Compound Chinese herbal medicine feed additive for promoting growth of megalobrama amblycephala and improving immunity and application of compound Chinese herbal medicine feed additive

Abstract

The invention belongs to the technical field of feed, and particularly relates to a compound Chinese herbal medicine feed additive for promoting growth of megalobrama amblycephala and improving immunity and application thereof. The invention discloses a compound Chinese herbal medicine feed additive which is prepared from the following components in parts by weight: 3-6 parts of radix astragali, 2-4 parts of fructus lycii, 1-2 parts of radix ginseng and 0.5-1.5 parts of rhizoma phragmitis. The compound Chinese herbal medicine feed additive can improve the survival rate of fishes, promote the growth of the fishes and improve the immunity and disease resistance of the fishes through the synergistic effect of all the components. The compound Chinese herbal medicine feed additive can be used for preparing fish feed, and experiments prove that after the compound Chinese herbal medicine feed additive is added into the fish feed, the growth of megalobrama amblycephala can be promoted, and the immunity and digestive function of the megalobrama amblycephala can be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of feed, and particularly relates to a compound Chinese herbal medicine feed additive capable of promoting the growth of Megalobrama amblycephala and improving immunity, and application thereof. Background Art

[0002] Aeromonas hydrophila causes bacterial septicemia in bream (Gallfish) from March to November, peaking from May to September. It can occur in water temperatures ranging from 9°C to 36°C. The infection can affect fish from fingerling to adult fish, with incidence rates reaching 100% and mortality rates exceeding 95% in severely affected fish farms. Symptoms include severe congestion and bleeding throughout the body, including in the upper and lower jaws, mouth, gill covers, eyes, fins, and flanks, with bleeding most severe in the abdomen and head. Even the muscles become congested and red. The fish also exhibit bulging eyes, a red and swollen anus, and a distended abdomen. Opening the abdominal cavity reveals large amounts of clear or bloody ascites. The liver, spleen, and kidneys are enlarged and severely bleeding, and the intestinal mucosa is bleeding and red, suggesting severe enteritis. Pathological changes under the microscope show severe congestion of small blood vessels and capillaries in various organs, diffuse red blood cell infiltration in multiple tissues; degeneration and necrosis of liver cells and pancreatic cells; degeneration and necrosis of renal tubular epithelial cells.

[0003] At present, chemical drugs (including antibiotics) are mainly used in the aquaculture of bighead carp to prevent and treat bacterial sepsis caused by Aeromonas hydrophila infection. This not only affects the quality and safety of aquatic products, but also damages the ecological environment, seriously hindering the green development of the aquaculture industry of bighead carp. Summary of the Invention

[0004] To address the above issues, the present invention provides a compound Chinese herbal feed additive for promoting the growth and immunity of Megalobrama amblycephala, and its use. The compound Chinese herbal feed additive comprises, by weight, 3 to 6 parts of astragalus, 2 to 4 parts of wolfberry, 1 to 2 parts of ginseng, and 0.5 to 1.5 parts of reed rhizome. This compound Chinese herbal feed additive can improve fish survival rate, promote fish growth, and enhance fish immunity and disease resistance. It can be used in the preparation of fish feed and has great application potential.

[0005] To achieve the above purpose, the specific technical solutions of the present invention are as follows: A first aspect of the present invention provides a compound Chinese herbal feed additive, which consists of 3 to 6 parts of astragalus, 2 to 4 parts of wolfberry, 1 to 2 parts of ginseng and 0.5 to 1.5 parts of reed root, based on weight.

[0006] Astragalus: sweet in taste, slightly warm in nature, it can invigorate Qi and strengthen the exterior, strengthen the body and eliminate evil. As the main medicine in the prescription, it has the effects of detoxification and pus discharge, relieving various inflammations, and improving immunity. It plays a major role in helping the amblycephalic bream resist Aeromonas hydrophila infection and reduce mortality.

[0007] Lycium barbarum: sweet in taste, neutral in nature, enters the liver and kidney meridians, has natural antioxidant and enzyme synthesis promoting biological activities, has significant effects and functions in improving the body's growth and development, digestion level, and improving immunity. As a ministerial drug in prescriptions, it has the effect of enhancing the efficacy of Astragalus.

[0008] Ginseng: sweet, slightly bitter, and slightly warm in nature; it enters the lung, spleen, heart, and kidney meridians, replenishes qi and blood, calms the mind and improves intelligence, restores yang and rescues adverse conditions, strengthens the spleen and lungs. As an adjuvant, it plays an important role in inhibiting the occurrence of inflammation, improving antioxidant capacity and immunity, etc.

