Low-oxygen-resistant low-fish meal compound feed, preparation method and application thereof

Abstract

The application belongs to the technical field of fishery culture, and provides a low fish meal compound feed with low oxygen tolerance, a preparation method and application thereof, the low fish meal compound feed contains the following raw materials: fish meal, soybean meal, soybean protein isolate, flour, chicken meal, cassava starch, fish oil, soybean oil, lecithin, calcium dihydrogen phosphate, compound vitamin, compound mineral substance, choline, vitamin C, DL-Met, Lys-HCL, Thr, astaxanthin and chrysoflagellate laminarin polysaccharide. Firstly, the compound plant protein such as soybean meal and soybean protein isolate and chicken meal are used to replace the traditional fish meal in the application, the amino acid composition is balanced, the nutritional proportion of the compound feed is optimized, and the proportion of fish meal is reduced to 20%; secondly, the compound antioxidant such as astaxanthin and chrysoflagellate laminarin polysaccharide is added in the compound feed, and the fish body antioxidant capacity can be significantly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fishery culture, and particularly relates to a low-oxygen-tolerant low-fishmeal compound feed and a preparation method and application thereof. BACKGROUND

[0002] As an important marine economic fish, Trachurus japonicus has the advantages of rapid growth, excellent meat quality and strong market demand, and the intensive culture scale has been continuously expanding in recent years. However, under the high-density culture mode, the dissolved oxygen content of the water body fluctuates significantly, especially in the summer high temperature and feeding peak period, low oxygen stress (dissolved oxygen < 3 mg / L) phenomenon is easy to occur, which leads to the decrease of fish feeding rate, metabolic disorder, and decrease of immunity, and even causes large-scale death, resulting in serious economic losses. The existing conventional feed is designed for normal dissolved oxygen environment, and the high protein ratio depends on fish meal as the main protein source, which not only increases the ammonia nitrogen excretion burden and accelerates the deterioration of water quality, but also cannot effectively remove the active oxygen induced by low oxygen due to the lack of antioxidant ingredients, resulting in tissue oxidative damage; at the same time, the feed lacks regulating ingredients for intestinal health, and the risk of intestinal flora imbalance increases under low oxygen environment, which directly affects the nutrient absorption efficiency.

[0003] In addition, the fish meal accounts for 30% to 50% in the traditional feed, and its excessive dependence aggravates the problems of depletion of marine resources, ecological imbalance and rising of breeding cost. It can be seen that the high dependence of traditional aquatic feed on fish meal has caused multiple challenges. Fish meal resources are facing depletion due to overfishing, and the price fluctuation threatens the stability of the aquaculture industry; fish meal production further aggravates the imbalance of marine ecology and destroys the structure of food chain; the fish meal accounts for more than 50% in the feed cost, which significantly compresses the profit space of the industry; at the same time, the nitrogen and phosphorus emissions of high fish meal feed are high, which aggravates the risk of water eutrophication. Under this background, it is an urgent need of the industry to reduce the fish meal dosage to below 20%. However, there is no feed in the existing technology that can solve the above two problems at the same time, therefore, there is an urgent need in the field to develop a new compound feed with low oxygen tolerance and low fish meal characteristics. SUMMARY

[0004] The purpose of the present application is to provide a low-oxygen-tolerant low-fishmeal compound feed and a preparation method and application thereof. The compound feed prepared by the present application can reduce the dependence on fish meal while ensuring the balanced nutrition of fish by scientifically proportioning the replacement protein sources such as plant protein and animal protein, promote the transformation of aquaculture to resource-saving and environment-friendly direction, and realize the dual goals of ecological protection and economic benefit.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The application provides a low-fish meal compound feed with low oxygen tolerance, which contains the following raw materials in parts by weight: 19-21 parts of fish meal, 20-22 parts of soybean meal, 4-6 parts of soybean protein isolate, 22-24 parts of flour, 13-15 parts of chicken meal, 0.5-1.5 parts of cassava starch, 2-3 parts of fish oil, 5-7 parts of soybean oil, 1-3 parts of lecithin, 0.5-1.5 parts of calcium dihydrogen phosphate, 0.5-1.5 parts of compound vitamin, 0.5-1.5 parts of compound mineral, 0.3-0.7 parts of choline, 0.3-0.7 parts of vitamin C, 0.3-0.6 parts of DL-Met, 0.7-0.9 parts of Lys-HCL, 0.3-0.5 parts of Thr, 0.03-0.07 parts of astaxanthin and 0.03-0.07 parts of chryso-phycus laminaria polysaccharide.

