Micropterus salmoides feed additive and application thereof in breeding of Micropterus salmoides

Abstract

The invention provides a micropterus salmoides feed additive and application thereof in micropterus salmoides culture, and belongs to the field of aquatic nutrition. The additive provided by the invention is composed of DHA and clostridium butyricum according to a mass ratio of 2: 1-30: 1. The invention further provides application of the additive in a largemouth bass feed and provides the largemouth bass feed, the obtained largemouth bass feed can significantly improve the growth performance and protein utilization efficiency of the largemouth bass, improve the meat quality and saturated fatty acid content of the largemouth bass, and improve the feed utilization rate of the largemouth bass. The abundance of the phylum and mycoplasma in the intestinal tract of the largemouth bass is improved, and the health level and economic benefits of the largemouth bass are comprehensively improved.

Description

Technical Field

[0001] The invention belongs to the field of aquatic nutrition, and in particular relates to a largemouth bass feed additive and application thereof in largemouth bass breeding. Background Art

[0002] Largemouth bass (Micropterus salmoides), commonly known as California bass, has the characteristics of rapid growth, easy fishing, short breeding cycle, tender meat, and no intramuscular spines. It is deeply loved by farmers and consumers. However, with the rapid development of intensive aquaculture, in order to further save costs and shorten the breeding cycle, high-energy feed is widely used in aquaculture. Long-term energy imbalance can cause a series of metabolic syndromes in fish, especially liver fat deposition and impaired liver function. Impaired liver function in fish often leads to negative effects such as systemic metabolic disorders and decreased disease resistance, which in turn leads to the outbreak of infectious diseases and syndromes, seriously endangering the green and healthy development of aquaculture. Unbalanced nutrient matching in feed is one of the main causes of liver lesions in farmed fish, which greatly restricts the development of aquaculture. Therefore, it is particularly important to develop functional feed additives and conduct reasonable nutritional intervention for largemouth bass aquaculture in view of liver damage caused by nutritional metabolic disorders of largemouth bass.

[0003] Exploring the protective effects of various functional feed additives on fish liver health is a hot topic in the field of aquatic nutrition. Studies have shown that aquatic feed additives have positive effects in improving feed conversion efficiency, promoting nutrient absorption, enhancing disease resistance, and reducing environmental pollution. In addition, feed additives can not only promote the healthy growth of fish, but also regulate and improve muscle quality through various channels, which has important practical application value for the aquaculture industry. However, at present, although the existing feed additives have a certain preventive and therapeutic effect on some common diseases of largemouth bass, the effect is not stable and significant enough, and usually only focuses on one aspect. The health care effects on other aspects such as intestinal health and liver function of largemouth bass are limited, and the health level of largemouth bass cannot be comprehensively improved. Therefore, providing comprehensive and effective largemouth bass feed additives is one of the key technical issues to be solved in the development of the largemouth bass aquaculture industry. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a largemouth bass feed additive and a largemouth bass feed, which can comprehensively and effectively improve the health level of largemouth bass and the economic benefits of breeding.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a largemouth bass feed additive, which consists of DHA and Clostridium butyricum in a mass ratio of 2:1 to 30:1.

[0007] The invention also provides application of the largemouth bass feed additive in largemouth bass breeding, wherein the addition amount of the feed additive accounts for 0.21-3.1% of the total mass of the feed.

[0008] Preferably, the largemouth bass feed additive improves the growth performance and protein utilization efficiency of largemouth bass, promotes protein deposition, and reduces fat deposition.

[0009] Preferably, the largemouth bass feed additive improves the hardness, chewiness, water holding capacity and saturated fatty acid content of largemouth bass muscles, and reduces the adhesion, juice loss and unsaturated fatty acid content of largemouth bass muscles.

[0010] Preferably, the largemouth bass feed additive increases the intestinal lipase activity of the largemouth bass and reduces the amylase activity of the largemouth bass.

[0011] Preferably, the largemouth bass feed additive increases the abundance of Firmicutes and Mycoplasma in the intestine of largemouth bass.

[0012] The present invention also provides a largemouth bass feed, which comprises, by weight, 35 parts of fish meal, 25 parts of soybean meal, 15 parts of corn protein powder, 10 parts of wheat flour, 3 parts of soybean lecithin, 5 parts of soybean oil, 1.5 parts of calcium dihydrogen phosphate, 1 part of mineral premix, 0.05 parts of vitamin C, 0.02 parts of antioxidant, 0.2 parts of choline chloride, 1.13-4.02 parts of microcrystalline cellulose, and 0.21-3.1 parts of feed additives; the feed additives are the above-mentioned feed additives.

[0013] Preferably, the mineral premix includes vitamin A, 500000 IU / kg; vitamin D3, 100000 IU / kg; vitamin E, 4000 mg / kg; vitamin K3, 1000 mg / kg; vitamin B1, 500 mg / kg; vitamin B2, 1000 mg / kg; vitamin B6, 1000 mg / kg; vitamin B12, 2.0 mg / kg; nicotinic acid, 4000 mg / kg; calcium pantothenate, 2000 mg / kg; biotin, 10.0 mg / kg; folic acid, 100 mg / kg; vitamin C, 15000 mg / kg; iron, 10000 mg / kg; copper, 300 mg / kg; zinc, 5000 mg / kg; manganese, 1200 mg / kg; iodine, 80 mg / kg; cobalt, 20 mg / kg; selenium, 30 mg / kg.

[0014] The invention also provides a method for preparing the largemouth bass feed, comprising the following steps: except for soybean oil, various feed raw materials are mixed uniformly; soybean oil and water are added to the raw material mixture, fully mixed, and granulated.

[0015] The present invention also provides the use of the largemouth bass feed in preparing a product for improving the growth performance of largemouth bass and / or improving the meat quality of largemouth bass and / or regulating the disorder of intestinal flora of largemouth bass.

