A compound herbal feed additive that promotes growth and enhances immunity in blunt snout bream and its application.

Abstract

This invention belongs to the field of feed technology, specifically relating to a compound herbal feed additive for promoting the growth and enhancing the immunity of bluntnose bream and its application. The invention discloses a compound herbal feed additive, which, by weight, consists of 3-6 parts Astragalus membranaceus, 2-4 parts Lycium barbarum, 1-2 parts Ginseng, and 0.5-1.5 parts Phragmites communis. This compound herbal feed additive can improve fish survival rate, promote fish growth, and enhance fish immunity and disease resistance through the synergistic effect of its components. The compound herbal feed additive can be used to prepare fish feed. Experiments have shown that adding this compound herbal feed additive to fish feed can promote the growth of bluntnose bream and improve its immunity and digestive function.

Description

Technical Field

[0001] This invention belongs to the field of feed technology, specifically relating to a compound herbal feed additive that promotes the growth of blunt snout bream and enhances its immunity, and its application. Background Technology

[0002] Bacterial septicemia caused by Aeromonas hydrophila infection in blunt snout bream occurs from March to November, with a peak period from May to September. It can occur at water temperatures ranging from 9°C to 36°C. The disease can affect fish from fry to adults, with severe outbreaks reaching 100% morbidity and over 95% mortality in fish farms. Symptoms include severe congestion and hemorrhage throughout the fish's body, including the upper and lower jaws, mouth, gill covers, eyes, fins, and sides of the body. Hemorrhage is most severe in the abdomen and head, even affecting the muscles, which become congested and reddish. The fish exhibit protruding eyes, a swollen and red anus, and abdominal distension. Dissection of the abdominal cavity reveals a large amount of clear or bloody ascites. The liver, spleen, and kidneys are enlarged and severely hemorrhagic, and the intestinal mucosa is hemorrhagic and red, exhibiting severe enteritis. Pathological changes under microscopy revealed severe congestion of small blood vessels and capillaries in various organs, diffuse erythrocyte infiltration in multiple tissues; degeneration and necrosis of hepatocytes and pancreatic cells; and degeneration and necrosis of renal tubular epithelial cells.

[0003] Currently, chemical drugs (including antibiotics) are mainly used in the farming of blunt snout bream to prevent bacterial septicemia caused by Aeromonas hydrophila infection. This not only affects the quality and safety of aquatic products, but also damages the ecological environment and seriously hinders the green development of blunt snout bream farming. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a compound herbal feed additive for promoting the growth and enhancing the immunity of blunt snout bream, and its application. By weight, the compound herbal feed additive comprises 3-6 parts Astragalus membranaceus, 2-4 parts Lycium barbarum, 1-2 parts Ginseng, and 0.5-1.5 parts Phragmites communis. This compound herbal feed additive can improve fish survival rate, promote fish growth, enhance fish immunity and disease resistance, and can be used in the preparation of fish feed, showing great application potential.

[0005] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0006] The first aspect of the present invention provides a compound herbal feed additive, which, by weight, is composed of 3 to 6 parts of Astragalus membranaceus, 2 to 4 parts of Lycium barbarum, 1 to 2 parts of Ginseng, and 0.5 to 1.5 parts of Phragmites communis.

[0007] Astragalus: It has a sweet taste and slightly warm nature. It invigorates qi, strengthens the exterior, supports the body's resistance, and eliminates pathogens. As the chief herb in the formula, it has the effects of detoxifying and draining pus, relieving various inflammations, and improving immunity. It plays a major role in resisting hydrophilic aeromonas infection in blunt snout bream and reducing mortality.

[0008] Goji berries: sweet in taste, neutral in nature, and enter the liver and kidney meridians. They have natural antioxidant and enzyme synthesis-promoting biological activities, and have significant effects on improving the body's growth and development, digestion, and immunity. As an auxiliary ingredient in the formula, they enhance the effects of astragalus.

[0009] Ginseng: It tastes sweet and slightly bitter, and is slightly warm in nature; it enters the lung, spleen, heart, and kidney meridians. It replenishes qi and blood, calms the mind and improves intelligence, restores yang and rescues from collapse, and strengthens the spleen and lungs. As an adjuvant, it plays an important role in inhibiting inflammation and improving antioxidant capacity and immunity.

[0010] Reed rhizome: It is cold in nature and sweet in taste. It enters the lung and stomach meridians and has the effects of clearing heat and purging fire, promoting body fluid and quenching thirst, relieving irritability, stopping vomiting, and promoting diuresis. It also has the effect of protecting the liver and is used as an adjuvant.

[0011] Furthermore, the compound herbal feed additive is prepared by the following steps: grinding each herbal raw material into powder and passing it through a 200-mesh sieve; weighing the powder of each herbal raw material after sieving according to the proportion; and mixing them to obtain a compound herbal feed additive.

[0012] A second aspect of the present invention provides the application of the above-described compound herbal feed additive in promoting fish growth.