[0009] Reed root: It is cold in nature and sweet in taste. It enters the lung and stomach meridians. It has the effects of clearing away heat and purging fire, promoting fluid production and quenching thirst, relieving restlessness, stopping vomiting, and promoting diuresis. It also has the effect of protecting the liver and is used as a guiding drug.

[0010] Furthermore, the compound Chinese herbal feed additive is prepared by the following steps: grinding each Chinese herbal raw material into powder and then passing it through a 200-mesh sieve, weighing the sieved powder of each Chinese herbal raw material according to proportion, and mixing them to obtain a compound Chinese herbal feed additive.

[0011] A second aspect of the present invention provides a use of the above-mentioned compound Chinese herbal medicine feed additive in promoting fish growth.

[0012] Furthermore, the compound Chinese herbal medicine feed additive is used to improve the survival rate of fish, promote fish weight gain, increase the growth rate of fish, or improve the digestion and absorption of feed by fish.

[0013] The third aspect of the present invention provides a use of the above-mentioned compound Chinese herbal medicine feed additive in improving the immunity of fish.

[0014] Furthermore, the compound Chinese herbal medicine feed additive is used to increase the activity of fish digestive enzymes or improve fish intestinal tissue.

[0015] A fourth aspect of the present invention provides a use of the above-mentioned compound Chinese herbal medicine feed additive in improving the disease resistance of fish to Aeromonas hydrophila infection.

[0016] A fifth aspect of the present invention provides a use of the above-mentioned compound Chinese herbal medicine feed additive in preparing fish feed.

[0017] A sixth aspect of the present invention provides a fish feed, which is composed of the above-mentioned compound Chinese herbal medicine feed additive and conventional aquatic feed.

[0018] Furthermore, based on the mass of the fish feed, the added mass of the compound Chinese herbal feed additive accounts for 1% to 4% of the fish feed.

[0019] A seventh aspect of the present invention provides a method for promoting fish growth or improving fish immunity or improving fish resistance to Aeromonas hydrophila infection, specifically comprising: feeding the above-mentioned fish feed to fish for more than 90 days.

[0020] Furthermore, the fish feed can improve the fish's head kidney, spleen, gills, liver or intestines. IgM 、 C3 、 TNF-ɑ and IL-1β Gene expression.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses a compound Chinese herbal medicine feed additive for promoting the growth and improving the immunity of Megalobrama amblycephala. The compound Chinese herbal medicine feed additive is composed of 3 to 6 parts of astragalus, 2 to 4 parts of wolfberry, 1 to 2 parts of ginseng, and 0.5 to 1.5 parts of reed rhizome by weight. The compound Chinese herbal medicine feed additive can improve the survival rate of fish, promote the growth of fish, and improve the immunity and disease resistance of fish through the synergistic effect of the various components. The compound Chinese herbal medicine feed additive can be used to prepare fish feed. Experiments have shown that after the compound Chinese herbal medicine feed additive is prepared into fish feed, it can significantly improve the intestinal and liver tissue structure of Megalobrama amblycephala, increase the activity of intestinal digestion-related enzymes, promote the growth of Megalobrama amblycephala, and significantly increase the expression of immune-related genes and the activity of immune-related enzymes in the serum of Megalobrama amblycephala, improve the disease resistance of Megalobrama amblycephala, and reduce the mortality rate after infection with Aeromonas hydrophila. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 Effects of different fish feeds on the growth performance and intestinal digestive enzyme activities of Megalobrama amblycephala; Figure a shows weight gain rate; Figure b shows specific growth rate; Figure c shows feed coefficient; Figure d shows intestinal trypsin activity; Figure e shows intestinal lipase activity; Figure f shows intestinal amylase activity; the same lowercase letters indicate no significant differences P> 0.05; different lowercase letters indicate significant differences P <0.05.

[0024] Figure 2 This is the intestinal tissue of the control group (fed with fish feed of Comparative Example 15). The blue line is the measurement line, and the yellow part indicates the measured length of the intestinal villi, the width of the intestinal villi and the thickness of the intestinal muscle layer.

[0025] Figure 3 This is the intestinal tissue of the T1 experimental group (fed with the fish feed of Example 6). The blue line is the measurement line, and the yellow part indicates the measured length and width of the intestinal villi and the thickness of the intestinal muscle layer.

[0026] Figure 4 This is the intestinal tissue of the T2 experimental group (fed with the fish feed of Example 5). The blue line is the measurement line, and the yellow part indicates the measured length and width of the intestinal villi and the thickness of the intestinal muscle layer.

[0027] Figure 5 This is the intestinal tissue of the T3 experimental group (fed with the fish feed of Example 7). The blue line is the measurement line, and the yellow part indicates the measured length and width of the intestinal villi and the thickness of the intestinal muscle layer.