[0007] Preferably, the compound vitamin contains the following raw materials in the following concentrations: 23-27 mg / kg of vitamin B1, 43-47 mg / kg of vitamin B2, 19-21 mg / kg of pyridoxine hydrochloride, 0.05-0.15 mg / kg of vitamin B12, 9-11 mg / kg of vitamin K3, 790-810 mg / kg of myo-inositol, 58-62 mg / kg of pantothenic acid, 195-205 mg / kg of nicotinic acid, 19-21 mg / kg of folic acid, 1.1-1.3 mg / kg of biotin, 30-34 mg / kg of retinyl acetate, 4-6 mg / kg of cholecalciferol, 115-125 mg / kg of alpha-tocopherol, 1950-2050 mg / kg of ascorbic acid, 2450-2550 mg / kg of choline chloride, and 145-155 mg / kg of ethoxyquin.

[0008] Preferably, the compound mineral salt contains the following raw materials in the following concentrations: 1.5-2.5 mg / kg of sodium fluoride, 0.6-1.0 mg / kg of potassium iodide, 48-52 mg / kg of cobalt chloride hexahydrate, 9-11 mg / kg of copper sulfate pentahydrate, 78-82 mg / kg of ferrous sulfate monohydrate, 48-52 mg / kg of zinc sulfate monohydrate, 58-62 mg / kg of manganese sulfate monohydrate, 1190-1210 mg / kg of magnesium sulfate heptahydrate, 2990-3010 mg / kg of calcium dihydrogen phosphate monohydrate, and 95-105 mg / kg of sodium chloride.

[0009] The application also provides the use of the low-fish meal compound feed in the preparation of a Trachurus japonicus feed.

[0010] The application also provides a preparation method of the low-fish meal compound feed, which comprises the following steps:

[0011] (1) crushing, sieving, mixing fish meal, soybean meal, soybean protein isolate, flour, chicken meal, cassava starch, calcium dihydrogen phosphate, compound vitamin, compound mineral, choline, vitamin C, DL-Met, Lys-HCL, Thr, astaxanthin and chrysolaminarin powder to obtain mixture 1;

[0012] (2) mixing fish oil, soybean oil, lecithin, water and mixture 1 to obtain mixture 2;

[0013] (3) granulating mixture 2, drying to obtain the expanded feed.

[0014] Preferably, the mesh size of the sieving in step (1) is 190-210 μm.

[0015] Preferably, the temperature of the drying in step (3) is 38-42 ℃, the diameter of the expanded feed is 2-3 mm, and the water content of the expanded feed is <10%.

[0016] The application also provides the use of the low-fish meal compound feed prepared by the preparation method in the preparation of Trachinotus ovatus feed.

[0017] The application has the following beneficial effects:

[0018] The application realizes the synergistic effect of low fish meal and low oxygen tolerance through multi-dimensional innovation. First, the nutrient ratio is optimized, the fish meal ratio is reduced to 20%, and compound plant proteins such as soybean meal and soybean protein isolate are used to replace traditional fish meal, and chicken meal is used to balance the amino acid composition; second, the functional ingredients are strengthened, and compound antioxidants such as astaxanthin and chrysolaminarin are added to significantly improve the antioxidant capacity of fish, and the survival rate under low oxygen is increased to more than 85% in the test. The application can alleviate the risk of eutrophication of aquaculture water, reduce the dependence on oxygenation equipment, and save energy consumption. The low fish meal feature reduces the cost of feed, relieves the pressure on resources, and improves the efficiency of aquaculture. The feed is especially suitable for deep-sea cage culture and other high-risk low-oxygen scenarios, provides technical support for the national green and healthy aquaculture strategy, has significant economic benefits and ecological value, and injects new impetus into the sustainable development of the aquaculture industry. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The transcription level of inflammation and antioxidant-related genes in the liver. DETAILED DESCRIPTION