[0013] Furthermore, the compound herbal feed additive is used to improve fish survival rate, promote fish weight gain, increase fish growth rate, or improve fish digestion and absorption of feed.

[0014] A third aspect of the present invention provides the application of the above-described compound herbal feed additive in improving the immunity of fish.

[0015] Furthermore, the compound herbal feed additive is used to improve the activity of digestive enzymes in fish or improve the intestinal tissue of fish.

[0016] The fourth aspect of this invention provides the application of the above-described compound herbal feed additive in improving the disease resistance of fish to Aeromonas hydrophila infection.

[0017] The fifth aspect of this invention provides the application of the above-described compound herbal feed additive in the preparation of fish feed.

[0018] The sixth aspect of the present invention provides a fish feed, which is composed of the above-mentioned compound herbal feed additive and conventional aquatic feed.

[0019] Furthermore, based on the mass of the fish feed, the compound herbal feed additive accounts for 1% to 4% of the fish feed.

[0020] The seventh aspect of the present invention provides a method for promoting fish growth, improving fish immunity, or enhancing fish resistance to Aeromonas hydrophila infection, specifically by feeding fish with the fish feed described above for more than 90 consecutive days.

[0021] Furthermore, the fish feed can improve the quality of the head, kidneys, spleen, gills, liver, or intestines of fish. IgM , C3 , TNF-α and IL-1β Gene expression.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] This invention discloses a compound herbal feed additive that promotes the growth and enhances the immunity of blunt snout bream. By weight, the compound herbal feed additive consists of 3-6 parts Astragalus membranaceus, 2-4 parts Lycium barbarum, 1-2 parts Ginseng, and 0.5-1.5 parts Phragmites communis. This compound herbal feed additive can improve fish survival rate, promote fish growth, and enhance fish immunity and disease resistance through the synergistic effect of its components. The compound herbal feed additive can be used to prepare fish feed. Experiments have shown that when this compound herbal feed additive is used to prepare fish feed, it can significantly improve the intestinal and liver tissue structure of blunt snout bream, increase the activity of intestinal digestive enzymes, promote the growth of blunt snout bream, and significantly increase the expression of immune-related genes and the activity of immune-related enzymes in serum, thereby enhancing the disease resistance of blunt snout bream and reducing the mortality rate after Aeromonas hydrophila infection. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The effects of different fish diets on the growth performance and intestinal digestive enzyme activity of blunt snout bream; Figure a shows the weight gain rate; Figure b shows the specific growth rate; Figure c shows the feed conversion ratio; Figure d shows the intestinal pancreatic enzyme activity; Figure e shows the intestinal lipase activity; Figure f shows the intestinal amylase activity; the same lowercase letters indicate no significant difference. P> 0.05; Different lowercase letters indicate significant differences. P <0.05.

[0026] Figure 2The intestinal tissue of blunt snout bream in the control group (fed with fish feed of control ratio 15) is shown. The blue lines are measurement lines, and the yellow parts indicate the measured length of intestinal villi, width of intestinal villi, and thickness of intestinal muscle layer.

[0027] Figure 3 The image shows the intestinal tissue of blunt snout bream in the T1 experimental group (fed the fish feed of Example 6). The blue lines are measurement lines, and the yellow parts indicate the measured length of the intestinal villi, the width of the intestinal villi, and the thickness of the intestinal muscle layer.

[0028] Figure 4 The image shows the intestinal tissue of blunt snout bream in the T2 experimental group (fed the fish feed of Example 5). The blue lines are measurement lines, and the yellow parts indicate the measured length of the intestinal villi, the width of the intestinal villi, and the thickness of the intestinal muscle layer.

[0029] Figure 5 The image shows the intestinal tissue of blunt snout bream in the T3 experimental group (fed the fish feed of Example 7). The blue lines are measurement lines, and the yellow parts indicate the measured length of the intestinal villi, the width of the intestinal villi, and the thickness of the intestinal muscle layer.

[0030] Figure 6 Statistical graphs showing the effects of different fish diets on the intestinal tissue of blunt snout bream; Graph A shows intestinal villus length; Graph B shows intestinal villus width; Graph C shows intestinal muscle layer thickness; identical lowercase letters indicate no significant difference. P> 0.05; Different lowercase letters indicate significant differences. P <0.05.

[0031] Figure 7 To illustrate the effects of different fish diets on the liver and spleen of blunt snout bream, C represents the control group fed with Example 15, T1 represents the experimental group fed with Example 6, T2 represents the experimental group fed with Example 5, and T3 represents the experimental group fed with Example 7; Figure a shows the liver of the control group; Figure b shows the liver of the T1 experimental group; Figure c shows the liver of the T2 experimental group; Figure d shows the liver of the T3 experimental group; Figure e shows the liver body mass index; Figure f shows the spleen body mass index; the same lowercase letters indicate no significant difference. P> 0.05; Different lowercase letters indicate significant differences. P <0.05, the black arrow indicates the sinusoidal space of the liver.