[0028] Figure 6 Statistical graphs showing the effects of different fish feeds on the intestinal tissue of Megalobrama amblycephala; Figure A shows the length of intestinal villi; Figure B shows the width of intestinal villi; Figure C shows the thickness of the intestinal muscle layer; the same lowercase letters indicate no significant difference P> 0.05; different lowercase letters indicate significant differences P <0.05.

[0029] Figure 7 Figure 2 shows the effects of different fish feeds on the liver and spleen of Megalobrama amblycephala. C represents the control group fed with Comparative 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 index; Figure f shows the spleen body index. The same lowercase letters indicate no significant differences. P> 0.05; different lowercase letters indicate significant differences P <0.05, black arrows indicate the sinusoidal space of the liver.

[0030] Figure 8 Figure 2 Effects of different fish diets on the activities of immune-related enzymes in the serum of Megalobrama amblycephala. Panel A shows the lysozyme (LYS) content; Panel B shows the acid phosphatase (ACP) activity; Panel C shows the alkaline phosphatase (AKP) activity; Panel D shows the catalase (CAT) activity; Panel E shows the superoxide dismutase (SOD) activity; and Panel F shows the glutathione peroxidase (GSH-PX) activity. Identical lowercase letters indicate no significant differences. P> 0.05; different lowercase letters indicate significant differences P <0.05.

[0031] Figure 9 Figure 2 is the effect of different fish feeds on the expression of immune-related genes in Megalobrama amblycephala, C represents the control group fed with Comparative 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 is IgM The relative expression of genes; Figure b is C3 Relative expression of genes; Figure c is TNF-ɑ The relative expression of genes; d is IL-1β Relative expression of genes; the same lowercase letters indicate no significant difference P> 0.05; different lowercase letters indicate significant differences P <0.05.

[0032] Figure 10 The figure shows the effect of adding compound Chinese herbal medicine to feed on the disease resistance of Megalobrama amblycephala, C represents the control group fed with Comparative 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 DESCRIPTION

[0033] 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 by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0034] Aeromonas hydrophila in the present invention ( Aeromonashydrophila ) was isolated and identified by the inventor's research group from a septicemic Megalobrama amblycephala. For strain information, see 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. At present, chemical drugs (including antibiotics) are mainly used in the farming of bighead carp to prevent and treat bacterial sepsis caused by Aeromonas hydrophila infection. This not only affects the quality and safety of aquatic products, but also damages the ecological environment, seriously hindering the green development of the bighead carp farming industry.

[0035] The present invention provides a compound Chinese herbal feed additive for promoting the growth and improving the immunity of Megalobrama amblycephala. The compound Chinese herbal feed additive comprises, by weight, 3 to 6 parts of astragalus, 2 to 4 parts of wolfberry, 1 to 2 parts of ginseng, and 0.5 to 1.5 parts of reed rhizome. This compound Chinese herbal feed additive can improve the survival rate of fish, 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.

[0036] Example 1: A compound Chinese herbal medicine feed additive is prepared by the following steps: Astragalus root ( Astragalus membranaceus )、wolfberry( Lycium barbarumL. )、Ginseng( Panax ginsengC. A. Mey. )、Reed root( Phragmitisrhizoma ) were ground into powder, passed through a 200-mesh sieve, and then mixed in a mass ratio of 4.5:3:1.5:1 to obtain the compound Chinese herbal feed additive. The composition and nutritional level of this compound Chinese herbal feed additive are shown in Table 1.

[0037] Table 1 Composition and nutritional levels of the compound Chinese herbal medicines.

[0038] Example 2: A compound Chinese herbal medicine feed additive is prepared by the following steps: Astragalus, wolfberry, ginseng and reed root are ground into powder and passed through a 200-mesh sieve, and then mixed in a mass ratio of 3:2:1:0.5 to obtain the compound Chinese herbal medicine feed additive.

[0039] Example 3: A compound Chinese herbal medicine feed additive is prepared by the following steps: Astragalus, wolfberry, ginseng and reed root are ground into powder and passed through a 200-mesh sieve, and then mixed in a mass ratio of 5:2:1.5:1 to obtain the compound Chinese herbal feed additive.

[0040] Example 4: A compound Chinese herbal medicine feed additive is prepared by the following steps: Astragalus, wolfberry, ginseng and reed root are ground into powder and passed through a 200-mesh sieve, and then mixed in a mass ratio of 6:4:2:1.5 to obtain the compound Chinese herbal feed additive.

[0041] Examples 1 to 4 have similar effects. For the convenience of subsequent discussion and reference, the following experiments are conducted using the compound Chinese herbal feed additive prepared in Example 1 as an example.