[0016] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0017] The present invention utilizes DHA and Clostridium butyricum, and obtains a feed additive for largemouth bass by combining DHA and Clostridium butyricum in a specific ratio, and further uses the feed additive to prepare largemouth bass feed, which can improve the growth performance and protein utilization efficiency of largemouth bass, promote protein deposition, and reduce fat deposition; improve the hardness, chewiness, water holding capacity and saturated fatty acid content of largemouth bass muscle, reduce the adhesion, juice loss and unsaturated fatty acid content of largemouth bass muscle; improve the intestinal lipase activity of largemouth bass, reduce the amylase activity of largemouth bass; and increase the abundance of Firmicutes and Mycoplasma in the intestine of largemouth bass. It can not only improve the health level of largemouth bass and reduce the demand for antibiotics, but also improve the environmental impact of aquaculture systems, which is helpful to promote the development of aquaculture industry in a more environmentally friendly, healthier and more sustainable direction. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effects of DHA algae oil and Clostridium butyricum on growth performance and feed utilization of largemouth bass;

[0019] Figure 2 The effects of DHA algae oil and Clostridium butyricum on muscle texture of largemouth bass;

[0020] Figure 3 The effects of DHA algae oil and Clostridium butyricum on water retention in the muscles of largemouth bass;

[0021] Figure 4 The effects of DHA algae oil and Clostridium butyricum on the digestive enzyme activities in the intestine of largemouth bass;

[0022] Figure 5 The effects of DHA algae oil and Clostridium butyricum on the composition of intestinal flora of largemouth bass;

[0023] Figure 6 The effects of DHA algae oil and Clostridium butyricum on the richness and species diversity of intestinal flora of largemouth bass;

[0024] Figure 7 The effects of DHA algae oil and Clostridium butyricum on the expression of genes related to muscle fat synthesis in largemouth bass;

[0025] Figure 8 The effect of DHA algae oil and Clostridium butyricum on the expression of genes related to muscle lipolysis in largemouth bass. DETAILED DESCRIPTION

[0026] The present invention provides a largemouth bass feed additive, which is composed of DHA and Clostridium butyricum in a mass ratio of 2:1 to 30:1, and the ratio is preferably 10:1 to 20:1. DHA is an n-3 polyunsaturated fatty acid containing six double bonds, which can act as an antioxidant and help maintain the integrity of cell membranes. Clostridium butyricum is an obligate anaerobic Gram-positive bacillus that can survive in harsh environments of high temperature, acidity and bile, and is not easily inactivated when passing through the digestive tract. The present invention uses a reasonable ratio of DHA and Clostridium butyricum and uses them as a largemouth bass feed additive, which can effectively protect the liver and intestinal health of largemouth bass, and ensure that largemouth bass better absorbs and utilizes nutrients through the health of the intestines and liver.

[0027] The amount of the feed additive added in the present invention accounts for 0.21-3.1% of the total mass of the feed, preferably 1-2%, and more preferably 1.1%, and the total mass of the feed includes the basic feed and the feed additive. The present invention can improve the growth performance and protein utilization efficiency of largemouth bass, promote protein deposition, and reduce fat deposition by adding the feed additive to the feed; improve the hardness, chewiness, water holding capacity and saturated fatty acid content of largemouth bass muscle, reduce the adhesion, juice loss and unsaturated fatty acid content of largemouth bass muscle; improve the intestinal lipase activity of largemouth bass, and reduce the amylase activity of largemouth bass; increase the abundance of Firmicutes and Mycoplasma in the intestine of largemouth bass, reduce the species richness and diversity of largemouth bass intestinal flora, and significantly change the flora structure; down-regulate genes related to fat synthesis (fas, srebp1), up-regulate genes related to fat oxidation and metabolism (accα, pparγ), and increase the relative expression of mRNA of genes related to fat decomposition (agtl, lpl, hsl, ppraα). It can not only improve the health level of largemouth bass and reduce the need for antibiotics, but also improve the environmental impact of largemouth bass farming systems, helping to promote the largemouth bass farming industry in a more environmentally friendly, healthier and more sustainable direction.

[0028] The present invention also provides a largemouth bass feed, which comprises, by weight, 35 parts of fish meal, 25 parts of soybean meal, 15 parts of corn gluten meal, 10 parts of wheat flour, 3 parts of soybean lecithin, 5 parts of soybean oil, 1.5 parts of calcium dihydrogen phosphate, 1 part of mineral premix, 0.05 parts of vitamin C, 0.02 parts of antioxidant, 0.2 parts of choline chloride, 3.13 parts of microcrystalline cellulose, and 1.1 parts of feed additives; the feed additive is the above feed additive. The antioxidant of the present invention is preferably ethoxyquinoline.

[0029] The present invention also provides a largemouth bass feed, which comprises, by weight, 35 parts of fish meal, 25 parts of soybean meal, 15 parts of corn protein powder, 10 parts of wheat flour, 3 parts of soybean lecithin, 5 parts of soybean oil, 1.5 parts of calcium dihydrogen phosphate, 1 part of mineral premix, 0.05 parts of vitamin C, 0.02 parts of antioxidant, 0.2 parts of choline chloride, 1.13-4.02 parts of microcrystalline cellulose, and 0.21-3.1 parts of feed additives; the feed additives are the above-mentioned feed additives.

[0030] Preferably, the mineral premix includes vitamin A, 500000 IU / kg; vitamin D3, 100000 IU / kg; vitamin E, 4000 mg / kg; vitamin K3, 1000 mg / kg; vitamin B1, 500 mg / kg; vitamin B2, 1000 mg / kg; vitamin B6, 1000 mg / kg; vitamin B12, 2.0 mg / kg; nicotinic acid, 4000 mg / kg; calcium pantothenate, 2000 mg / kg; biotin, 10.0 mg / kg; folic acid, 100 mg / kg; vitamin C, 15000 mg / kg; iron, 10000 mg / kg; copper, 300 mg / kg; zinc, 5000 mg / kg; manganese, 1200 mg / kg; iodine, 80 mg / kg; cobalt, 20 mg / kg; selenium, 30 mg / kg.

[0031] The present invention also provides a method for preparing the above-mentioned largemouth bass feed, comprising the following steps: except for soybean oil, various feed raw materials are mixed uniformly; soybean oil and water are added to the raw material mixture, fully mixed, and granulated. The amount of water added in the present invention is 25-35% of the total weight of the feed raw materials, preferably 30%.

[0032] The present invention also provides the use of the largemouth bass feed in preparing a product for improving the growth performance of largemouth bass and / or improving the meat quality of largemouth bass and / or regulating the disorder of intestinal flora of largemouth bass.

[0033] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] In the specific embodiment of the present invention, DHA was purchased from Beijing Zhuoyuan Hangcheng Technology Development Co., Ltd., and Clostridium butyricum was purchased from Guangdong Microbiological Culture Collection Center, and the strain collection number is GDMCC No.1.2756.

[0035] Before statistical analysis, all test data were analyzed for homogeneity of variance using the Levene method, and the Shapiro-Wilk method was used for normal distribution test. All results were analyzed using one-way ANOVA, followed by Tukey's multiple comparison test, and all results were expressed as mean ± standard deviation (SD), with P < 0.05 as statistically significant. The results of intestinal flora sequencing were analyzed using the Omicsmart dynamic online analysis platform (http: / / www.omicsmart.com).

[0036] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0037] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0038] Example 1

[0039] A largemouth bass feed additive, comprising DHA and Clostridium butyricum in a mass ratio of 10:1.

[0040] Example 2

[0041] A largemouth bass feed additive, comprising DHA and Clostridium butyricum in a mass ratio of 2:1.

[0042] Example 3

[0043] A largemouth bass feed additive, comprising DHA and Clostridium butyricum in a mass ratio of 20:1.

