Composition containing astragalus polysaccharide and acanthopanax senticosus extract and application of composition in larimichthys crocea juvenile fish feed

Abstract

The invention relates to the technical field of breeding feed. The invention provides a composition containing astragalus polysaccharide and an acanthopanax senticosus extract, which is characterized in that the mass ratio of the astragalus polysaccharide to the acanthopanax senticosus extract is 10: (1-10), and the composition can be used for preparing feed with the effects of resisting oxidation, relieving inflammation, increasing growth rate, improving intestinal structure and enhancing digestive enzyme activity. The invention also provides a larimichthys crocea juvenile fish feed containing 0.1-0.2% of the composition, and by using the feed combined with the astragalus polysaccharide and the acanthopanax senticosus extract, the growth rate, the survival rate and the stress recovery of the larimichthys crocea juvenile fish are obviously improved; the anti-inflammatory effect is effectively achieved by inhibiting a TLR / MyD88 pathway; the intestinal structure is obviously improved; the digestive enzyme activity is enhanced; the activity of antioxidant enzyme is improved and the concentration of malonaldehyde is reduced, so that the antioxidant defense capability is enhanced.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture feed, in particular to a composition containing astragalus polysaccharide and acanthopanax senticosus extract and application of the composition in feed for juvenile large yellow croaker. Background Art

[0002] In recent years, the aquaculture industry has faced major challenges due to the inevitable outbreaks of diseases, especially viral and bacterial infections, caused by feeding carnivorous fish with fresh-frozen fish and artificially formulated feeds with unbalanced nutritional content. Although the use of toxic chemicals and antibiotics for treatment was initially effective, it inadvertently contributed to the emergence of drug-resistant pathogens, prompting many countries to restrict or outright ban their use. As a result, the aquaculture industry has been forced to seek innovative and sustainable solutions to mitigate these pressing issues.

[0003] Large yellow croaker (Larimichthys crocea) is the main economic marine fish species in Chinese aquaculture, accounting for the largest share of marine fish production, with an annual production of 257,700 tons in 2022. However, the continued growth of aquaculture activities, as well as increasing stocking densities, continued aquatic environmental degradation and recurring extreme weather, have driven a sharp increase in disease outbreaks. The critical early life stages of large yellow croaker, namely larvae and juveniles, are particularly prone to high mortality, which is a key bottleneck that needs to be overcome in large yellow croaker farming. These juveniles are susceptible to oxidative stress and intestinal damage caused by environmental stressors.

[0004] In summary, the development of reliable and effective immunomodulators is highly desirable, which have the potential to enhance the innate defenses of fish by enhancing antioxidant capacity and immune responses, thereby reducing the incidence of diseases. Summary of the invention

[0005] The purpose of the present invention is to provide a composition containing astragalus polysaccharide and Acanthopanax senticosus extract and its application in feed for juvenile large yellow croaker. By using the feed combined with astragalus polysaccharide and Acanthopanax senticosus extract, the growth rate, survival rate and stress resilience of juvenile large yellow croaker are significantly improved; the anti-inflammatory effect is effectively achieved by inhibiting the TLR / MyD88 pathway; the intestinal structure is significantly improved and the digestive enzyme activity is enhanced; the antioxidant enzyme activity is increased and the malondialdehyde concentration is reduced, thereby enhancing the antioxidant defense ability.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a composition containing astragalus polysaccharide and acanthopanax senticosus extract, wherein the mass ratio of the astragalus polysaccharide to the acanthopanax senticosus extract is 10:(1-10).

[0008] The invention provides application of the composition in preparing feed with antioxidant effect.

[0009] The invention provides application of the composition in preparing feed with the effect of alleviating inflammation.

[0010] The invention provides application of the composition in preparing feed with the effects of improving intestinal structure and enhancing digestive enzyme activity.

[0011] The invention provides application of the composition in preparing feed with the efficacy of improving growth rate and survival rate.