[0032] Figure 8 The effects of different fish diets on the serum immune-related enzyme activities of blunt snout bream; Figure A shows lysozyme (LYS) content; Figure B shows acid phosphatase (ACP) activity; Figure C shows alkaline phosphatase (AKP) activity; Figure D shows catalase (CAT) activity; Figure E shows superoxide dismutase (SOD) activity; Figure F shows glutathione peroxidase (GSH-PX) activity; identical lowercase letters indicate no significant difference. P>0.05; Different lowercase letters indicate significant differences. P <0.05.

[0033] Figure 9 To illustrate the effects of different fish diets on the expression of immune-related genes in blunt snout bream, C represents the control group fed with Example 15, T1 represents the experimental group fed with Example 6, T2 represents the experimental group fed with Example 5, and T3 represents the experimental group fed with Example 7; Figure a shows... IgM Relative gene expression levels; Figure b shows... C3 Relative gene expression levels; Figure c shows... TNF-α Relative gene expression levels; d is... IL-1β Relative gene expression levels; identical lowercase letters indicate no significant difference. P> 0.05; Different lowercase letters indicate significant differences. P <0.05.

[0034] Figure 10 The effect of adding compound traditional Chinese medicine to feed on the disease resistance of blunt snout bream was investigated. C represents the control group fed with example 15, T1 represents the experimental group fed with example 6, T2 represents the experimental group fed with example 5, and T3 represents the experimental group fed with example 7. Detailed Implementation

[0035] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0036] Aeromonas hydrophila in this invention ( Aeromonashydrophila The strain was isolated and identified by the inventors' research group from septicemic crucian carp. For strain information, please refer to Xia H, Liu L, Zhou W, et al. Immune response to Aeromonas hydrophila and molecular characterization of polymericimmunoglobulin receptor in juvenile Megalobrama amblycephala . Fish ShellfishImmunol. 2024;153:109821.

[0037] Currently, chemical drugs (including antibiotics) are mainly used in the farming of blunt snout bream to prevent bacterial septicemia caused by Aeromonas hydrophila infection. This not only affects the quality and safety of aquatic products, but also damages the ecological environment and seriously hinders the green development of blunt snout bream farming.

[0038] This invention provides a compound herbal feed additive that promotes the growth and enhances the immunity of blunt snout bream. By weight, the compound herbal feed additive consists of 3-6 parts Astragalus membranaceus, 2-4 parts Lycium barbarum, 1-2 parts Ginseng, and 0.5-1.5 parts Phragmites communis. This compound herbal feed additive can improve fish survival rate, promote fish growth, and enhance fish immunity and disease resistance through the synergistic effect of its components, and can be used to prepare fish feed.

[0039] Example 1: A compound traditional Chinese medicine feed additive, prepared by the following steps:

[0040] Astragalus ( Astragalus membranaceus ), wolfberry ( Lycium barbarum L. ), Ginseng ( Panax ginsengC. A. Mey. ), reed rhizome ( Phragmitis rhizoma After being ground into powder, the mixture was passed through a 200-mesh sieve and then mixed in a mass ratio of 4.5:3:1.5:1 to obtain the compound herbal feed additive. The composition and nutritional level of this compound herbal feed additive are shown in Table 1.

[0041] Table 1. Composition and nutritional levels of compound Chinese herbal medicines.

[0042]

[0043] Example 2: A compound traditional Chinese medicine feed additive, prepared by the following steps:

[0044] Astragalus, wolfberry, ginseng, and reed rhizome are ground into powder and passed through a 200-mesh sieve. They are then mixed in a mass ratio of 3:2:1:0.5 to obtain the compound Chinese herbal feed additive.

[0045] Example 3: A compound traditional Chinese medicine feed additive, prepared by the following steps:

[0046] Astragalus, wolfberry, ginseng, and reed rhizome are ground into powder and passed through a 200-mesh sieve. They are then mixed in a mass ratio of 5:2:1.5:1 to obtain the compound Chinese herbal feed additive.

[0047] Example 4: A compound traditional Chinese medicine feed additive, prepared by the following steps:

[0048] Astragalus, wolfberry, ginseng, and reed rhizome are ground into powder and passed through a 200-mesh sieve. They are then mixed in a mass ratio of 6:4:2:1.5 to obtain the compound Chinese herbal feed additive.

[0049] Examples 1-4 have similar effects. For the convenience of subsequent discussion and reference, the compound Chinese herbal medicine feed additive prepared in Example 1 is used as an example to conduct the following experiments.

[0050] Example 5: A fish feed prepared by the following steps:

[0051] 2000g of soybean meal (CP44%), 4000g of rapeseed meal, and 1550g of wheat were pulverized and passed through a 200-mesh sieve. Then, 200g of the compound herbal feed additive prepared in Example 1, 1100g of fish meal (CP67%), 600g of soybean oil, 300g of compound feed premix, 230g of bentonite, 17g of choline chloride, 1g of preservative, and 2g of antioxidant were added. After thorough mixing, a fish feed was prepared using a pellet mill. The mass percentage of the compound herbal feed additive prepared in Example 1 in this fish feed was 2%.