[0020] The application provides a low-fish meal compound feed with low-oxygen resistance, which contains the following raw materials in parts by weight: 19-21 parts of fish meal, 20-22 parts of soybean meal, 4-6 parts of soybean protein isolate, 22-24 parts of flour, 13-15 parts of chicken meal, 0.5-1.5 parts of cassava starch, 2-3 parts of fish oil, 5-7 parts of soybean oil, 1-3 parts of lecithin, 0.5-1.5 parts of calcium dihydrogen phosphate, 0.5-1.5 parts of compound vitamin, 0.5-1.5 parts of compound mineral substance, 0.3-0.7 parts of choline, 0.3-0.7 parts of vitamin C, 0.3-0.6 parts of DL-Met, 0.7-0.9 parts of Lys-HCL, 0.3-0.5 parts of Thr, 0.03-0.07 parts of astaxanthin and 0.03-0.07 parts of chryso-phycus laminarin polysaccharide.

[0021] In the application, the low-fish meal compound feed preferably contains the following raw materials in parts by weight: 20 parts of fish meal, 21.14 parts of soybean meal, 5 parts of soybean protein isolate, 22.64 parts of flour, 14 parts of chicken meal, 1 part of cassava starch, 2.5 parts of fish oil, 6 parts of soybean oil, 2 parts of lecithin, 1 part of calcium dihydrogen phosphate, 1 part of compound vitamin, 1 part of compound mineral substance, 0.5 part of choline, 0.5 part of vitamin C, 0.43 part of DL-Met, 0.78 part of Lys-HCL, 0.42 part of Thr, 0.05 part of astaxanthin and 0.05 part of chryso-phycus laminarin polysaccharide.

[0022] In the present application, the complex vitamin preferably contains the following concentrations of raw materials: 23-27 mg / kg vitamin B1, 43-47 mg / kg vitamin B2, 19-21 mg / kg pyridoxal hydrochloride, 0.05-0.15 mg / kg vitamin B12, 9-11 mg / kg vitamin K3, 790-810 mg / kg myo-inositol, 58-62 mg / kg pantothenic acid, 195-205 mg / kg nicotinic acid, 19-21 mg / kg folic acid, 1.1-1.3 mg / kg biotin, 30-34 mg / kg retinyl acetate, 4-6 mg / kg cholecalciferol, 115-125 mg / kg alpha-tocopherol, 1950-2050 mg / kg ascorbic acid, 2450-2550 mg / kg choline chloride, 145-155 mg / kg ethoxyquin; further preferably contains the following concentrations of raw materials: 25 mg / kg vitamin B1, 45 mg / kg vitamin B2, 20 mg / kg pyridoxal hydrochloride, 0.1 mg / kg vitamin B12, 10 mg / kg vitamin K3, 800 mg / kg myo-inositol, 60 mg / kg pantothenic acid, 200 mg / kg nicotinic acid, 20 mg / kg folic acid, 1.2 mg / kg biotin, 32 mg / kg retinyl acetate, 5 mg / kg cholecalciferol, 120 mg / kg alpha-tocopherol, 2000 mg / kg ascorbic acid, 2500 mg / kg choline chloride, 150 mg / kg ethoxyquin.