[0012] The invention also provides a large yellow croaker fry feed, comprising the following components in percentage by mass: 37-43% of fish meal, 27-32% of soybean meal, 20-24% of flour, 2-3% of fish oil, 0.8-1.2% of soybean lecithin, 2-3% of brewer's yeast, 0.5-1.5% of complex vitamins, 0.5-1.4% of complex mineral salts and 0.1-0.2% of the composition.

[0013] The present invention also provides the use of the large yellow croaker fry feed in improving the growth rate of large yellow croaker fry, relieving inflammation of large yellow croaker fry, enhancing the antioxidant capacity of large yellow croaker fry, improving the intestinal structure of large yellow croaker fry and enhancing the activity of digestive enzymes.

[0014] By adopting the above technical solution, the present invention has the following beneficial effects:

[0015] The technical solution of the present invention significantly improves the growth rate, survival rate and stress resilience of yellow croaker fry by using a feed combined with astragalus polysaccharide and Acanthopanax senticosus extract; effectively inhibits the TLR / MyD88 pathway to achieve anti-inflammatory effects; significantly improves intestinal structure and enhances digestive enzyme activity; also increases antioxidant enzyme activity and reduces malondialdehyde concentration, thereby enhancing antioxidant defense capabilities. By comprehensively improving the immune function and environmental stress resistance of yellow croaker fry, the incidence of diseases is reduced and the survival rate is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The statistical data of digestive enzyme activities of juvenile yellow croaker in different treatment groups ( Figure 1 A represents the activity of α-amylase, B represents the activity of lipase, and C represents the activity of trypsin);

[0017] Figure 2 The midgut morphology of juvenile large yellow croaker in different treatment groups (n=4, scale bar is 100μm);

[0018] Figure 3 The length of mid-intestinal villi of juvenile large yellow croaker in different treatment groups (n=4);

[0019] Figure 4 The relative expression levels of inflammatory cytokines in the head kidney of juvenile large yellow croaker in different treatment groups;

[0020] Figure 5 The relative expression levels of apoptosis-related genes in the head kidney of large yellow croaker fry in different treatment groups. DETAILED DESCRIPTION

[0021] The present invention provides a composition containing Astragalus membranaceus polysaccharides (APS) and Eleutherococcus senticosus extract (ESE), wherein the mass ratio of the Astragalus membranaceus polysaccharides to the Eleutherococcus senticosus extract is preferably 10:(1-10), further preferably 10:(3-7), and further preferably 10:5.

[0022] In the present invention, the astragalus polysaccharide and Acanthopanax senticosus extract are both purchased from Beijing Central Technology Co., Ltd., wherein the astragalus polysaccharide contains 60% of polysaccharide in net content, and the active ingredient of the Acanthopanax senticosus extract is 1.2%-1.5% of eleutheroside.

[0023] The invention provides application of the composition in preparing feed with antioxidant effect.

[0024] The invention provides application of the composition in preparing feed with the effect of alleviating inflammation.

[0025] The invention provides application of the composition in preparing feed with the effects of improving intestinal structure and enhancing digestive enzyme activity.

[0026] The invention provides application of the composition in preparing feed with the effect of improving growth rate.

[0027] The invention also provides a large yellow croaker fry feed, comprising the following components: fish meal, soybean meal, flour, fish oil, soybean lecithin, brewer's yeast, complex vitamins, complex mineral salts and the above combination.

[0028] In the present invention, the large yellow croaker fry feed preferably includes the following components in percentage by weight: 37-43% fish meal, 27-32% soybean meal, 20-23% flour, 2-3% fish oil, 0.8-1.2% soybean lecithin, 2-3% brewer's yeast, 0.5-1.5% complex vitamins, 0.5-1.4% complex mineral salts and 0.1-0.2% of the above composition; further preferably includes 38-42% fish meal, 27.5-31% soybean meal, 21.3-22% flour Flour, 2.2-2.7% fish oil, 0.93-1.1% soy lecithin, 2.3-2.9% brewer's yeast, 0.8-1.2% complex vitamins, 0.75-1.1% complex mineral salts and 0.12-0.17% of the above composition; further preferably comprises 40% fish meal, 30% soybean meal, 21.75% flour, 2.6% fish oil, 1% soy lecithin, 2.5% brewer's yeast, 1% complex vitamins, 1% complex mineral salts and 0.15% of the above composition.