[0052] Example 6: A fish feed prepared by the following steps:

[0053] 2000g of soybean meal (CP44%), 4000g of rapeseed meal, and 1650g of wheat were pulverized and passed through a 200-mesh sieve. Then, 100g of the compound herbal feed additive prepared in Example 1, 1100g of fish meal (CP67%), 600g of soybean oil, 300g of compound feed premix, 230g of bentonite, 17g of choline chloride, 1g of preservative, and 2g of antioxidant were added. After thorough mixing, a fish feed was prepared using a pellet mill. The mass percentage of the compound herbal feed additive prepared in Example 1 in this fish feed was 1%.

[0054] Example 7: A fish feed prepared by the following steps:

[0055] 2000g of soybean meal (CP44%), 4000g of rapeseed meal, and 1350g of wheat were pulverized and passed through a 200-mesh sieve. Then, 400g of the compound herbal feed additive prepared in Example 1, 1100g of fish meal (CP67%), 600g of soybean oil, 300g of compound feed premix, 230g of bentonite, 17g of choline chloride, 1g of preservative, and 2g of antioxidant were added. After thorough mixing, a fish feed was prepared using a pellet mill. The mass percentage of the compound herbal feed additive prepared in Example 1 in this fish feed was 4%.

[0056] Comparative Example 1: A compound traditional Chinese medicine feed additive, prepared by the following steps:

[0057] Astragalus, wolfberry, ginseng and reed rhizome are ground into powder and passed through a 200-mesh sieve. They are then mixed in a mass ratio of 1:1:1:1 to obtain the compound Chinese herbal feed additive.

[0058] Comparative Example 2: A compound traditional Chinese medicine feed additive, prepared by the following steps:

[0059] Goji berries, ginseng, and reed rhizome are ground into powder and passed through a 200-mesh sieve. They are then mixed in a mass ratio of 3:1.5:1 to obtain the compound herbal feed additive.

[0060] Comparative Example 3: A compound traditional Chinese medicine feed additive, prepared by the following steps:

[0061] Astragalus, ginseng, and reed rhizome were ground into powder and passed through a 200-mesh sieve. They were then mixed in a mass ratio of 4.5:1.5:1 to obtain the compound herbal feed additive.

[0062] Comparative Example 4: A compound traditional Chinese medicine feed additive, prepared by the following steps:

[0063] Astragalus, wolfberry and reed rhizome are ground into powder and passed through a 200-mesh sieve. They are then mixed in a mass ratio of 4.5:3:1 to obtain the compound Chinese herbal feed additive.

[0064] Comparative Example 5: A compound traditional Chinese medicine feed additive, prepared by the following steps:

[0065] Astragalus, wolfberry and ginseng are ground into powder and passed through a 200-mesh sieve. They are then mixed in a mass ratio of 4.5:3:1.5 to obtain the compound Chinese herbal feed additive.

[0066] Comparative Example 6: A fish feed prepared by the following steps:

[0067] 2000g of soybean meal (CP44%), 4000g of rapeseed meal, and 1550g of wheat were pulverized and passed through a 200-mesh sieve. Then, 200g of the compound herbal feed additive prepared in Comparative Example 1, 1100g of fish meal (CP67%), 600g of soybean oil, 300g of compound feed premix, 230g of bentonite, 17g of choline chloride, 1g of preservative, and 2g of antioxidant were added. After thorough mixing, a fish feed was prepared using a pellet mill. The mass percentage of the compound herbal feed additive prepared in Comparative Example 1 in this fish feed was 2%.

[0068] Comparative Example 7: A fish feed prepared by the following steps:

[0069] 2000g of soybean meal (CP44%), 4000g of rapeseed meal, and 1550g of wheat were crushed and passed through a 200-mesh sieve. Then, 200g of the compound herbal feed additive prepared in Comparative Example 2, 1100g of fish meal (CP67%), 600g of soybean oil, 300g of compound feed premix, 230g of bentonite, 17g of choline chloride, 1g of preservative, and 2g of antioxidant were added. After thorough mixing, a fish feed was prepared using a pellet mill. The mass percentage of the compound herbal feed additive prepared in Comparative Example 2 in this fish feed was 2%.

[0070] Comparative Example 8: A fish feed prepared by the following steps:

[0071] 2000g of soybean meal (CP44%), 4000g of rapeseed meal, and 1550g of wheat were pulverized and passed through a 200-mesh sieve. Then, 200g of the compound herbal feed additive prepared in Comparative Example 3, 1100g of fish meal (CP67%), 600g of soybean oil, 300g of compound feed premix, 230g of bentonite, 17g of choline chloride, 1g of preservative, and 2g of antioxidant were added. After thorough mixing, a fish feed was prepared using a pellet mill. The mass percentage of the compound herbal feed additive prepared in Comparative Example 3 in this fish feed was 2%.