[0042] Example 5: A fish feed is prepared by the following steps: 2000g soybean meal (CP44%), 4000g rapeseed meal, and 1550g wheat were ground separately and passed through a 200-mesh sieve. 200g of the compound Chinese herbal feed additive prepared in Example 1, 1100g fish meal (CP67%), 600g 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 granulator. The weight proportion of the compound Chinese herbal feed additive prepared in Example 1 in the fish feed was 2%.

[0043] Example 6: A fish feed is prepared by the following steps: 2000g soybean meal (CP44%), 4000g rapeseed meal, and 1650g wheat were ground separately and passed through a 200-mesh sieve. 100g of the compound Chinese herbal feed additive prepared in Example 1, 1100g fish meal (CP67%), 600g 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 granulator. The weight proportion of the compound Chinese herbal feed additive prepared in Example 1 in the fish feed was 1%.

[0044] Example 7: A fish feed is prepared by the following steps: 2000g soybean meal (CP44%), 4000g rapeseed meal, and 1350g wheat were ground separately and passed through a 200-mesh sieve. 400g of the compound Chinese herbal feed additive prepared in Example 1, 1100g fish meal (CP67%), 600g 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 granulator. The weight proportion of the compound Chinese herbal feed additive prepared in Example 1 in the fish feed was 4%.

[0045] Comparative Example 1: A compound Chinese herbal medicine feed additive is prepared by the following steps: Astragalus, wolfberry, ginseng and reed root are ground into powder and passed through a 200-mesh sieve, and then mixed in a mass ratio of 1:1:1:1 to obtain the compound Chinese herbal feed additive.

[0046] Comparative Example 2: A compound Chinese herbal medicine feed additive is prepared by the following steps: The wolfberry, ginseng and reed root are ground into powder and passed through a 200-mesh sieve, and then mixed in a mass ratio of 3:1.5:1 to obtain the compound Chinese herbal feed additive.

[0047] Comparative Example 3: A compound Chinese herbal medicine feed additive is prepared by the following steps: Astragalus, ginseng and reed root are ground into powder and passed through a 200-mesh sieve, and then mixed in a mass ratio of 4.5:1.5:1 to obtain the compound Chinese herbal medicine feed additive.

[0048] Comparative Example 4: A compound Chinese herbal medicine feed additive is prepared by the following steps: The astragalus root, wolfberry and reed root are ground into powder and passed through a 200-mesh sieve, and then mixed in a mass ratio of 4.5:3:1 to obtain the compound Chinese herbal medicine feed additive.

[0049] Comparative Example 5: A compound Chinese herbal medicine feed additive is prepared by the following steps: The astragalus root, wolfberry and ginseng are ground into powder and passed through a 200-mesh sieve, and then mixed in a mass ratio of 4.5:3:1.5 to obtain the compound Chinese herbal medicine feed additive.

[0050] Comparative Example 6: A fish feed is prepared by the following steps: 2000g soybean meal (CP44%), 4000g rapeseed meal, and 1550g wheat were crushed and passed through a 200-mesh sieve, and then 200g of the compound Chinese herbal feed additive prepared in Comparative Example 1, 1100g fish meal (CP67%), 600g soybean oil, 300g compound feed premix, 230g bentonite, 17g choline chloride, 1g preservative and 2g antioxidant were added. After thorough mixing, a fish feed was prepared using a granulator. The mass proportion of the compound Chinese herbal feed additive prepared in Comparative Example 1 in the fish feed was 2%.

[0051] Comparative Example 7: A fish feed is prepared by the following steps: 2000g soybean meal (CP44%), 4000g rapeseed meal, and 1550g wheat were crushed and passed through a 200-mesh sieve, and then 200g of the compound Chinese herbal feed additive prepared in Comparative Example 2, 1100g fish meal (CP67%), 600g soybean oil, 300g compound feed premix, 230g bentonite, 17g choline chloride, 1g preservative and 2g antioxidant were added. After thorough mixing, a fish feed was prepared using a granulator. The mass proportion of the compound Chinese herbal feed additive prepared in Comparative Example 2 in the fish feed was 2%.

[0052] Comparative Example 8: A fish feed is prepared by the following steps: 2000g soybean meal (CP44%), 4000g rapeseed meal, and 1550g wheat were crushed and passed through a 200-mesh sieve, and then 200g of the compound Chinese herbal feed additive prepared in Comparative Example 3, 1100g fish meal (CP67%), 600g soybean oil, 300g compound feed premix, 230g bentonite, 17g choline chloride, 1g preservative and 2g antioxidant were added. After thorough mixing, a fish feed was prepared using a granulator. The mass proportion of the compound Chinese herbal feed additive prepared in Comparative Example 3 in the fish feed was 2%.