[0044] Example 4

[0045] A largemouth bass feed additive, comprising DHA and Clostridium butyricum in a mass ratio of 30:1.

[0046] Example 5

[0047] A largemouth bass feed: 35wt% of fish meal, 25wt% of soybean meal, 15wt% of corn gluten meal, 10wt% of wheat flour, 3wt% of soybean lecithin, 5wt% of soybean oil, 1.5wt% of calcium dihydrogen phosphate, 1wt% of mineral premix, 0.05wt% of vitamin C, 0.02wt% of antioxidant, 0.2wt% of choline chloride, 3.13wt% of microcrystalline cellulose, and 1.1wt% of the feed additive of Example 1. The antioxidant is ethoxyquin; the mineral premix is: vitamin A, 500000IU / kg; vitamin D3, 100000IU / kg; vitamin E, 4000mg / kg; vitamin K3, 1000mg / kg; vitamin B1, 500mg / kg; vitamin B2, 1000mg / kg; vitamin B6, 1000mg / kg; vitamin B12, 2.0mg / kg; niacin, 4000mg / kg; calcium pantothenate, 2000mg / kg; biotin, 10.0mg / kg; folic acid, 100mg / kg; vitamin C, 15000mg / kg; iron, 10000mg / kg; copper, 300mg / kg; zinc, 5000mg / kg; manganese, 1200mg / kg; iodine, 80mg / kg; cobalt, 20mg / kg; selenium, 30mg / kg.

[0048] Preparation method of largemouth bass feed: accurately weigh various feed raw materials according to the formula, mix the other raw materials evenly except soybean oil, then add soybean oil and water (30% of the total weight) and mix them thoroughly with a blender, finally use a feed granulator to process it into granules with a diameter of 2.5 mm, dry it naturally and store it at -20°C.

[0049] Example 6

[0050] A largemouth bass feed: 35wt% of fish meal, 25wt% of soybean meal, 15wt% of corn gluten meal, 10wt% of wheat flour, 3wt% of soybean lecithin, 5wt% of soybean oil, 1.5wt% of calcium dihydrogen phosphate, 1wt% of mineral premix, 0.05wt% of vitamin C, 0.02wt% of antioxidant, 0.2wt% of choline chloride, 2.13wt% of microcrystalline cellulose, and 2.1wt% of the feed additive of Example 2. The antioxidant is ethoxyquin; the mineral premix is: vitamin A, 500000IU / kg; vitamin D3, 100000IU / kg; vitamin E, 4000mg / kg; vitamin K3, 1000mg / kg; vitamin B1, 500mg / kg; vitamin B2, 1000mg / kg; vitamin B6, 1000mg / kg; vitamin B12, 2.0mg / kg; niacin, 4000mg / kg; calcium pantothenate, 2000mg / kg; biotin, 10.0mg / kg; folic acid, 100mg / kg; vitamin C, 15000mg / kg; iron, 10000mg / kg; copper, 300mg / kg; zinc, 5000mg / kg; manganese, 1200mg / kg; iodine, 80mg / kg; cobalt, 20mg / kg; selenium, 30mg / kg.

[0051] Preparation method of largemouth bass feed: accurately weigh various feed raw materials according to the formula, mix the other raw materials evenly except soybean oil, then add soybean oil and water (25% of the total weight) and mix them thoroughly with a blender, finally use a feed granulator to process it into granules with a diameter of 2.5 mm, dry it naturally and store it at -20°C.

[0052] Example 7

[0053] A largemouth bass feed: 35wt% of fish meal, 25wt% of soybean meal, 15wt% of corn gluten meal, 10wt% of wheat flour, 3wt% of soybean lecithin, 5wt% of soybean oil, 1.5wt% of calcium dihydrogen phosphate, 1wt% of mineral premix, 0.05wt% of vitamin C, 0.02wt% of antioxidant, 0.2wt% of choline chloride, 4.02wt% of microcrystalline cellulose, and 0.21wt% of the feed additive of Example 1. The antioxidant is ethoxyquin; the mineral premix is: vitamin A, 500000IU / kg; vitamin D3, 100000IU / kg; vitamin E, 4000mg / kg; vitamin K3, 1000mg / kg; vitamin B1, 500mg / kg; vitamin B2, 1000mg / kg; vitamin B6, 1000mg / kg; vitamin B12, 2.0mg / kg; niacin, 4000mg / kg; calcium pantothenate, 2000mg / kg; biotin, 10.0mg / kg; folic acid, 100mg / kg; vitamin C, 15000mg / kg; iron, 10000mg / kg; copper, 300mg / kg; zinc, 5000mg / kg; manganese, 1200mg / kg; iodine, 80mg / kg; cobalt, 20mg / kg; selenium, 30mg / kg.

[0054] Preparation method of largemouth bass feed: accurately weigh various feed raw materials according to the formula, mix the raw materials except soybean oil evenly, then add soybean oil and water (35% of the total weight) and mix them thoroughly with a blender, finally use a feed granulator to process it into granules with a diameter of 2.5 mm, dry it naturally and store it at -20°C.

[0055] Example 8

[0056] A largemouth bass feed: 35wt% of fish meal, 25wt% of soybean meal, 15wt% of corn gluten meal, 10wt% of wheat flour, 3wt% of soybean lecithin, 5wt% of soybean oil, 1.5wt% of calcium dihydrogen phosphate, 1wt% of mineral premix, 0.05wt% of vitamin C, 0.02wt% of antioxidant, 0.2wt% of choline chloride, 1.13wt% of microcrystalline cellulose, and 3.1wt% of the feed additive of Example 3. The antioxidant is ethoxyquin; the mineral premix is: vitamin A, 500000IU / kg; vitamin D3, 100000IU / kg; vitamin E, 4000mg / kg; vitamin K3, 1000mg / kg; vitamin B1, 500mg / kg; vitamin B2, 1000mg / kg; vitamin B6, 1000mg / kg; vitamin B12, 2.0mg / kg; niacin, 4000mg / kg; calcium pantothenate, 2000mg / kg; biotin, 10.0mg / kg; folic acid, 100mg / kg; vitamin C, 15000mg / kg; iron, 10000mg / kg; copper, 300mg / kg; zinc, 5000mg / kg; manganese, 1200mg / kg; iodine, 80mg / kg; cobalt, 20mg / kg; selenium, 30mg / kg.

[0057] Preparation method of largemouth bass feed: accurately weigh various feed raw materials according to the formula, mix the other raw materials evenly except soybean oil, then add soybean oil and water (30% of the total weight) and mix them thoroughly with a blender, finally use a feed granulator to process it into granules with a diameter of 2.5 mm, dry it naturally and store it at -20°C.