[0072] Comparative Example 9: A fish feed prepared by the following steps:

[0073] 2000g of soybean meal (CP44%), 4000g of rapeseed meal, and 1550g of wheat were crushed and passed through a 200-mesh sieve. Then, 200g of the compound herbal feed additive prepared in Comparative Example 4, 1100g of fish meal (CP67%), 600g of soybean oil, 300g of compound feed premix, 230g of bentonite, 17g of choline chloride, 1g of preservative, and 2g of antioxidant were added. After thorough mixing, a fish feed was prepared using a pellet mill. The mass percentage of the compound herbal feed additive prepared in Comparative Example 4 in this fish feed was 2%.

[0074] Comparative Example 10: A fish feed prepared by the following steps:

[0075] 2000g of soybean meal (CP44%), 4000g of rapeseed meal, and 1550g of wheat were pulverized and passed through a 200-mesh sieve. Then, 200g of the compound herbal feed additive prepared in Comparative Example 5, 1100g of fish meal (CP67%), 600g of soybean oil, 300g of compound feed premix, 230g of bentonite, 17g of choline chloride, 1g of preservative, and 2g of antioxidant were added. After thorough mixing, a fish feed was prepared using a pellet mill. The mass percentage of the compound herbal feed additive prepared in Comparative Example 5 in this fish feed was 2%.

[0076] Comparative Example 11: A fish feed prepared by the following steps:

[0077] After pulverizing Astragalus membranaceus, pass it through a 200-mesh sieve to obtain Astragalus membranaceus powder.

[0078] 2000g soybean meal (CP44%), 4000g rapeseed meal, and 1350g wheat were crushed and passed through a 200-mesh sieve. Then, 200g astragalus powder, 1100g fish meal (CP67%), 600g soybean oil, 300g compound feed premix, 230g bentonite, 17g choline chloride, 1g preservative, and 2g antioxidant were added and mixed thoroughly. The mixture was then granulated to produce a fish feed. The astragalus powder contained in this fish feed accounted for 2% of the total mass.

[0079] Comparative Example 12: A fish feed prepared by the following steps:

[0080] After crushing the goji berries, pass them through a 200-mesh sieve to obtain goji berry powder.

[0081] 2000g soybean meal (CP44%), 4000g rapeseed meal, and 1350g wheat were crushed and passed through a 200-mesh sieve. Then, 200g wolfberry powder, 1100g fish meal (CP67%), 600g soybean oil, 300g compound feed premix, 230g bentonite, 17g choline chloride, 1g preservative, and 2g antioxidant were added and mixed thoroughly. The mixture was then granulated to produce a fish feed. The wolfberry powder contained in this fish feed accounted for 2% of the total mass.

[0082] Comparative Example 13: A fish feed prepared by the following steps:

[0083] Ginseng is crushed and passed through a 200-mesh sieve to obtain ginseng powder.

[0084] 2000g soybean meal (CP44%), 4000g rapeseed meal, and 1350g wheat were crushed and passed through a 200-mesh sieve. Then, 200g ginseng powder, 1100g fish meal (CP67%), 600g soybean oil, 300g compound feed premix, 230g bentonite, 17g choline chloride, 1g preservative, and 2g antioxidant were added and mixed thoroughly. The mixture was then granulated to produce a fish feed. The ginseng powder contained in this fish feed accounted for 2% of the total mass.

[0085] Comparative Example 14: A fish feed prepared by the following steps:

[0086] After crushing the reed roots, pass them through a 200-mesh sieve to obtain reed root powder.

[0087] 2000g soybean meal (CP44%), 4000g rapeseed meal, and 1350g wheat were crushed and passed through a 200-mesh sieve. Then, 200g reed rhizome powder, 1100g fish meal (CP67%), 600g soybean oil, 300g compound feed premix, 230g bentonite, 17g choline chloride, 1g preservative, and 2g antioxidant were added and mixed thoroughly. The mixture was then granulated to produce a fish feed. The reed rhizome powder contained in this fish feed accounted for 2% of the total mass.

[0088] Comparative Example 15: A fish feed prepared by the following steps:

[0089] 2000g soybean meal (CP44%), 4000g rapeseed meal, and 1750g wheat were crushed and passed through a 200-mesh sieve. Then, 1100g fish meal (CP67%), 600g soybean oil, 300g compound feed premix, 230g bentonite, 17g choline chloride, 1g preservative, and 2g antioxidant were added and mixed thoroughly. The mixture was then granulated to produce a fish feed.

[0090] Experiment Example 1: Effects of Compound Traditional Chinese Medicine Feed Additives on Fish Survival Rate

[0091] The experimental fish, *Brucea buergeriana*, weighing 42-46g, were obtained from the fish breeding base of Hunan University of Arts and Sciences. Before sampling, the experimental fish were temporarily housed in a laboratory recirculating aquaculture system for two weeks. During the rearing process, the fish were aerated 24 hours a day to maintain sufficient dissolved oxygen in the water, ranging from 5.8 μg / mL to 6.2 μg / mL, at a water temperature of 26℃ to 30℃, and were fed an appropriate amount of adult floating feed.