[0029] In the present invention, the multivitamin preferably includes 245000-255000 IU / kg vitamin A, 720-780 mg / kg riboflavin, 380-420 mg / kg pyridoxine-hydrochloride, 0.8-1.2 mg / kg vitamin B12, 220-270 mg / kg ammonium sulfate, 220-280 mg / kg vitamin K, 100-150 mg / kg folic acid, 8-12 mg / kg biotin, 2-3 mg / kg vitamin E, 7800-8300 mg / kg inositol, 1200 -1300mg / kg calcium pantothenate, 1700-2200mg / kg niacin, 7500-8200mg / kg choline chloride, 44000-45500IU / kg vitamin D3, 6500-7500mg / kg vitamin C, further preferably including 248000-253000IU / kg vitamin A, 730-760mg / kg riboflavin, 390-410mg / kg pyridoxine-hydrochloride, 0.9-1.1mg / kg vitamin B12, 230-260mg / kg ammonium sulfate , 230-270 mg / kg vitamin K, 110-140 mg / kg folic acid, 9-11 mg / kg biotin, 2.2-2.8 mg / kg vitamin E, 7900-8200 mg / kg inositol, 1220-1280 mg / kg calcium pantothenate, 1800-2100 mg / kg niacin, 7800-8100 mg / kg choline chloride, 44500-45200 IU / kg vitamin D3, 6800-7200 mg / kg vitamin C, and more preferably 250000 IU / kg vitamin A, 750mg / kg riboflavin, 400mg / kg pyridoxine hydrochloride, 1mg / kg vitamin B12, 250mg / kg ammonium sulfate, 250mg / kg vitamin K, 125mg / kg folic acid, 10mg / kg biotin, 2.5mg / kg vitamin E, 8000mg / kg inositol, 1250mg / kg calcium pantothenate, 2000mg / kg niacin, 8000mg / kg choline chloride, 45000IU / kg vitamin D3, 7000mg / kg vitamin C.

[0030] In the present invention, the composite mineral salt preferably includes 0.03-0.05 g / kg zinc sulfate heptahydrate, 36-39 g / kg calcium carbonate, 4.5-5.8 g / kg potassium chloride, 0.03-0.05 g / kg potassium iodide, 2-3 g / kg sodium chloride, 0.015-0.025 g / kg copper sulfate pentahydrate, 0.015-0.025 g / kg cobalt sulfate heptahydrate, 0.6-1.2 g / kg iron sulfate heptahydrate, 0.02-0.04 g / kg manganese sulfate monohydrate, 3-4 g / kg magnesium sulfate heptahydrate, 9-11.5 g / kg calcium dihydrogen phosphate dihydrate, and further preferably includes 0.035-0.045 g / kg zinc sulfate heptahydrate, 37-38.3 g / kg calcium carbonate, 5-5.6 g / kg potassium chloride, 0.035-0.045 g / kg potassium iodide, 2.4 -2.8g / kg sodium chloride, 0.018-0.022g / kg copper sulfate pentahydrate, 0.018-0.022g / kg cobalt sulfate heptahydrate, 0.8-1.1g / kg ferric sulfate heptahydrate, 0.025-0.035g / kg manganese sulfate monohydrate, 3.2-3.8g / kg magnesium sulfate heptahydrate, 9.2-10.5g / kg calcium dihydrogen phosphate dihydrate, further preferably including 0.04g / kg zinc sulfate heptahydrate, 37.9g / kg calcium carbonate, 5.3g / kg potassium chloride, 0.04g / kg potassium iodide, 2.6g / kg sodium chloride, 0.02g / kg copper sulfate pentahydrate, 0.02g / kg cobalt sulfate heptahydrate, 0.9g / kg ferric sulfate heptahydrate, 0.03g / kg manganese sulfate monohydrate, 3.5g / kg magnesium sulfate heptahydrate, 9.8g / kg calcium dihydrogen phosphate dihydrate.