[0058] Test Example 1 Determination of changes in functional component content

[0059] Largemouth bass was used as the research object to study the effects of the feed additive of the present invention on the growth performance, feed utilization and liver health of largemouth bass: the breeding experiment set up three treatment groups, namely the experimental group (DHA_CB algae group), the control group and the DHA added group (DHA algae oil group). In order to ensure that the blank control group and the DHA group had the same nitrogen and fat content as the experimental group feed, soybean oil was used for adjustment and then microcrystalline cellulose was used for leveling. The feed formula and nutritional level (air-dried basis) of each group are shown in Table 1.

[0060] Table 1 Feed formula and nutritional level of each group

[0061] Raw materials (wt%) Control group DHA algae oil group DHA_CB Algae and Bacteria Group Domestic fish meal 35.00 35.00 35.00 Soybean meal 25.00 25.00 25.00 Corn Gluten Meal 15.00 15.00 15.00 wheat flour 10.00 10.00 10.00 Soy lecithin 3.00 3.00 3.00 Soybean Oil 6.00 5.00 5.00 Calcium dihydrogen phosphate 1.50 1.50 1.50 Mineral premix 1.00 1.00 1.00 Vitamin C 0.05 0.05 0.05 Antioxidants 0.02 0.02 0.02 Choline chloride 0.20 0.20 0.20 Microcrystalline Cellulose 3.23 3.23 3.13 DHA Algae Oil 0.00 1.00 1.00 Clostridium butyricum 0.00 0.00 0.10 total 100.00 100.00 100.00 Protein g / kg 45.34 45.35 45.87 Fat g / kg 11.13 11.17 11.26

[0062] Note: Mineral premix is: vitamin A, 500000IU / kg; vitamin D3, 100000IU / kg; vitamin E, 4000mg / kg; vitamin K3, 1000mg / kg; vitamin B1, 500mg / kg; vitamin B2, 1000mg / kg; vitamin B6, 1000mg / kg; vitamin B12, 2.0mg / kg; nicotinic acid, 4000mg / kg; calcium pantothenate, 2000mg / kg; biotin, 10.0mg / kg; folic acid, 100mg / kg; vitamin C, 15000mg / kg; iron, 10000mg / kg; copper, 300mg / kg; zinc, 5000mg / kg; manganese, 1200mg / kg; iodine, 80mg / kg; cobalt, 20mg / kg; selenium, 30mg / kg. The preparation method of the feed is the same as that of Example 5.

[0063] The feeding experiment was conducted at the research and experimental base of Zhanjiang Animal Science Specialty Agricultural Expo Park. The experimental fish were purchased from Shiziling Xiangshui Fish Fry Farm in Gaozhou City. A total of 270 fry were selected with a body weight of (4.00±0.01) g and randomly divided into 3 groups, each with 3 replicates, and each replicate contained 30 fish. The experimental period was 8 weeks, during which they were fed twice a day (8:00 and 16:00), based on apparent satiation, and the feeding amount was adjusted in time according to the feeding situation of the fry and weather conditions. At the same time, the food intake, number of deaths and corresponding body weight were recorded every day. During the breeding period, the water quality conditions were maintained as follows: pH value between 7.8 and 8.2, water temperature between 28.4 and 31.2℃, ammonia nitrogen mass concentration less than 0.03 mg / L, dissolved oxygen content higher than 6.7 mg / L, seawater salinity between 26 and 28, and continuous aeration.

[0064] After the breeding experiment, the feed consumption during the breeding period was counted, and all fish were fasted overnight. Before sampling, the largemouth bass were anesthetized with 50 mg / L MS-222 (Sigma). The total weight of each tank was weighed and the number of fish was counted to calculate the weight gain rate (WGR), protein efficiency ratio (PER) and feed conversion ratio (FCR). Three whole fish in each tank were randomly selected, and their weight and length were recorded and stored at -20°C. In addition, two fish in each tank were randomly selected and sterile dissected. The liver, muscle and intestinal tissues were quickly frozen in liquid nitrogen and stored at -80°C for subsequent determination of conventional nutrients and analysis of bacterial flora. The dorsal skin was peeled, and then the whole muscle was cut from the base of the pectoral fin to the base of the caudal fin from the head to the tail. The muscles were then divided and the muscles above the lateral line and below the dorsal fin were used for texture detection. The muscles on the other side were placed in cryovials, placed in liquid nitrogen, and then stored at -80°C for the determination of molecular and enzyme activity indicators. The muscles above the midline and below the lateral line were placed in a -80°C refrigerator for the determination of water holding capacity and other routine chemical parameters. Texture and water holding capacity were determined within 48 hours after the sample was collected.

[0065] (1) Determination of conventional nutrients

[0066] Ash, crude protein and crude fat were determined for feed, whole fish, liver and muscle tissue: moisture content was calculated by drying at 105°C to constant weight. Ash content was determined by 550°C muffle furnace burning method. Crude protein was determined by Dumas nitrogen analyzer (Primacs SN100). Crude fat was determined by Soxhlet extraction (36680-Extractor, BUCHI, Switzerland). Each sample was repeated twice. If the relative deviation of the two times was too large, increase the number of repetitions.

[0067] Effects of DHA algae oil and Clostridium butyricum on growth performance and feed utilization of largemouth bass Figure 1As shown. In the figure, A represents the weight gain rate of each group, B represents the feed coefficient of each group, C represents the protein efficiency ratio of each group, D represents the ash content of the whole fish in each group, E represents the protein content of the whole fish in each group, and F represents the fat content of the whole fish in each group. It can be concluded from the figure that the weight gain rate and protein efficiency ratio of the DHA algae oil group and the DHA_CB algae group were significantly higher than those of the control group (P<0.05) (Figure A&C); the feed coefficient of the DHA algae oil group and the DHA_CB algae group was significantly lower than that of the control group (P<0.05) (Figure B); there was no significant difference in the ash content of the fish among the groups (P>0.05) (Figure D); the protein content of the fish in the DHA_CB algae group was significantly higher than that in the DHA algae oil group, and the protein content of the fish in the DHA algae oil group was significantly lower than that in the control group (P<0.05) (Figure E); the fat content of the fish in the DHA_CB algae group was significantly lower than that in the DHA algae oil group and the control group (P<0.05) (Figure F). The above results show that the addition of DHA algae oil and DHA_CB algae can significantly improve the growth performance and protein utilization efficiency of fish, especially DHA_CB algae is more effective in promoting protein deposition and reducing fat deposition, without affecting the water content of the fish.