[0053] Comparative Example 9: A fish feed is prepared by the following steps: 2000g soybean meal (CP44%), 4000g rapeseed meal, and 1550g wheat were crushed and passed through a 200-mesh sieve, and then 200g of the compound Chinese herbal feed additive prepared in Comparative Example 4, 1100g fish meal (CP67%), 600g soybean oil, 300g compound feed premix, 230g bentonite, 17g choline chloride, 1g preservative and 2g antioxidant were added. After thorough mixing, a fish feed was prepared using a granulator. The mass proportion of the compound Chinese herbal feed additive prepared in Comparative Example 4 in the fish feed was 2%.

[0054] Comparative Example 10: A fish feed is prepared by the following steps: 2000g soybean meal (CP44%), 4000g rapeseed meal, and 1550g wheat were crushed and passed through a 200-mesh sieve, and then 200g of the compound Chinese herbal feed additive prepared in Comparative Example 5, 1100g fish meal (CP67%), 600g soybean oil, 300g compound feed premix, 230g bentonite, 17g choline chloride, 1g preservative and 2g antioxidant were added. After thorough mixing, a fish feed was prepared using a granulator. The mass proportion of the compound Chinese herbal feed additive prepared in Comparative Example 5 in the fish feed was 2%.

[0055] Comparative Example 11: A fish feed is prepared by the following steps: The astragalus was crushed and passed through a 200-mesh sieve to obtain astragalus powder.

[0056] 2000g soybean meal (CP44%), 4000g rapeseed meal, and 1350g wheat were ground separately and passed through a 200-mesh sieve. 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. After thorough mixing, a fish feed was prepared using a pelletizer. The weight proportion of the astragalus powder in the fish feed was 2%.

[0057] Comparative Example 12: A fish feed is prepared by the following steps: The wolfberries were crushed and passed through a 200-mesh sieve to obtain wolfberry powder.

[0058] 2000g soybean meal (CP44%), 4000g rapeseed meal, and 1350g wheat were ground separately and passed through a 200-mesh sieve. 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. After thorough mixing, a fish feed was prepared using a pelletizer. The weight proportion of wolfberry powder in the fish feed was 2%.

[0059] Comparative Example 13: A fish feed is prepared by the following steps: Ginseng was crushed and passed through a 200-mesh sieve to obtain ginseng powder.

[0060] 2000g soybean meal (CP44%), 4000g rapeseed meal, and 1350g wheat were ground separately and passed through a 200-mesh sieve. 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. After thorough mixing, a fish feed was prepared using a pelletizer. The weight ratio of ginseng powder in the fish feed was 2%.

[0061] Comparative Example 14: A fish feed is prepared by the following steps: The reed rhizome was crushed and passed through a 200-mesh sieve to obtain reed rhizome powder.

[0062] 2000g soybean meal (CP44%), 4000g rapeseed meal, and 1350g wheat were respectively ground and passed through a 200-mesh sieve, and then 200g reed root powder, 1100g fish meal (CP67%), 600g soybean oil, 300g compound feed premix, 230g bentonite, 17g choline chloride, 1g preservative, and 2g antioxidant were added. After being fully mixed, a fish feed was prepared using a pelletizer. The mass proportion of the reed root powder in the fish feed was 2%.

[0063] Comparative Example 15: A fish feed is prepared by the following steps: 2000g soybean meal (CP44%), 4000g rapeseed meal, and 1750g wheat were respectively ground and passed through a 200-mesh sieve, and then 1100g fish meal (CP67%), 600g soybean oil, 300g compound feed premix, 230g bentonite, 17g choline chloride, 1g preservative, and 2g antioxidant were added. After being fully mixed, a fish feed was prepared using a pelletizer.

[0064] Experimental Example 1: Effects of compound Chinese herbal feed additives on fish survival rate The experimental fish, Megalobrama amblycephala, weighing 42-46 g, were obtained from the fish breeding base of Hunan University of Arts and Science. Prior to sampling, the fish were maintained in a laboratory recirculating aquaculture system for two weeks. During this period, the fish were aerated 24 hours a day to maintain sufficient dissolved oxygen in the water (5.8-6.2 μg / mL). The water temperature was maintained between 26°C and 30°C, and the fish were fed an appropriate amount of adult fish floating feed.

[0065] After the experimental fish were temporarily acclimated for two weeks, the fish feeds of Examples 5 to 7 of the present invention and Comparative Examples 6 to 15 were fed to the amblycephala at 8:30 am and 17:00 pm every day. The feeding mass was 2% of the experimental fish body weight each time. The water was changed once a day, and the water change volume was 1 / 3 of the total volume of the aquaculture water. The dirt in the aquaculture tank was cleaned, and the aquaculture tank was aerated and oxygenated for 24 hours. The feeding was continued for 90 days. After 90 days of aquaculture, 30 amblycephala were randomly selected and intraperitoneally injected with 0.1 mL of the viable bacteria count of 1×10 7 The fish were infected with Aeromonas hydrophila liquid with a concentration of 100 CFU / mL, and the cumulative mortality rate of the amblycephala bream 14 days after infection was calculated to explore the effects of different compound Chinese herbal feed additives on fish.