[0031] The present invention also provides the use of the large yellow croaker fry feed in improving the growth rate of large yellow croaker fry, alleviating inflammation of large yellow croaker fry, enhancing the antioxidant capacity of large yellow croaker fry, improving the intestinal structure of large yellow croaker fry and enhancing the activity of digestive enzymes.

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] Example 1

[0034] A composition containing astragalus polysaccharide and acanthopanax senticosus extract, wherein the mass ratio of the astragalus polysaccharide to the acanthopanax senticosus extract is 10:5.

[0035] Example 2

[0036] A composition containing astragalus polysaccharide and acanthopanax senticosus extract, wherein the mass ratio of the astragalus polysaccharide to the acanthopanax senticosus extract is 10:10.

[0037] Example 3

[0038] A composition containing astragalus polysaccharide and acanthopanax senticosus extract, wherein the mass ratio of the astragalus polysaccharide to the acanthopanax senticosus extract is 10:1.

[0039] Example 4

[0040] A feed for juvenile yellow croaker, comprising the following components: 40% fish meal, 30% soybean meal, 21.75% flour, 2.6% fish oil, 1% soybean lecithin, 2.5% brewer's yeast, 1% complex vitamins, 1% complex mineral salts, and 0.15% of the composition described in Example 1;

[0041] The multivitamin comprises 250000 IU / kg vitamin A, 750 mg / kg riboflavin, 400 mg / kg pyridoxine-hydrochloride, 1 mg / kg vitamin B12, 250 mg / kg ammonium sulfate, 250 mg / kg vitamin K, 125 mg / kg folic acid, 10 mg / kg biotin, 2.5 mg / kg vitamin E, 8000 mg / kg inositol, 1250 mg / kg calcium pantothenate, 2000 mg / kg niacin, 8000 mg / kg choline chloride, 450 00IU / kg vitamin D3, 7000mg / kg vitamin C; the composite mineral salt is 0.04g / kg zinc sulfate heptahydrate, 37.9g / kg calcium carbonate, 5.3g / kg potassium chloride, 0.04g / kg potassium iodide, 2.6g / kg sodium chloride, 0.02g / kg copper sulfate pentahydrate, 0.02g / kg cobalt sulfate heptahydrate, 0.9g / kg iron sulfate heptahydrate, 0.03g / kg manganese sulfate monohydrate, 3.5g / kg magnesium sulfate heptahydrate, and 9.8g / kg calcium dihydrogen phosphate dihydrate.

[0042] Example 5

[0043] A feed for juvenile yellow croaker, comprising: 39% fish meal, 27.9% soybean meal, 24% flour, 2.7% fish oil, 1.1% soybean lecithin, 2.7% brewer's yeast, 1.3% complex vitamins, 1.2% complex mineral salts, and 0.1% of the composition described in Example 1;

[0044] The multivitamin comprises 250000 IU / kg vitamin A, 750 mg / kg riboflavin, 400 mg / kg pyridoxine-hydrochloride, 1 mg / kg vitamin B12, 250 mg / kg ammonium sulfate, 250 mg / kg vitamin K, 125 mg / kg folic acid, 10 mg / kg biotin, 2.5 mg / kg vitamin E, 8000 mg / kg inositol, 1250 mg / kg calcium pantothenate, 2000 mg / kg niacin, 8000 mg / kg choline chloride, 450 00IU / kg vitamin D3, 7000mg / kg vitamin C; the composite mineral salt is 0.04g / kg zinc sulfate heptahydrate, 37.9g / kg calcium carbonate, 5.3g / kg potassium chloride, 0.04g / kg potassium iodide, 2.6g / kg sodium chloride, 0.02g / kg copper sulfate pentahydrate, 0.02g / kg cobalt sulfate heptahydrate, 0.9g / kg iron sulfate heptahydrate, 0.03g / kg manganese sulfate monohydrate, 3.5g / kg magnesium sulfate heptahydrate, and 9.8g / kg calcium dihydrogen phosphate dihydrate.