[0068] The conventional nutritional composition of liver and muscle of largemouth bass is shown in Table 2. From the conventional nutritional composition of liver, it can be seen that there is no significant difference in water content among the groups (P>0.05), the crude ash content of DHA algae oil group is significantly higher than that of control group, the crude protein content of control group is significantly higher than that of DHA algae oil group and DHA_CB algae group, and the crude fat content of DHA_CB algae group is significantly higher than that of control group and DHA algae oil group (P<0.05); from the conventional nutritional composition of muscle, it can be seen that there is no significant difference in crude ash and crude protein content among the groups (P>0.05), the water content of DHA_CB algae group is significantly lower than that of control group and DHA algae oil group, and the crude fat content of DHA_CB algae group is significantly higher than that of control group and DHA algae oil group (P<0.05). The DHA_CB algae group showed higher crude fat content and lower muscle water content, indicating that DHA_CB algae has a potential significant effect in affecting fat metabolism and water metabolism, and may affect fat accumulation by regulating metabolic pathways or nutrient absorption.

[0069] Table 2 Effects of DHA algae oil and Clostridium butyricum on the general nutritional composition of liver and muscle of largemouth bass

[0070] parameter Control group DHA algae oil group DHA_CB Algae and Bacteria Group ANOVA Linear Trend Curve Trend liver(%) Moisture 68.97±0.25 69.30±0.28 69.82±0.67 0.133 0.038 0.133 Ash <![CDATA[1.11±0.04 a ]]> <![CDATA[1.13±0.03 ab ]]> <![CDATA[1.20±0.03 b ]]> 0.034 0.013 0.034 Crude Protein <![CDATA[12.78±0.05 b ]]> <![CDATA[11.82±0.05 a ]]> <![CDATA[11.81±0.06 a ]]> 0.000 0.002 0.000 Crude fat <![CDATA[2.87±0.04 a ]]> <![CDATA[3.02±0.11 a ]]> <![CDATA[3.29±0.07 b ]]> 0.002 0.000 0.002 muscle(%) Moisture <![CDATA[77.52±0.07 b ]]> <![CDATA[77.17±0.24 b ]]> <![CDATA[76.01±0.31 a ]]> 0.000 0.001 0.000 Ash 1.51±0.04 1.49±0.03 1.52±0.08 0.838 0.759 0.838 Crude Protein 19.89±0.27 20.14±0.23 19.62±0.07 0.062 0.279 0.062 Crude fat <![CDATA[1.05±0.04 a ]]> <![CDATA[1.11±0.04 a ]]> <![CDATA[2.00±0.07 b ]]> 0.000 0.001 0.000

[0071] (2) Texture determination

[0072] The texture of the back muscle was measured by using a texture analyzer (TMS-PRO, FTC, USA) in TPA mode to test muscle hardness, elasticity, chewiness, adhesion and cohesion. Parameter settings: cylindrical probe diameter 8 mm; starting force 0.1 N; force sensor with a range of 25 N; detection down speed 30 mm / min; down distance 60% of the thickness of the meat slice. Six points were selected for each piece of fish for measurement, and the average value was used for statistical data.

[0073] Effects of DHA algae oil and Clostridium butyricum on muscle texture of largemouth bass Figure 2 As shown in the figure, A represents the hardness of each muscle group, B represents the elasticity of each muscle group, C represents the cohesion of each muscle group, D represents the adhesion of each muscle group, E represents the chewiness of each muscle group, and F represents the recovery of each muscle group. It can be concluded from the figure that there is no significant difference in muscle elasticity, cohesion, and recovery among the groups (P>0.05) (Figure B&C&F); the muscle hardness and chewiness of the DHA algae oil group and the DHA_CB algae group are significantly higher than those of the control group (P<0.05) (Figure A&E); the muscle adhesion of the DHA_CB algae group is significantly lower than that of the control group and the DHA algae oil group (P<0.05) (Figure D). The above research results show that DHA algae oil and DHA_CB algae improve the hardness and chewiness of fish meat to a certain extent, especially DHA_CB algae has a significant effect on reducing muscle adhesion. Such characteristics may make fish meat have better texture performance during processing and consumption, which meets the needs of some consumers for muscle hardness and low adhesion.

[0074] (3) Determination of water holding capacity

[0075] Muscle water holding capacity includes water loss rate, fat loss rate and juice loss rate, which is an important indicator for evaluating muscle quality. Weigh two pieces of filter paper, record the mass, peel, weigh a piece of muscle mass, wrap the muscle with filter paper, put it in a centrifuge tube, balance the centrifuge tube (put the filter paper on the top), centrifuge at 4000g for 10 minutes, take out the muscle, weigh the filter paper (absorbed the moisture and fat of the muscle), put the filter paper in a weighing bottle, dry (dry water), bake at 75℃ for 2 days, take out the weighing bottle and put it in the room temperature atmosphere for 2 hours and then weigh the dry paper again.

[0076] Effects of DHA algae oil and Clostridium butyricum on water retention in muscles of largemouth bass Figure 3As shown in the figure, A represents the juice loss rate of each group, B represents the water loss rate of each group, C represents the water holding capacity of each group, and D represents the fat loss rate of each group. It can be concluded from the figure that there is no significant difference in the fat loss rate among the groups (P>0.05) (Figure D); the juice loss rate of the DHA_CB algae group is significantly lower than that of the control group (P<0.05) (Figure A); the water loss rate of the DHA_CB algae group is significantly lower than that of the control group and the DHA algae oil group, while the water holding capacity of the DHA_CB algae group is just the opposite (P<0.05) (Figure B&C). DHA_CB algae has a positive effect on the quality characteristics of fish meat, especially in reducing juice loss and water loss rate and enhancing water holding capacity. These characteristics help to improve the juiciness and overall quality of fish meat, indicating that DHA_CB algae may have significant potential in improving the water holding capacity of fish meat and improving taste.

[0077] (4) Measurement of muscle fatty acid content

[0078] Weigh 5.0g of uniform sample into a 250mL flat-bottom flask, accurately add 1.0mL of tridecanoic acid internal standard solution, add about 100mg of pyrogallic acid and a few zeolites, then add 2mL of 95% ethanol and 4mL of water, mix well, add 10mL of 8.3mol / L hydrochloric acid solution, mix well and put it in a 70-80℃ water bath for hydrolysis for 40min (shake the flask every 10min to mix the particles adhering to the flask wall into the solution). Cool the hydrolyzate to room temperature, add 10mL of 95% ethanol and mix well, transfer to a separatory funnel, rinse the flask and stopper with 50mL of ether-petroleum ether mixture, add the rinse liquid into the separatory funnel, cover and shake for 5min, let stand for 10min, collect the ether layer extract into a 250mL flask. Repeat the extraction of the hydrolyzate 3 times. Finally, rinse the separatory funnel with a mixture of ether and petroleum ether, collect the liquid into a 250mL flask, and evaporate it to dryness. Add 8mL of 2% potassium hydroxide methanol solution to the residual fat extract and shake well. Reflux in a water bath at 80°C until the oil droplets dissolve. After cooling, add 7mL of 15% boron trifluoride methanol solution and shake well. Reflux in a water bath at 80°C for 2min. After cooling, add 2.0mL of saturated sodium chloride again. Accurately add 5mL of n-hexane solution and shake. After standing, pipette the upper clear liquid into a sample bottle for analysis.