[0066] Cumulative mortality (%) = total number of fish that died in 14 days / 30 × 100%; Survival rate (%) = total number of fish surviving on day 14 / 30 × 100%; The test results are shown in Table 2.

[0067] Table 2 Fish mortality rate of compound Chinese herbal feed additives Since Examples 5 to 7 can significantly improve the survival rate of fish, the present invention uses fish feed not containing the compound Chinese herbal feed additive (Comparative Example 15) as a control group and fish feed containing different amounts of the compound Chinese herbal feed additive prepared in Examples 5 to 7 as an experimental group for in-depth research.

[0068] 1. Experimental Methods 1. Determination of growth indicators The initial body weight of the experimental fish was weighed at the beginning of the experiment. After 90 days of feeding, the final body weight, liver weight, and spleen weight of the experimental fish were weighed, and the feed consumption during the breeding process was recorded. The growth-related indicators of the experimental fish were calculated using the following formula.

[0069] Weight gain rate (WGR) = (W1-W0) / W0 × 100%; Specific growth rate (SGR) = (lnW1–lnW0) / T × 100%; Feed coefficient (FCR) = feed consumption / (W1-W0) × 100%; Hepatosomatic index (HSI) = G1 / W1 × 100%; Spleen index (SPI) = G2 / W1 × 100%; W0 represents the average initial fish weight at the beginning of the experiment, W1 represents the average fish weight at the end of the experiment, T represents the culture time (d), G1 represents the average liver weight of the experimental fish, and G2 represents the average spleen weight of the experimental fish.

[0070] 2. Observation of liver and intestinal tissue sections After feeding the fish with the fish feed prepared in Examples 5 to 7 and Comparative Example 15 for 90 days, five amblycephalic bream were randomly selected from the control group and each experimental group. The fish were anesthetized with MS-222 and blood was collected from the tail vein via a syringe. 3 mm to 5 mm of liver and intestinal tissue were collected and fixed in Bouin's test solution. The samples were dehydrated, wax-impregnated, embedded, sectioned, spread, dried, stained with HE, and mounted. After the mounting resin dried, the samples were observed under a microscope and microphotographed.

[0071] 3. Determination of serum antioxidant and immune-related enzyme activities After 90 days of feeding with the fish feed prepared in Examples 5-7 and Comparative Example 15, five amblycephala 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 4000 rpm for 10 min at 4°C to separate the serum, which was then temporarily stored in a 4°C refrigerator. A kit purchased from Nanjing Jiancheng Bioengineering Research Institute was used to assay the serum for lysozyme (Lys), total superoxide dismutase (T-SOD), catalase (CAT), acid phosphatase (ACP), alkaline phosphatase (AKP), and glutathione peroxidase (GSH-PX) activity using a microplate reader according to the instructions in the kit instructions.

[0072] 4. Determination of intestinal digestion-related enzyme activity Five Megalobrama amblycephala were randomly selected from each experimental group and the control group. After anesthesia with MS-222 and blood collection, 2 g of intestinal tissue was collected from each group and added with pre-cooled physiological saline (4°C) at a ratio of 1:9 (W / V). The tissue was homogenized in an ice-water bath for 30 min. The homogenate was then centrifuged at 3000 r / min at 4°C for 10 min. The supernatant was separated and the activities of amylase (AMS), lipase (LPS), and trypsin (TRS) in the intestine of Megalobrama amblycephala were determined using a microplate reader according to the instructions of the kit provided by Nanjing Jiancheng Bioengineering Research Institute.

[0073] 5. Statistics on mortality of Megalobrama amblycephala infected with Aeromonas hydrophila Aeromonas hydrophila was isolated from diseased bream ( Aeromonas hydrophila ), pick a single clone of the strain and inoculate it into Luriabroth (LB) liquid medium, and culture it at 28°C with shaking overnight. Through preliminary experiments, the half-lethal concentration of Aeromonas hydrophila for Megalobrama amblycephala was about 1×10 7 After feeding the fish feed prepared in Examples 5 to 7 and Comparative Example 15 for 90 days, 0.1 mL (1×10 7 The experimental group was infected with Aeromonas hydrophila (CFU / mL), and the cumulative mortality of Megalobrama agglomerata in different experimental groups on the 1st, 3rd, 5th, 7th and 14th day after infection was calculated.