[0092] After two weeks of temporary rearing and acclimatization, the bluntnose bream were fed the fish feeds described in Examples 5-7 and Comparative Examples 6-15 of this invention at 8:30 AM and 5:00 PM daily. Each feeding consisted of 2% of the fish's body weight. The water was changed once daily, replacing one-third of the total water volume. Waste in the rearing tanks was cleaned, and the tanks were aerated 24 hours a day. This feeding and rearing continued for 90 days. After 90 days, 30 bluntnose bream were randomly selected and injected intraperitoneally with 0.1 mL of a live bacteria count of 1×10⁻⁶. 7 The fish were infected with CFU / mL Aeromonas hydrophila culture, and the cumulative mortality rate of blunt snout bream 14 days after infection was counted to explore the effects of different compound herbal feed additives on fish.

[0093] Cumulative mortality rate (%) = Total number of fish that died in 14 days / 30 × 100%;

[0094] Survival rate (%) = Total number of fish surviving after 14 days / 30 × 100%;

[0095] The test results are shown in Table 2.

[0096] Table 2. Fish mortality rate with compound traditional Chinese medicine feed additives

[0097]

[0098] Since Examples 5-7 can significantly improve the survival rate of fish, this invention uses fish feed without added compound Chinese herbal medicine feed additives (Comparative Example 15) as the control group and fish feed with different amounts of added compound Chinese herbal medicine feed additives prepared in Examples 5-7 as the experimental group to conduct in-depth research.

[0099] I. Experimental Methods

[0100] 1. Measurement of growth indicators

[0101] At the start of the experiment, the initial weight of the experimental fish was measured. After 90 days of rearing, the final weight of the experimental fish, the weight of the liver, and the weight of the spleen were measured. The amount of feed consumed during the rearing process was also recorded. The growth-related indicators of the experimental fish were calculated using the following formula.

[0102] Weight gain rate (WGR) = (W1 - W0) / W0 × 100%;

[0103] Special growth rate (SGR) = (lnW1 – lnW0) / T × 100%;

[0104] Feed coefficient (FCR) = Feed consumption / (W1 - W0) × 100%;

[0105] Hepatosomatic index (HSI) = G1 / W1 × 100%;

[0106] Spleen index (SPI) = G2 / W1 × 100%;

[0107] W0 represents the initial average weight of the fish at the start of the experiment, W1 represents the average weight of the fish at the end of the experiment, T represents the culture time (d), G1 represents the average weight of the liver of the experimental fish, and G2 represents the average weight of the spleen of the experimental fish.

[0108] 2. Observation of liver and intestinal tissue sections

[0109] Fish were fed with the fish diets prepared in Examples 5-7 and Comparative Example 15 for 90 days. Five silver carp were randomly selected from the control group and each experimental group. After anesthesia with MS-222, blood was collected from the tail vein with a syringe. Liver and intestinal tissues of 3-5 mm were taken and fixed in Bouin's solution. After dehydration, paraffin infiltration, embedding, sectioning, spreading, drying, HE staining, and mounting, the slides were observed under a microscope and photographed.

[0110] 3. Determination of serum antioxidant and immune-related enzyme activities

[0111] Fish were fed with the diets prepared in Examples 5-7 and Comparative Example 15 for 90 days. Five blunt snout bream were randomly selected from the experimental and control groups. After anesthesia with MS-222, 1 mL of blood was collected from the tail vein using a sterile syringe. The blood was then centrifuged at 4°C and 4000 r / min for 10 min to separate the serum, which was then stored at 4°C for later use. Using a kit purchased from Nanjing Jiancheng Biotechnology Institute, and following the instructions, the levels of lysozyme (Lys), total superoxide dismutase (T-SOD), catalase (CAT), acid phosphatase (ACP), alkaline phosphatase (AKP), and glutathione peroxidase (GSH-PX) in the serum of the blunt snout bream were detected using an ELISA reader.

[0112] 4. Assay of intestinal digestion-related enzyme activity

[0113] Five blunt snout bream were randomly selected from each experimental group and control group. After anesthesia with MS-222 and blood collection, 2g of intestinal tissue was taken from each group and added to pre-cooled physiological saline (4°C) at a ratio of 1:9 (w / v). The mixture was homogenized in an ice-water bath for 30 min, and then centrifuged at 3000 r / min for 10 min at 4°C. The supernatant was separated, and the activities of amylase (AMS), lipase (LPS), and trypsin (TRS) in the intestine of blunt snout bream were measured using a microplate reader according to the instructions of the kit from Nanjing Jiancheng Bioengineering Institute.