[0045] Example 6

[0046] A feed for juvenile yellow croaker, comprising: 43% fish meal, 30% soybean meal, 20% flour, 2.1% fish oil, 0.9% soybean lecithin, 2.1% brewer's yeast, 0.9% complex vitamins, 0.8% complex mineral salts and 0.2% of the composition described in Example 1;

[0047] The multivitamin comprises 250000 IU / kg vitamin A, 750 mg / kg riboflavin, 400 mg / kg pyridoxine-hydrochloride, 1 mg / kg vitamin B12, 250 mg / kg ammonium sulfate, 250 mg / kg vitamin K, 125 mg / kg folic acid, 10 mg / kg biotin, 2.5 mg / kg vitamin E, 8000 mg / kg inositol, 1250 mg / kg calcium pantothenate, 2000 mg / kg niacin, 8000 mg / kg choline chloride, 450 00IU / kg vitamin D3, 7000mg / kg vitamin C; the composite mineral salt is 0.04g / kg zinc sulfate heptahydrate, 37.9g / kg calcium carbonate, 5.3g / kg potassium chloride, 0.04g / kg potassium iodide, 2.6g / kg sodium chloride, 0.02g / kg copper sulfate pentahydrate, 0.02g / kg cobalt sulfate heptahydrate, 0.9g / kg iron sulfate heptahydrate, 0.03g / kg manganese sulfate monohydrate, 3.5g / kg magnesium sulfate heptahydrate, and 9.8g / kg calcium dihydrogen phosphate dihydrate.

[0048] Test Example 1

[0049] (I) Preparation of experimental feed

[0050] Five experimental diets with equal nitrogen and lipid content were set up, including the control group, APS diet group, 0.05% ESE group, 0.1% ESE group and 0.15% ESE group. The control group was a diet without APS and ESE; the APS diet group was a diet with 0.1% APS added; the 0.05% ESE group was a diet with 0.1% APS and 0.05% ESE added; the 0.1% ESE group was a diet with 0.1% APS and 0.1% ESE added; and the 0.15% ESE group was a diet with 0.1% APS and 0.15% ESE added. The specific composition of the experimental diets in each group is shown in Table 1.

[0051] Table 1 Composition of experimental diets in different groups

[0052]

[0053]

[0054] The multivitamins used in the feeds are 250000 IU / kg vitamin A, 750 mg / kg riboflavin, 400 mg / kg pyridoxine-hydrochloride, 1 mg / kg vitamin B12, 250 mg / kg ammonium sulfate, 250 mg / kg vitamin K, 125 mg / kg folic acid, 10 mg / kg biotin, 2.5 mg / kg vitamin E, 8000 mg / kg inositol, 1250 mg / kg calcium pantothenate, 2000 mg / kg niacin, 8000 mg / kg chloride. Choline, 45000IU / kg vitamin D3, 7000mg / kg vitamin C; complex mineral salts are 0.04g / kg zinc sulfate heptahydrate, 37.9g / kg calcium carbonate, 5.3g / kg potassium chloride, 0.04g / kg potassium iodide, 2.6g / kg sodium chloride, 0.02g / kg copper sulfate pentahydrate, 0.02g / kg cobalt sulfate heptahydrate, 0.9g / kg iron sulfate heptahydrate, 0.03g / kg manganese sulfate monohydrate, 3.5g / kg magnesium sulfate heptahydrate, and 9.8g / kg calcium dihydrogen phosphate dihydrate.