[0074] 6. qRT-PCR detection of immune-related gene expression in Megalobrama amblycephala The fish were fed with the fish feed prepared in Examples 5 to 7 and Comparative Example 15 for 90 days. Five Megalobrama amblycephala were taken from the control group and the experimental group, respectively. After anesthesia with MS-222 and blood sampling from the tail vein, 100 mg each of the head kidney, liver, spleen, intestine, and gill were collected and ground into powder in a mortar using liquid nitrogen. RNA from various tissues of Megalobrama amblycephala was extracted using an RNA extraction kit (Simgen) according to its instructions, and reverse transcription was performed to obtain a cDNA template. IL-1β ), tumor necrosis factor-α (Tumor necrosis factor - Alpha, TNF-ɑ ), Immunoglobulin M IgM ) and complement factor C3 (Complement factor C3, C3 ) gene sequence to design real-time fluorescence quantitative PCR (qRT-PCR) primers. β-actinThe gene was used as an internal reference gene, and the qRT-PCR kit (TaKaRa) was used to detect the expression of the gene in Megalobrama amblycephala according to its instructions. IL-1β 、 TNF-ɑ 、 IgM and C3 Gene expression in different tissues is determined by 2 -ΔΔCt The relative expression levels of immune-related genes in various tissues of Megalobrama amblycephala were calculated using the method.

[0075] Table 3 Primers for real-time fluorescence quantitative PCR amplification of immune-related genes in Megalobrama amblycephala 7. Data Analysis The experimental data were expressed as mean ± standard deviation, and one-way analysis of variance was performed using SPSS 15.0 statistical software. P <0.05 was considered significant difference. P <0.01 was considered to be extremely significant.

[0076] 2. Experimental Results 1. Determination of nutritional levels of different fish feeds Table 4 Nutritional level determination As can be seen from Table 4, there is no significant difference in crude protein, crude fat and total energy between Example 5, Example 6, Example 7 and Comparative Example 15 (control group), and the total nitrogen and total energy are balanced.

[0077] 2. Effects of different fish feeds on growth performance and intestinal digestive enzyme activity of Megalobrama amblycephala The weight gain rate refers to the percentage of weight gain of fish in 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 breeding efficiency. The 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. The feed coefficient refers to the amount of feed required for fish to achieve a certain weight gain. The lower the feed coefficient, the higher the feed utilization efficiency and the lower the breeding cost.

[0078] like Figure 1 As shown in Figures a to c, the weight gain rate and specific growth rate of the experimental group's Megalobrama amblycephala were higher than those of the control group, and the weight gain rate and specific growth rate of the Megalobrama amblycephala fed the group of Example 5 were the highest. The feed conversion ratios of all experimental groups were lower than those of the control group, and the feed conversion ratio of the group fed Example 5 was the lowest.

[0079] The higher the activity of amylase, lipase and trypsin in the intestines of the amblycephalic bream, the more efficient the amblycephalic bream is in digesting starchy feed and breaking down fat and protein, thereby 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 the amblycephalic bream.

[0080] like Figure 1 As shown in Figures d to f, the intestinal trypsin and lipase activities of the group of Megalobrama amblycephala fed Example 5 and Example 6 were significantly higher than those of the control group, with the group fed Example 6 having the highest TRS and LPS activities. The intestinal TRS activity of the group fed Example 7 was significantly lower than that of the control group, while the intestinal LPS activity was not significantly different from that of the control group. The intestinal amylase activity of the group fed Example 5 was significantly higher than that of the control group, with the intestinal AMS activity being the highest.

[0081] 3. Effects of different fish feeds on intestinal tissue of Megalobrama amblycephala like Figures 2 to 6 As shown, the length of the intestinal villi and the thickness of the intestinal muscle layer of the amblycephalus in all experimental groups were significantly higher than those in the control group, and the length of the intestinal villi of the experimental group fed with Example 6 was the longest, and the thickness of the intestinal muscle layer of the experimental group fed with Example 5 was the thickest. The width of the intestinal villi of the amblycephalus fed with Example 6 was significantly greater than that of the control group, and the width of the intestinal villi of the amblycephalus fed with Examples 5 and 7 was less than that of the control group.

[0082] 4. Effects of different fish feeds on the liver and spleen of Megalobrama amblycephala The liver-body index refers to the ratio of the liver weight to the body weight of a fish. It reflects the relative size of the liver and is usually used to assess the nutritional reserves and health status of the fish. A higher liver-body index may mean that more nutrients are stored in the liver; the spleen-body index refers to the ratio of the spleen weight to the body weight of a fish. It reflects the relative size of the spleen and is usually used to assess the immune status of the fish. A higher spleen-body index indicates that the fish's immune system is more active.