[0114] 5. Mortality statistics of blunt snout bream after infection with Aeromonas hydrophila

[0115] Aeromonas hydrophila was isolated from diseased blunt snout bream. Aeromonas hydrophilaSingle clones of the strain were selected and inoculated into Luriabroth (LB) liquid medium and cultured overnight at 28°C with shaking. Preliminary experiments indicated that the median lethal concentration (LD50) of *Aeromonas hydrophila* for *Brucea bream* was approximately 1 × 10⁻⁶. 7 CFU / mL. After feeding the fish with the diets prepared in Examples 5-7 and Comparative Example 15 for 90 days, the blunt snout bream were intraperitoneally injected with 0.1 mL (1×10⁻⁶ CFU / mL) of Aeromonas hydrophila. 7 The researchers infected the fish with Aeromonas hydrophila (CFU / mL) and calculated the cumulative mortality rate of different experimental groups of crucian carp on days 1, 3, 5, 7 and 14 after infection.

[0116] 6. qRT-PCR detection of immune-related genes in blunt snout bream

[0117] Fish were fed with the feeds prepared in Examples 5-7 and Comparative Example 15 for 90 days. Five blunt snout bream were collected from each of the control and experimental groups. After anesthesia with MS-222 and blood collection from the tail vein, 100 mg each of head kidney, liver, spleen, intestine, and gills were collected. These were ground into powder in a mortar using liquid nitrogen. RNA was extracted from each tissue using an RNA extraction kit (Simgen) according to its instructions, and cDNA templates were obtained by reverse transcription. Based on the blunt snout bream interleukin-1β (IL-1β), IL-1β Tumor necrosis factor-alpha (TNF-α) TNF-α Immunoglobulin M (Immunoglobulin M) IgM ) and complement factor C3 C3 ) Design primers for real-time quantitative PCR (qRT-PCR) based on gene sequences, in order to β-actin The gene was used as an internal reference gene. The qRT-PCR kit (TaKaRa) was used to detect the blunt snout bream according to its instructions. IL-1β , TNF-α , IgM and C3 Gene expression in different tissues, through 2 -ΔΔCt The relative expression levels of immune-related genes in various tissues of blunt snout bream were calculated.

[0118] Table 3 Primers for real-time quantitative PCR amplification of immune-related genes in blunt snout bream

[0119]

[0120] 7. Data Analysis

[0121] Experimental data are expressed as mean ± standard deviation, and one-way ANOVA was performed using SPSS 15.0 statistical software. P <0.05 indicates a significant difference. P <0.01 indicates a highly significant difference.

[0122] II. Experimental Results

[0123] 1. Determination of nutritional levels in different fish feeds

[0124] Table 4 Nutritional Level Measurement

[0125]

[0126] As can be seen from Table 4, there were no significant differences in crude protein, crude fat and total energy between Examples 5, 6 and 7 and Comparative Example 15 (control group), and total nitrogen and total energy were in balance.

[0127] 2. Effects of different fish diets on growth performance and intestinal digestive enzyme activity in blunt snout bream

[0128] Weight gain rate refers to the percentage increase in fish weight within a certain period of time. It reflects the growth rate of fish. The higher the weight gain rate, the faster the fish grows and the better the aquaculture benefits. Specific growth rate refers to the logarithmic growth rate of fish weight per unit time. It can more accurately reflect the growth performance of fish and is especially suitable for comparing fish with different initial weights. Feed conversion ratio (FCR) refers to the amount of feed required for fish to reach a certain weight gain. The lower the FCR, the higher the feed utilization efficiency and the lower the aquaculture cost.

[0129] like Figure 1 As shown in Figures a to c, the weight gain rate and specific growth rate of the blunt snout bream in the experimental groups were higher than those in the control group. The blunt snout bream fed with Example 5 had the highest weight gain rate and specific growth rate. The feed conversion ratio of all experimental groups was lower than that of the control group, and the feed conversion ratio of the group fed with Example 5 was the lowest.

[0130] The higher the activity of amylase, lipase and trypsin in the intestine of blunt snout bream, the more efficiently the bream can digest starchy feeds and break down fats and proteins, thus better absorbing and utilizing the energy, nutrients, fat and protein components in the feed. This can be used to comprehensively evaluate the digestive function and nutrient absorption capacity of blunt snout bream.

[0131] like Figure 1As shown in Figures d-f, the intestinal trypsin and lipase activities of the blunt snout bream in feeding Examples 5 and 6 were significantly higher than those in the control group, with the highest TRS and LPS activities in the feeding Example 6 group. The intestinal TRS activity of the blunt snout bream in the feeding Example 7 group was significantly lower than that in the control group, while the intestinal LPS activity showed no significant difference from the control group. The intestinal amylase activity of the blunt snout bream in all experimental groups was significantly higher than that in the control group, with the highest AMS activity in the feeding Example 5 group.