[0055] The above-mentioned dry ingredients were crushed and filtered with a 200 μm sieve, and the sieve was collected; then they were weighed and mixed according to the formula of each group to obtain a mixture; then fish oil, soybean lecithin and water were added to the mixture in turn, and stirred vigorously to mix evenly; then the obtained mixture was made into an expanded feed with a diameter of 2 mm, and dried in a ventilated oven at 40°C until the water content of the feed was reduced to less than 10%. Finally, the experimental feed was stored at -20°C. (two)

[0057] Large yellow croaker juveniles were obtained from a commercial seedling farm (Ningde Lingling Fishery Technology Co., Ltd.) in Ningde, Fujian Province, China, and placed in seawater floating cages with a size of 4.0 m × 3.0 m × 2.5 m for a two-week adaptation period, in order to adapt the fish to the experimental conditions. Before starting the feeding experiment, all fish were fasted for 24 h. Subsequently, 1200 large yellow croaker juveniles with an initial average body weight of 13.55 ± 0.56 g were selected and randomly distributed in 20 smaller floating cages, each with a size of 1 m × 1 m × 1.5 m, with 60 large yellow croaker juveniles in each cage. The experimental feed types of each group were then randomly assigned to four of the cages. During the 8-week experiment, feeding was carried out twice a day at 7 am and 6 pm to ensure that their nutritional requirements were met. During this period, the water temperature was maintained in the optimal range of 27 °C to 29 °C, and the dissolved oxygen level was always maintained above 6.0 mg / L.

[0058] After the 8-week feeding experiment, the juvenile yellow croakers in each group were fasted for 24 hours and then anesthetized with 100 mg / L eugenol solution. The weight of the juvenile yellow croakers in different treatment groups was measured, and the weight gain rate (WGR), specific growth rate (SGR), feed efficiency (FE) and survival rate (SR) were calculated. The specific results are shown in Table 2. The data are expressed as mean and standard deviation.

[0059] Weight gain rate (%) = (final weight - initial weight) / initial weight × 100;

[0060] Specific growth rate (%) = (ln final weight - ln initial weight) / experimental days × 100;

[0061] Feed efficiency = (final weight - initial weight) / feed intake;

[0062] Survival rate (%) = final number of fish / initial number of fish × 100.

[0063] Table 2 Growth performance of juvenile yellow croaker in different treatment groups

[0064]

[0065] According to Table 2, compared with the control group, the 0.05% ESE group, 0.1% ESE group and 0.15% ESE group significantly increased the weight gain rate, specific growth rate and feed efficiency of large yellow croaker fry (P<0.05), among which the 0.05% ESE group had the most obvious improvement effect. Although the APS diet group was higher than the control group in these indicators, these differences did not reach statistical significance (P>0.05). In addition, compared with the control group, the group with APS alone and the group with APS and ESE added significantly increased the survival rate of large yellow croaker fry (P<0.05). (three)

[0067] After the above growth performance indicators were determined, 10 large yellow croaker fry were randomly selected from each cage, and the liver and head kidney tissues were collected and immediately frozen in liquid nitrogen to further evaluate the antioxidant markers and gene expression levels. In addition, the intestines of these 10 large yellow croaker fry were quickly removed and frozen in liquid nitrogen for later analysis of digestive enzyme activity. Finally, 3 large yellow croaker fry were randomly selected from each cage to extract midgut samples, which were preserved in 4% paraformaldehyde for intestinal structural histology and morphological analysis.

[0068] 1. Detection of antioxidant capacity and digestive enzyme activity

[0069] Take the intestinal or liver tissue obtained above, and then homogenize the intestinal or liver tissue in ice-cold saline solution at a ratio of 10 volumes of saline to 1 weight of tissue to obtain a mixture; then rotate the mixture in a centrifuge at 4°C at 2000 times per minute for 30 minutes to obtain intestinal tissue supernatant or liver tissue supernatant, respectively. These supernatants were then used to evaluate the activity of digestive enzymes and antioxidant parameters. The analysis of antioxidant capacity included the specific activity of peroxidase (CAT), superoxide dismutase (SOD), total antioxidant capacity (T-AOC) and the concentration of malondialdehyde (MDA); the intestinal samples were also analyzed for digestive enzyme activity, and the indicators included trypsin, lipase and α-amylase. The above determinations were all carried out according to the instructions of the kit, and each detection kit was purchased from Nanjing Jiancheng Bioengineering Institute. The determination results are shown in Figure 1 and Table 3.