[0083] like Figure 7 As shown, the sinusoidal space in the liver of the amblycephalus in the control group was widened, the sinusoidal space in the liver of the amblycephalus in the experimental group fed with Example 6 was narrowed, and the sinusoidal space in the liver of the amblycephalus in the experimental groups fed with Examples 5 and 7 disappeared. The liver body index of the amblycephalus in all experimental groups was significantly higher than that in the control group. The spleen body index of the amblycephalus in the experimental groups fed with Examples 5 and 6 was significantly higher than that in the control group, and the spleen body index and liver body index of the experimental group fed with Example 5 were the highest, but the spleen body index of the amblycephalus in the experimental group fed with Example 7 was significantly lower than that in the control group.

[0084] 5. Effects of different fish feeds on the activity of immune-related enzymes in the serum of Megalobrama amblycephala like Figure 8As shown in the results, the serum lysozyme content, superoxide dismutase, catalase, glutathione peroxidase, acid phosphatase and alkaline phosphatase activities of the experimental group of Megalobrama amblycephala were significantly higher than those of the control group. In the experimental group fed with Example 6, the Lys content, SOD, CAT and GSH-PX activities in the serum of Megalobrama amblycephala were the highest. The ACP and AKP activities in the serum of Megalobrama amblycephala gradually increased with the increase in the amount of the compound Chinese herbal feed additive, and the ACP and AKP activities in the serum of Megalobrama amblycephala fed with Example 7 were the highest.

[0085] 6. Effects of different fish feeds on the expression of immune-related genes in Megalobrama amblycephala like Figure 9 As shown in the figure, the experimental group of Megalobrama amblycephala IgM 、 C3 、 TNF-ɑ and IL-1β The expression levels of the gene in the head kidney, spleen, gill, liver and intestine were significantly higher than those in the control group. IgM The gene expression level was highest in the experimental group fed with Example 5, and in the experimental group fed with Example 6, the gene expression level in the spleen and gills was IgM The gene expression level is highest in the head kidney, spleen, gills and intestines. C3 The gene expression level was highest in the experimental group fed with Example 5, with C3 The gene expression level was the highest in the experimental group fed with Example 6. TNF-ɑ The gene expression level was highest in the experimental group fed with Example 5, with the highest expression in gills and liver. TNF-ɑ The gene expression level was the highest in the experimental group fed with Example 6. IL-6 The gene expression level was highest in the experimental group fed with Example 5, with the highest expression in the liver and intestine. IL-6 The gene expression level was the highest in the experimental group fed with Example 6.

[0086] 7. Effects of different fish feeds on disease resistance of Megalobrama amblycephala like Figure 10 As shown, after infection with Aeromonas hydrophila, the cumulative mortality of the amblycephala in the control and experimental groups gradually increased, reaching a maximum on the 7th day after infection. However, the mortality of the amblycephala in the experimental groups was significantly lower than that in the control group. On the 7th day after infection with Aeromonas hydrophila, there was no significant difference in the cumulative mortality between the experimental groups fed with Example 5 and Example 7, and the cumulative mortality of the amblycephala in the experimental group fed with Example 5 was the lowest.

[0087] It should be noted that when numerical ranges are mentioned in the present invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints may be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes preferred embodiments. Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0088] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A compound Chinese herbal medicine feed additive, characterized in that: Calculated by weight, the compound Chinese herbal feed additive consists of 3 to 6 parts of astragalus, 2 to 4 parts of wolfberry, 1 to 2 parts of ginseng and 0.5 to 1.5 parts of reed root.

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

3. Use of the compound Chinese herbal medicine feed additive according to claim 1 in promoting fish growth.

4. The use according to claim 3, characterized in that The compound Chinese herbal medicine feed additive is used for promoting weight gain of fish, increasing the growth rate of fish or improving the digestion and absorption of feed by fish.

5. Use of the compound Chinese herbal medicine feed additive according to claim 1 in improving the immunity of fish.

6. Use of the compound Chinese herbal medicine feed additive according to claim 1 for improving the disease resistance of fish to Aeromonas hydrophila infection.

7. Use of the compound Chinese herbal medicine feed additive according to claim 1 in preparing fish feed.

8. A fish feed, characterized in that: The fish feed consists of the compound Chinese herbal medicine feed additive according to claim 1 and conventional aquatic feed.

9. The fish feed according to claim 8, characterized in that Based on the mass of the fish feed, the added mass of the compound Chinese herbal feed additive accounts for 1% to 4% of the fish feed.

10. A method for promoting fish growth or improving fish immunity or improving fish resistance to Aeromonas hydrophila infection, characterized in that: Feed the fish feed according to claim 8 to fish for more than 90 days.

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