[0079] On-line: The fatty acid composition was determined by GC-7890B gas chromatograph (Agilent), and the content of each fatty acid was quantified by normalization method and expressed as a percentage (%). Gas chromatography conditions: chromatographic column DB-Fast FAME (90m×0.25mm×0.25μm); injection port temperature: 250℃; split injection: split ratio 20:1; carrier gas: nitrogen; constant pressure: 44psi; FID detector temperature: 250℃; hydrogen flow: 30mL / mim; air flow: 300mL / mim; tail nitrogen flow: 25mL / mim; heating program: initial temperature 75℃, hold for 1min; increase to 200℃ at 35℃ / min, hold for 14min; then increase to 210℃ at 2.5℃ / min, hold for 5min; finally increase to 230℃ at 12℃ / min, hold for 20min. The effects of DHA algae oil and Clostridium butyricum on the fatty acid composition of largemouth bass muscle are shown in Table 3.

[0080] Table 3 Effects of DHA algae oil and Clostridium butyricum on fatty acid composition of largemouth bass muscle

[0081] fatty acid(%) Control group DHA algae oil group DHA_CB Algae and Bacteria Group ANOVA Linear Trend Curve Trend C14:0 0.99±0.03 0.94±0.02 0.93±0.04 0.116 0.053 0.116 C15:0 <![CDATA[0.22±0.00 a ]]> <![CDATA[0.25±0.00 b ]]> <![CDATA[0.25±0.00 b ]]> 0.000 0.001 0.000 C16:0 <![CDATA[17.47±0.32 a ]]> <![CDATA[18.82±0.18 b ]]> <![CDATA[18.39±0.10 b ]]> 0.000 0.028 0.000 C17:0 <![CDATA[0.32±0.00 a ]]> <![CDATA[0.37±0.01 b ]]> <![CDATA[0.37±0.00 b ]]> 0.000 0.001 0.000 C18:0 <![CDATA[4.74±0.0.07 a ]]> <![CDATA[5.64±0.04 b ]]> <![CDATA[5.45±0.18 b ]]> 0.000 0.008 0.000 <![CDATA[ 1 SFA]]> <![CDATA[23.74±0.36 a ]]> <![CDATA[26.02±0.15 c ]]> <![CDATA[25.39±0.24 b ]]> 0.000 0.015 0.000 C16:1 1.81±0.07 1.68±0.06 1.76±0.0.08 0.111 0.480 0.111 C17:1 0.21±0.00 0.20±0.01 0.21±0.01 0.221 0.687 0.221 C18:1 <![CDATA[20.57±0.15 b ]]> <![CDATA[18.80±0.26 a ]]> <![CDATA[19.11±0.39 a ]]> 0.000 0.006 0.000 C20:1 0.73±0.01 0.74±0.03 0.72±0.02 0.235 0.230 0.235 <![CDATA[ 2 MUFA]]> <![CDATA[23.32±0.11 b ]]> <![CDATA[21.42±0.36 a ]]> <![CDATA[21.80±0.49 a ]]> 0.000 0.010 0.000 <![CDATA[ 3 PUFA]]> 52.90±0.29 52.56±0.46 52.75±0.11 0.358 0.525 0.358 C18:2 <![CDATA[37.35±0.25 b ]]> <![CDATA[32.77±0.25 a ]]> <![CDATA[33.05±0.28 a ]]> 0.000 0.001 0.000 C20:2 <![CDATA[1.40±0.05 a ]]> <![CDATA[1.49±0.01 b ]]> <![CDATA[1.38±0.01 a ]]> 0.002 0.797 0.002 C20:3 <![CDATA[0.80±0.03 b ]]> <![CDATA[0.63±0.02 a ]]> <![CDATA[0.60±0.02 a ]]> 0.000 0.000 0.000 C20:4 <![CDATA[1.07±0.02 a ]]> <![CDATA[1.15±0.04 b ]]> <![CDATA[1.11±0.04 ab ]]> 0.016 0.183 0.016 n-6PUFA <![CDATA[40.61±0.34 b ]]> <![CDATA[36.05±0.31 a ]]> <![CDATA[36.14±0.23 a ]]> 0.000 0.000 0.000 C18:3 <![CDATA[3.33±0.03 b ]]> <![CDATA[2.80±0.02 a ]]> <![CDATA[2.83±0.02 a ]]> 0.000 0.001 0.000 C20:5 0.58±0.02 0.60±0.02 0.58±0.03 0.398 0.953 0.398 C22:6 (DHA) <![CDATA[8.38±0.20 a ]]> <![CDATA[13.12±0.19 b ]]> <![CDATA[13.20±0.22 b ]]> 0.000 0.000 0.000 n-3PUFA <![CDATA[12.29±0.17 a ]]> <![CDATA[16.52±0.20 b ]]> <![CDATA[16.61±0.23 b ]]> 0.000 0.000 0.000 <![CDATA[ 4 n-3 / n-6]]> <![CDATA[0.30±0.01 a ]]> <![CDATA[0.46±0.00 b ]]> <![CDATA[0.46±0.01 b ]]> 0.000 0.000 0.000

[0082] As shown in Table 3, the muscle saturated fatty acid content of the DHA algae oil group was significantly higher than that of the other groups (P<0.05), and the muscle saturated fatty acid content of the DHA_CB algae group was significantly higher than that of the control group (P<0.05), which indicated that the addition of DHA algae oil and DHA_CB algae would increase the saturated fatty acid content in muscle; the muscle monounsaturated fatty acid content of the control group was significantly higher than that of the DHA algae oil group and the DHA_CB algae group (P<0.05), indicating that the supplementation of DHA may inhibit the accumulation of monounsaturated fatty acids; the muscle n-6 polyunsaturated fatty acid content of the control group was significantly higher than that of the DHA algae oil group and the DHA_CB algae group (P<0.05 ), the muscle n-3 polyunsaturated fatty acid content of the control group was significantly lower than that of the DHA algae oil group and the DHA_CB algae group (P<0.05), and there was no significant difference in the muscle polyunsaturated fatty acid content among the groups (P>0.05). The muscle n-3 / n-6 polyunsaturated fatty acid ratio of the control group was significantly lower than that of the DHA algae oil group and the DHA_CB algae group (P<0.05), which indicated that the supplementation of DHA algae oil and DHA_CB algae could increase the accumulation of n-3 polyunsaturated fatty acids and reduce the content of n-6 polyunsaturated fatty acids; the muscle DHA content of the control group was significantly lower than that of the DHA algae oil group and the DHA_CB algae group (P<0.05). The addition of DHA algae oil and DHA_CB algae significantly improved the content of n-3 polyunsaturated fatty acids and DHA in fish meat, and significantly increased the ratio of n-3 / n-6 polyunsaturated fatty acids. These changes help to improve the nutritional quality of fish meat, especially DHA_CB algae and DHA algae oil have significant advantages in increasing the content of beneficial fatty acids and improving the ratio of fatty acids.