[0070] Table 3 Antioxidant capacity of juvenile yellow croaker in different treatment groups

[0071]

[0072] According to the results in Table 3, the CAT activity and T-AOC levels of juvenile yellow croaker fed with the 0.05% ESE diet were significantly increased, significantly higher than those of all other diet groups (P<0.05). Except for the 0.05% ESE group, the APS diet group and the ESE-added group showed increased CAT activity and T-AOC, but the difference did not reach statistical significance compared with the control group (P>0.05). Compared with the control group, the APS diet group and the ESE-added group both led to a significant decrease in MDA levels (P<0.05); the APS diet group and the ESE-added group also showed an increase in SOD activity, but this increase did not reach statistical significance compared with the control group (P>0.05).

[0073] like Figure 1 As shown in the results, the APS diet and the ESE-added diet resulted in a significant increase in lipase activity (P<0.05). In addition, compared with the control group, the trypsin activity of the yellow croaker fry fed with the 0.05% ESE group and the 0.1% ESE group was significantly increased (P<0.05). There was no significant change in the trypsin activity of the yellow croaker fry fed with the control group, the APS diet, and the 0.15% ESE group; there was no significant difference in the α-amylase activity of the yellow croaker fry fed with the diets of the various treatment groups.

[0074] 2. Observation of midgut morphology

[0075] The midgut specimens of each group were fixed with 4% paraformaldehyde and then dehydrated in ethanol solutions with increasing concentrations (75% ethanol solution for 4 h, 85% ethanol solution for 2 h, 90% ethanol solution for 2 h, 95% ethanol solution for 1 h, and 100% ethanol solution for 1 h). After dehydration, they were embedded in paraffin and cut using a rotary microtome to obtain 5 μm midgut slices, which were then stained with hematoxylin and eosin. These prepared slices were then examined and photographed using an optical microscope (ECLIPSE Ci, Tokyo, Japan). The length of the villi depicted in the images was quantitatively determined using the ImagePro Plus6.0 software application.

[0076] The results are as follows Figure 2 and Figure 3 As shown, in all experimental groups, the intestinal tissue morphology remained normal histologically without any obvious pathological changes, indicating that the doses of APS and ESE had no toxic or harmful effects. In addition, the villi of the yellow croaker fry fed with the 0.05% ESE group were significantly longer (P<0.05), significantly higher than those of the other treatment groups. This also shows that the combined use of APS and Acanthopanax senticosus extracts enhanced intestinal function.

[0077] 3. Transcription levels of genes related to inflammation, apoptosis and anti-oxidation in the head kidney

[0078] Head kidney samples of juvenile yellow croaker in each cage were taken and RNA was extracted using Trizol reagent (TaKaRa, Dalian, China) according to the instructions. Subsequently, the concentration and purity of the extracted RNA were quantified using Nanodrop 2000 spectrophotometer (Thermo, Wasserhaus, USA) and further confirmed by 1% agarose gel electrophoresis. Primescript TM Total RNA was reverse transcribed into cDNA using a RT assay kit (TaKaRa, Dalian, China) and prepared for qRT-PCR analysis. The qRT-PCR procedure was based on “Dietary Tribonema sp. supplementation increased growth performance, antioxidant capacity, immunity and improved hepatic health in golden pompano (Trachinotus ovatus)” (Zhao, Wei Fang, Hao-Hang Gao, Bao-Yan Dai, Chen-Min Liu, Zhen-Zhou Zhang, Cheng-Wu Niu, Jin. Dietary Tribonema sp. supplementation increased growth performance, antioxidant capacity, immunity and improved hepatic health in golden pompano (Trachinotus ovatus) [J]. Aquaculture, 2020, 529 (1).). The specific primer sequences used for qRT-PCR are shown in Table 4. 2 -ΔΔCt Methods The expression levels of target genes were calculated, and all results were normalized with the expression of the housekeeping gene β-actin to ensure consistency and accuracy.