[0132] 3. Effects of different fish diets on the intestinal tissue of blunt snout bream

[0133] like Figures 2-6 As shown, the length of the intestinal villi and the thickness of the intestinal muscle layer in all experimental groups were significantly higher than those in the control group. The experimental group fed with Example 6 had the longest intestinal villi, while the experimental group fed with Example 5 had the thickest intestinal muscle layer. The width of the intestinal villi in the bream fed with Example 6 was significantly greater than that in the control group, while the width of the intestinal villi in the bream fed with Examples 5 and 7 was smaller than that in the control group.

[0134] 4. Effects of different fish diets on the liver and spleen of blunt snout bream

[0135] The liver body index is the ratio of a fish's liver weight to its body weight. It reflects the relative size of the liver and is usually used to assess a fish's nutritional reserves and health status. A higher liver body index may mean that the liver stores more nutrients. The spleen body index is the ratio of a fish's spleen weight to its body weight. It reflects the relative size of the spleen and is usually used to assess a fish's immune status. A higher spleen body index indicates that the fish's immune system is more active.

[0136] like Figure 7 As shown, the sinusoidal spaces in the livers of blunt snout bream in the control group were widened, while those in the experimental group fed with Example 6 were narrowed, and those in the experimental groups fed with Examples 5 and 7 disappeared. The liver body mass index of blunt snout bream in all experimental groups was significantly higher than that in the control group. The spleen body mass index of blunt snout bream was significantly higher in the experimental groups fed with Examples 5 and 6 than in the control group, with the spleen and liver body mass indices being the highest in the experimental group fed with Example 5. However, the spleen body mass index of blunt snout bream in the experimental group fed with Example 7 was significantly lower than that in the control group.

[0137] 5. Effects of different fish diets on the activity of serum immune-related enzymes in blunt snout bream

[0138] like Figure 8As shown, the serum lysozyme content, superoxide dismutase, catalase, glutathione peroxidase, acid phosphatase, and alkaline phosphatase activities in the experimental group of blunt snout bream were significantly higher than those in the control group. In the experimental group fed Example 6, the serum Lys content, SOD, CAT, and GSH-PX activities were the highest. The serum ACP and AKP activities in blunt snout bream gradually increased with the increase of the compound herbal feed additive, with the highest ACP and AKP activities observed in the serum of blunt snout bream fed Example 7.

[0139] 6. Effects of different fish diets on the expression of immune-related genes in blunt snout bream

[0140] like Figure 9 As shown, the bluntnose bream in the experimental group IgM , C3 , TNF-α and IL-1β The gene expression levels in the head kidney, spleen, gills, liver, and intestine were significantly higher than those in the control group. In the head kidney, liver, and intestine... IgM Gene expression levels were highest in the experimental group fed Example 5, and lower in the spleen and gills of the experimental group fed Example 6. IgM Gene expression levels were highest in the head kidney, spleen, gills, and intestines. C3 Gene expression levels were highest in the experimental group fed with Example 5, and in the liver C3 Gene expression levels were highest in the experimental group fed according to Example 6. (Head kidney, spleen, and intestine) TNF-α Gene expression levels were highest in the experimental group fed Example 5, and also highest in the gills and liver. TNF-α Gene expression levels were highest in the experimental group fed according to Example 6. In the head kidney, spleen, and gills... IL-6 Gene expression levels were highest in the experimental group fed Example 5, particularly in the liver and intestines. IL-6 Gene expression levels were highest in the experimental group fed Example 6.

[0141] 7. Effects of different fish diets on the disease resistance of blunt snout bream

[0142] like Figure 10 As shown, after *Aeromonas hydrophila* infection of blunt snout bream, the cumulative mortality rate of both the control and experimental groups gradually increased, reaching its maximum on day 7 post-infection. However, the mortality rate of the experimental groups was significantly lower than that of the control group. On day 7 after infection with *Aeromonas hydrophila*, there was no significant difference in the cumulative mortality rate between the experimental groups fed with *Example 5* and *Example 7*, and the cumulative mortality rate of the experimental group fed with *Example 5* was the lowest.

[0143] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0144] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A compound traditional Chinese medicine feed additive, characterized in that, The compound herbal feed additive is used to promote fish growth, improve fish immunity, and enhance fish resistance to Aeromonas hydrophila infection. By weight, the compound herbal feed additive consists of 3 to 6 parts Astragalus membranaceus, 2 to 4 parts Lycium barbarum, 1 to 2 parts Ginseng, and 0.5 to 1.5 parts Phragmites communis.

2. The compound herbal feed additive according to claim 1, characterized in that, The compound herbal feed additive is prepared by the following steps: grinding each herbal raw material into powder and passing it through a 200-mesh sieve; weighing the powder of each herbal raw material after sieving according to the proportion; and mixing them to obtain the compound herbal feed additive.

3. The application of the compound herbal feed additive of claim 1 in the preparation of fish feed.

4. A fish feed, characterized in that, The fish feed consists of the compound herbal feed additive as described in claim 1 and conventional aquatic feed.

5. The fish feed according to claim 4, characterized in that, Based on the mass of the fish feed, the compound herbal feed additive accounts for 1% to 4% of the fish feed.

Images