 parts of monocalcium phosphate, 30 parts of mineral premix, 25 parts of vitamin premix, 1200 parts of bentonite, and 9 parts of antimicrobial peptide.

3. The grass carp feed according to claim 1 comprises, by weight, 1000 parts of soybean meal containing 43% protein, 1462 parts of rapeseed meal, 1200 parts of rapeseed cake, 1800 parts of fish meal, 800 parts of corn germ meal, 1800 parts of flour, 350 parts of soybean oil, 30 parts of L-lysine hydrochloride, 20 parts of DL-methionine, 3 parts of ethoxyquin, 30 parts of choline chloride, 250 parts of calcium dihydrogen phosphate, 30 parts of mineral premix, 25 parts of vitamin premix, 1200 parts of bentonite, and 18 parts of antimicrobial peptide.

4. The grass carp feed according to claim 1 comprises, by weight, 1000 parts of soybean meal containing 43% protein, 1462 parts of rapeseed meal, 1200 parts of rapeseed cake, 1800 parts of fish meal, 800 parts of corn germ meal, 1800 parts of flour, 350 parts of soybean oil, 30 parts of L-lysine hydrochloride, 20 parts of DL-methionine, 3 parts of ethoxyquin, 30 parts of choline chloride, 250 parts of calcium dihydrogen phosphate, 30 parts of mineral premix, 25 parts of vitamin premix, 1200 parts of bentonite, and 27 parts of antimicrobial peptides.

5. The grass carp feed according to claim 1, wherein the vitamin premix comprises, by weight, 50-100 parts of vitamin B1, 100-150 parts of vitamin B2, 80-150 parts of vitamin B6, 5-15 parts of vitamin B12, 300-400 parts of vitamin D, 2000-3000 parts of vitamin E, 3000-4000 parts of vitamin K; 300-400 parts of nicotinamide, 300-500 parts of calcium pantothenate, 2000-3000 parts of VC phosphate, 500-600 parts of inositol, 10-20 parts of folic acid, 50-100 parts of biotin, 10-15 parts of ethoxyquinoline, 2000-3000 parts of rice husk powder, and 5-10 parts of zeolite powder; Optionally, the vitamin premix includes, by weight, 82 parts of vitamin B1, 128 parts of vitamin B2, 93 parts of vitamin B6, 10 parts of vitamin B12, 329 parts of vitamin D, 2480 parts of vitamin E, 3280 parts of vitamin K; 350 parts of niacinamide, 400 parts of calcium pantothenate, 2600 parts of VC phosphate, 530 parts of inositol, 14 parts of folic acid, 86 parts of biotin, 15 parts of ethoxyquin, 2798 parts of rice husk powder, and 5 parts of zeolite powder.

6. The grass carp feed according to claim 1, wherein the mineral premix comprises, by weight, 30-50 parts of calcium iodate, 200-300 parts of sulfuric acid pentahydrate, 1300-1800 parts of ferrous sulfate monohydrate, 700-800 parts of zinc sulfate monohydrate, 100-200 parts of manganese sulfate monohydrate, 3000-4000 parts of magnesium sulfate monohydrate, 10-20 parts of sodium selenite, 30-50 parts of cobalt chloride, 2000-3000 parts of zeolite powder and 1000-1100 parts of rice husk powder; Optionally, the mineral premix includes, by weight, 50 parts of calcium iodate, 240 parts of sulfuric acid pentahydrate, 1500 parts of ferrous sulfate monohydrate, 740 parts of zinc sulfate monohydrate, 135 parts of manganese sulfate monohydrate, 3500 parts of magnesium sulfate monohydrate, 15 parts of sodium selenite, 40 parts of cobalt chloride, 2700 parts of zeolite powder and 1080 parts of rice husk powder.

7. A method for improving the antioxidant defense ability of grass carp, comprising feeding the grass carp feed as claimed in any one of claims 1 to 7 at 9:00 am and 17:00 pm every day until the young fish are full; and At 20:00 every night, 1 / 3 of the aeration water in the breeding tank was replaced regularly, and the excrement at the bottom of the tank was cleaned with a sewage suction device. Feeding was continued for 60 days.

8. A method for enhancing the intestinal barrier of grass carp, comprising feeding the grass carp feed according to any one of claims 1 to 7 to the young fish at 9:00 a.m. and 17:00 p.m. every day until the young fish are full; and At 20:00 every night, 1 / 3 of the aeration water in the breeding tank was replaced regularly, and the excrement at the bottom of the tank was cleaned with a sewage suction device. Feeding was continued for 60 days.

9. A method for increasing the IL-22 level in serum of grass carp, comprising feeding the grass carp feed as claimed in any one of claims 1 to 7 to the young fish at 9:00 a.m. and 17:00 p.m. every day until the young fish are full; and At 20:00 every night, 1 / 3 of the aeration water in the breeding tank was replaced regularly, and the excrement at the bottom of the tank was cleaned with a sewage suction device. Feeding was continued for 60 days.

10. The use of the grass carp feed according to any one of claims 1 to 6, wherein the use is selected from at least one of the following: Promote the growth of grass carp; Improve the disease resistance of grass carp; Improve the intestinal morphology of grass carp; Enhance the integrity and function of the intestinal barrier of grass carp; Improve the serum T-SOD level of grass carp; Improve the antioxidant defense capacity of grass carp; Promote the expression of claudin c gene in the intestine of grass carp; Promote the expression of occludin gene in the intestine of grass carp; Promote the expression of ZO-2 gene in the intestine of grass carp; Promote the expression of Mucin-2 gene in the intestine of grass carp; Increase the level of IL-22 in grass carp serum; Reduce LPS levels in grass carp serum; Reduce the intestinal permeability of grass carp; Preparation of preparations for preventing enteritis; Preparation of preparations for treating enteritis; Improve the intestinal microbial composition of grass carp; Improve the intestinal microbial diversity of grass carp; Regulate metabolic pathways in grass carp; Regulating interactions between intestinal microorganisms in grass carp.

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