[0079] Table 4 qRT-PCR specific primer sequences

[0080]

[0081]

[0082] Among the proinflammatory cytokines, the mRNA levels of toll-like receptor 2A (tlr12α), toll-like receptor 5A (tlr15α), interleukin 8 (il-8), and tumor necrosis factor-α (tnf-α) in the juvenile yellow croaker fed with APS diet, 0.05% ESE, 0.1% ESE, and 0.15% ESE were significantly downregulated (P<0.05). In addition, compared with the control group and the APS diet group, the transcription levels of myeloid differentiation factor 88 (myd88) and toll-like receptor 1 (tlr1) in the juvenile yellow croaker in the groups supplemented with ESE diet were significantly inhibited (P<0.05) (e.g. Figure 4 shown).

[0083] Among the anti-inflammatory cytokines, the mRNA expression levels of interleukin 10 (IL-10) and transforming growth factor-β (TGF-β) in the group supplemented with ESE diet were significantly upregulated compared with the control group and APS diet group (P<0.05). Figure 4 shown).

[0084] Among apoptosis-related genes, the mRNA level of the anti-apoptotic factor B-cell lymphoma-2 (bcl2) in the group supplemented with ESE diet was significantly upregulated compared with the control group and APS diet group (P<0.05). Compared with the control group, the transcription levels of pro-apoptotic factors, especially caspase 3, caspase 8 and caspase 9, were significantly downregulated in the group supplemented with APS diet and ESE diet compared with the control group (P<0.05). In addition, the expression level of the pro-apoptotic factor Bcl2-associated X protein (bax) in the group supplemented with ESE diet was significantly decreased compared with the control group and APS diet group (P<0.05). Figure 5 shown).

[0085] In summary, it can be seen that when APS is used alone, it has a certain promoting effect on the growth of yellow croaker fry. When APS is combined with ESE, a synergistic effect is achieved, and the growth promotion effect is further improved. When APS is used in combination with 0.05% ESE, the intestinal structure of yellow croaker fry is significantly improved and the activity of digestive enzymes is enhanced, achieving the optimal growth rate and feed efficiency. In addition, the synergistic application of APS and ESE improves the antioxidant defense capacity of fry by increasing the activity of antioxidant enzymes. The strategic regulation using APS and ESE combined with the key apoptosis regulators can reduce inflammation-induced cell apoptosis, ensure the health of yellow croaker fry, and exert anti-inflammatory effects by inhibiting the TLR / MyD88 signaling pathway. The synergistic effect of APS and ESE in the above functions is dose-dependent, and excessive supplementation does not further enhance these functions. Considering the relevant results of growth and physiological indicators, the combined use of 0.1% APS and 0.05% ESE has the best effect on improving the growth rate, survival rate and resilience to stress factors of yellow croaker fry.

[0086] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition containing astragalus polysaccharide and Acanthopanax senticosus extract, characterized in that: The mass ratio of the astragalus polysaccharide to the Acanthopanax senticosus extract is 10:(1-10).

2. Use of the composition according to claim 1 in preparing feed with antioxidant effect.

3. Use of the composition according to claim 1 in preparing feed having the effect of alleviating inflammation.

4. Use of the composition according to claim 1 in preparing feed having the effects of improving intestinal structure and enhancing digestive enzyme activity.

5. Use of the composition according to claim 1 in preparing feed having the effect of improving growth rate.

6. A feed for juvenile large yellow croaker, characterized in that: The invention comprises the following components in percentage by weight: 37-43% fish meal, 27-32% soybean meal, 20-24% flour, 2-3% fish oil, 0.8-1.2% soybean lecithin, 2-3% brewer's yeast, 0.5-1.5% complex vitamins, 0.5-1.4% complex mineral salts and 0.1-0.2% of the composition according to claim 1.

7. Use of the large yellow croaker fry feed according to claim 6 in improving the growth rate of large yellow croaker fry, alleviating inflammation of large yellow croaker fry, enhancing the antioxidant capacity of large yellow croaker fry, improving the intestinal structure of large yellow croaker fry and enhancing the activity of digestive enzymes.

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