[0083] (5) Determination of intestinal digestive enzyme activity

[0084] The samples were added to 9 volumes of normal saline at a weight ratio of 1:9, mechanically homogenized with a tissue disruptor in an ice-water bath, centrifuged at 3500 r / min at 4°C for 10 min, and the supernatant was collected in a 1.5 mL centrifuge tube for analysis of trypsin (TRP, JL22682), lipase (LPS, JL22695), and amylase (AMS, JL37039) activities using purchased commercial kits in strict accordance with the instructions of the kits.

[0085] Effects of DHA algae oil and Clostridium butyricum on the activities of digestive enzymes in the intestine of largemouth bass Figure 4As shown in the figure, A represents the intestinal lipase activity of each group, B represents the intestinal amylase activity of each group, and C represents the intestinal trypsin activity of each group. The intestinal lipase activity of the DHA_CB algae group was significantly higher than that of the control group and the DHA algae oil group (P<0.05) (Figure A); the amylase activity of the control group was significantly higher than that of the DHA algae oil group and the DHA_CB algae group (P<0.05) (Figure B); the intestinal trypsin activity of the DHA algae oil group was significantly higher than that of the other groups (P<0.05), and the trypsin activity of the control group was significantly higher than that of the DHA_CB algae group (P<0.05) (Figure C). The effects of DHA_CB algae and DHA algae oil on the activities of different digestive enzymes were different. DHA_CB algae significantly increased lipase activity, which helped the digestion and absorption of fat, while DHA algae oil significantly enhanced trypsin activity, indicating that it had a stronger support effect on protein digestion. In addition, DHA supplementation had an inhibitory effect on amylase activity, which may affect the digestion process of carbohydrates.

[0086] (6) Intestinal flora analysis

[0087] The genomic DNA of the sample was extracted according to the CTAB or SDS method, and the integrity of the DNA was detected by agarose gel electrophoresis. The appropriate amount of the extracted sample was placed in a centrifuge tube and diluted to 1 ng / μl with sterile water. In order to ensure the amplification efficiency and accuracy, the diluted genomic DNA was used as a template, and the V3+V4 target domain of 16S rDNA was amplified by PCR technology using specific primers with Barcode and high-efficiency fidelity enzymes. The PCR was performed as follows: 94℃2min; 98℃10s, 62℃30s, 68℃30s, 30 cycles; and finally 68℃5min. The primer sequences corresponded to 5'-CCTACGGGNGGCW GCAG-3' (341F, SEQID No.1) and 5'-GGACTACHVGGGTATCTAAT-3' (806R, SEQ ID No.2). Then, the PCR product was purified by 2% agarose gel electrophoresis, and the target band was cut and recovered, and quantified using QuantiFluorTM fluorometer. The purified amplified products were mixed in equal amounts, connected to sequencing adapters, and sequencing libraries were constructed and sequenced on the llumina PE250 machine. After filtering the noise sequences and checking the chimeras, the data clustering was operated at a similarity threshold of 97%, and then the data were assigned to the same operational taxonomic units (OTUs). OTUs rarefaction curves and saturation curves were drawn to evaluate the sequencing depth. The Ace, Chao1, Shannon and Simpson indexes of each sample were statistically analyzed by Alpha diversity analysis; the differences in species diversity of each sample were compared by Beta diversity analysis (NMDS analysis, PCOA analysis); the biomarkers with statistical differences between different groups were found by inter-group difference significance analysis (LEFSe analysis); based on the KEGG database, the sample gene function was predicted by Picrust2 function prediction.

[0088] Effects of DHA algae oil and Clostridium butyricum on the composition of intestinal flora of largemouth bass Figure 5As shown. The results showed that the control group, DHA algae oil group and DHA_CB algae and fungi group were mainly composed of Firmicutes, Proteobacteria, Bacteroidota, Actinobacteriota, Planctomycetota, Patescibacteria, Chloroflexi, Spirochaetota, Acidobacteriota and Nitrospirota. Among them, Firmicutes was the dominant phylum, with relative abundances of 35.53%, 22.19% and 68.27% in each group, respectively. Followed by Proteobacteria (28.63%, 47.98%, 21.58%) and Bacteroidetes (12.39%, 7.03%, 1.29%). This indicates that the addition of DHA_CB algae may significantly promote the growth of Firmicutes, and DHA algal oil may help promote the growth of Proteobacteria. In general, the addition of DHA may have an inhibitory effect on Bacteroidetes.

[0089] The community composition at the genus level was mainly composed of Mycoplasma, Aeromonas, Acinetobacter, Klebsiella, Brevinema, Streptococcus, Plesiomonas, Ferruginibacter, Candidatus Jettenia, and Ralstonia. Among them, Mycoplasma was the dominant genus, with relative abundances of 27.21%, 20.18%, and 66.85% in each group, respectively. Aeromonas (0.09%, 16.46%, and 15.58%) and Acinetobacter (11.25%, 7.33%, and 0.26%) followed. This indicated that DHA_CB algae may significantly promote the growth of Mycoplasma and inhibit the growth of Acinetobacter. The relative abundance of Aeromonas was higher in the DHA algae oil group and the DHA_CB algae group (16.46% and 15.58%, respectively), while it was almost absent in the control group, indicating that DHA supplementation is beneficial to the growth of Aeromonas.

[0090] The results showed that DHA_CB algae significantly increased the abundance of Firmicutes and Mycoplasma, which are associated with healthy intestinal function, help maintain intestinal microecological balance, and improve host immune function. The increased abundance of Proteobacteria and Aeromonas in the DHA algae oil group may reflect the promotion of DHA algae oil on specific bacterial communities, which are related to nutrient metabolism and pathogen inhibition. These changes in bacterial communities may help improve the intestinal health and overall growth performance of fish by improving intestinal microecological balance and metabolic function.

[0091] Richness and species diversity of intestinal flora of largemouth bass Figure 6 As shown in the results, the Shannon and Simpson indices of the DHA_CB algae group were significantly lower than those of the control group and the DHA algae oil group (P<0.05); the Sobs, Chao1, and Ace indices of the DHA_CB algae group were significantly lower than those of the control group (P<0.05). The PCoA analysis results showed that the DHA_CB algae group was separated from the samples of the control group and the DHA algae oil group, indicating that the addition of Clostridium butyricum affected the composition of the intestinal microbial community. The above results indicate that DHA_CB algae significantly reduced the species richness and diversity of the intestinal flora of largemouth bass and significantly changed the flora structure. The reduction in flora diversity may reflect the expansion or enhancement of the dominance of specific flora, especially the addition of Clostridium butyricum had a significant effect on the overall flora composition. These results suggest that DHA_CB algae have a potential regulatory role in the intestinal microbial community, which may optimize the intestinal environment by selectively promoting specific beneficial flora, thereby helping to improve the health and growth performance of fish.

[0092] (7) Real-time fluorescence quantitative PCR

[0093] Total RNA from muscle tissue was extracted using the Trizol method, and then the integrity of RNA was evaluated by agarose gel electrophoresis, and RNA was reverse transcribed into cDNA using a cDNA synthesis kit. Finally, fluorescent dye MIX was added and real-time fluorescence quantitative PCR was performed. The qPCR reaction system was 10 μL: 0.4 μL (10 μM) of each forward and reverse primer, 1 μL cDNA, 3.2 μL enzyme-free water, and 5 μL SYBR Green Master Mix. The qPCR reaction conditions were: 95, 10 min; 95, 5 sec; 60, 15 sec, for a total of 40 cycles, and finally the melting curve was analyzed to determine the specificity of the primers. The average value of the internal reference gene β-actin was used to calibrate the target gene, and the gene expression results were determined by relative quantitative methods (2 -ΔΔCt ) for analysis.

[0094] Effects of DHA algae oil and Clostridium butyricum on the expression of genes related to fat synthesis in muscles of largemouth bass Figure 7As shown. The results showed that the relative expression of muscle fas and srebp1 mRNA in the DHA algae oil group was significantly lower than that in the other groups (P<0.05), and the relative expression of muscle fas and srebp1 mRNA in the DHA_CB algae group was significantly lower than that in the control group (P<0.05); the relative expression of muscle accα and pparγ mRNA in the DHA_CB algae group was significantly higher than that in the DHA algae oil group (P<0.05), and there was no significant difference between the relative expression of muscle accα and pparγ mRNA in the DHA algae oil group and the control group (P>0.05). DHA_CB algae has a more significant effect in regulating lipid metabolism genes. Not only did it downregulate genes related to fat synthesis (fas, srebp1), but it also upregulated genes related to fat oxidation and metabolism (accα, pparγ). This may lead to reduced fat synthesis and increased lipolysis, thereby reducing fat accumulation. DHA algae oil mainly downregulated the expression of genes related to fat synthesis (fas, srebp1), but had no significant effect on fat oxidation genes (accα, pparγ). This shows that DHA algae oil is effective in inhibiting fat synthesis, but has limited effect in promoting fat decomposition.

[0095] Effects of DHA algae oil and Clostridium butyricum on the expression of genes related to lipolysis in muscles of largemouth bass Figure 8 As shown in the data, the relative expression levels of muscle agtl, lpl, hsl, and ppraα mRNA in the DHA_CB algae group were significantly higher than those in the other groups (P<0.05), and the relative expression levels of muscle agtl, lpl, hsl, and ppraα mRNA in the control group were significantly higher than those in the DHA algae oil group (P<0.05). These genes (agtl, lpl, hsl, and ppraα) are closely related to fat hydrolysis, lipid metabolism, and oxidative metabolism. Their high expression levels indicate that DHA_CB algae have a positive effect on promoting fat decomposition and fatty acid oxidation. The relative expression level of muscle cpt1 mRNA in the control group was significantly higher than that in the other groups (P<0.05), and the relative expression level of muscle cpt1 mRNA in the DHA_CB algae group was significantly higher than that in the DHA algae oil group (P>0.05). The higher expression of cpt1 suggests that the control group may have a stronger fatty acid oxidation ability. In summary, DHA_CB algae have a comprehensive and significant effect on the regulation of lipid metabolism gene expression, and it has advantages in promoting fat hydrolysis and fatty acid oxidation. DHA algae oil may have an inhibitory effect on lipid metabolism gene expression, especially in the expression of genes related to fat decomposition and fatty acid oxidation, which may lead to insufficient fat metabolism capacity.

[0096] 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 largemouth bass feed additive, characterized in that: The additive consists of DHA and Clostridium butyricum in a mass ratio of 2:1 to 30:

1.

2. The use of the largemouth bass feed additive according to claim 1 in largemouth bass breeding, characterized in that: The added amount of the feed additive accounts for 0.21-3.1% of the total mass of the feed.

3. The use according to claim 2, characterized in that: The largemouth bass feed additive improves the growth performance and protein utilization efficiency of the largemouth bass, promotes protein deposition, and reduces fat deposition.

4. The use according to claim 2, characterized in that: The largemouth bass feed additive improves the hardness, chewiness, water holding capacity and saturated fatty acid content of largemouth bass muscles, and reduces the adhesion, juice loss and unsaturated fatty acid content of largemouth bass muscles.

5. The use according to claim 2, characterized in that: The largemouth bass feed additive increases the intestinal lipase activity of the largemouth bass and reduces the amylase activity of the largemouth bass.

6. The use according to claim 2, characterized in that: The largemouth bass feed additive increases the abundance of Firmicutes and Mycoplasma in the largemouth bass intestine.

7. A largemouth bass feed, characterized in that: The feed comprises, by weight, 35 parts of fish meal, 25 parts of soybean meal, 15 parts of corn protein meal, 10 parts of wheat flour, 3 parts of soybean lecithin, 5 parts of soybean oil, 1.5 parts of calcium dihydrogen phosphate, 1 part of mineral premix, 0.05 parts of vitamin C, 0.02 parts of antioxidant, 0.2 parts of choline chloride, 1.13-4.02 parts of microcrystalline cellulose, and 0.21-3.1 parts of feed additives; the feed additive is the feed additive according to claim 1.

8. The largemouth bass feed according to claim 7, characterized in that: The mineral premix comprises vitamin A, 500000 IU / kg; vitamin D3, 100000 IU / kg; vitamin E, 4000 mg / kg; vitamin K3, 1000 mg / kg; vitamin B1, 500 mg / kg; vitamin B2, 1000 mg / kg; vitamin B6, 1000 mg / kg; vitamin B12, 2.0 mg / kg; nicotinic acid, 4000 mg / kg; calcium pantothenate, 2000 mg / kg; biotin, 10.0 mg / kg; folic acid, 100 mg / kg; vitamin C, 15000 mg / kg; iron, 10000 mg / kg; copper, 300 mg / kg; zinc, 5000 mg / kg; manganese, 1200 mg / kg; iodine, 80 mg / kg; cobalt, 20 mg / kg; and selenium, 30 mg / kg.

9. The method for preparing the largemouth bass feed according to any one of claims 7 to 8, characterized in that: The following steps are involved: The various feed raw materials except soybean oil are mixed uniformly; soybean oil and water are added to the raw material mixture, mixed thoroughly, and granulated.

10. Use of the largemouth bass feed according to any one of claims 7 to 8 in preparing a product for improving the growth performance of largemouth bass and / or improving the meat quality of largemouth bass and / or regulating the intestinal flora disorder of largemouth bass.

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