[0023] In the present application, the complex mineral salt preferably contains the following concentrations of raw materials: 1.5-2.5 mg / kg sodium fluoride, 0.6-1.0 mg / kg potassium iodide, 48-52 mg / kg cobalt chloride hexahydrate, 9-11 mg / kg copper sulfate pentahydrate, 78-82 mg / kg ferrous sulfate monohydrate, 48-52 mg / kg zinc sulfate monohydrate, 58-62 mg / kg manganese sulfate monohydrate, 1190-1210 mg / kg magnesium sulfate heptahydrate, 2990-3010 mg / kg calcium phosphate monohydrate, and 95-105 mg / kg sodium chloride; further preferably contains the following concentrations of raw materials: 2 mg / kg sodium fluoride, 0.8 mg / kg potassium iodide, 50 mg / kg cobalt chloride hexahydrate, 10 mg / kg copper sulfate pentahydrate, 80 mg / kg ferrous sulfate monohydrate, 50 mg / kg zinc sulfate monohydrate, 60 mg / kg manganese sulfate monohydrate, 1200 mg / kg magnesium sulfate heptahydrate, 3000 mg / kg calcium phosphate monohydrate, and 100 mg / kg sodium chloride.

[0024] The present application also provides the use of the low-fish meal compound feed in the preparation of a Trachurus japonicus feed.

[0025] The present application also provides a preparation method of the low-fish meal compound feed, comprising the following steps:

[0026] (1) crushing, sieving, mixing fish meal, soybean meal, soybean protein isolate, flour, chicken meal, cassava starch, calcium dihydrogen phosphate, compound vitamin, compound mineral, choline, vitamin C, DL-Met, Lys-HCL, Thr, astaxanthin and ecklonia maxima polysaccharide powder to obtain mixture 1;

[0027] (2) mixing fish oil, soybean oil, lecithin, water and mixture 1 to obtain mixture 2;

[0028] (3) granulating mixture 2 and drying to obtain the expanded feed.

[0029] In the present application, the mesh size of the sieve in step (1) is preferably 190-210 μm, and more preferably 200 μm.

[0030] In the present application, the temperature of the drying in step (3) is preferably 38-42℃, and more preferably 40℃, the diameter of the expanded feed is preferably 2-3 mm, and more preferably 2.5 mm, and the moisture content of the expanded feed is preferably <10%, and more preferably 8%.

[0031] The application also provides the use of the low-fish meal compound feed prepared by the preparation method in the preparation of Trachurus japonicus feed.

[0032] The technical solutions provided by the present application will be described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0033] Example 1 Raw material composition of experimental feed

[0034] Five experimental diets with equal nitrogen and lipid content were set, including D1 group, D2 group, D3 group, D4 group and D5 group. Among them, D1 group is the control group, normal fish meal level group; D2 group is the low fish meal level group; D3 group is the low fish meal supplemented with 0.05% astaxanthin diet; D4 group is the low fish meal supplemented with 0.05% ecklonia maxima polysaccharide diet; D5 group is the low fish meal supplemented with 0.05% astaxanthin and 0.05% ecklonia maxima polysaccharide diet. The composition of each group of experimental feed is shown in Table 1, and the nutrient content of each group of experimental feed is shown in Table 2.

[0035] Table 1 Composition of experimental feed in different groups

[0036]

[0037]

[0038] Table 2 Nutrient content of experimental feed in different groups

[0039] Nutritional level (%) D1 D2 D3 D4 D5 Moisture content 9.53 9.64 9.39 9.72 9.66 Protein content 39.70 39.59 39.62 39.74 39.63 Fat content 15.29 15.36 15.33 15.38 15.34 Ash 10.26 10.43 10.19 10.28 10.35

[0040] Note: The compound vitamins used in each feed are 25 mg / kg vitamin B1, 45 mg / kg vitamin B2, 20 mg / kg pyridoxine hydrochloride, 0.1 mg / kg vitamin B12, 10 mg / kg vitamin K3, 800 mg / kg inositol, 60 mg / kg pantothenic acid, 200 mg / kg nicotinic acid, 20 mg / kg folic acid, 1.20 mg / kg biotin, 32 mg / kg retinyl acetate, 5 mg / kg cholecalciferol, 120 mg / kg alpha-tocopherol, 2000 mg / kg ascorbic acid, 2500 mg / kg choline chloride, 150 mg / kg ethoxyquin.

[0041] The compound mineral salts are 2 mg / kg sodium fluoride, 0.8 mg / kg potassium iodide, 50 mg / kg cobalt chloride hexahydrate, 10 mg / kg copper sulfate pentahydrate, 80 mg / kg ferrous sulfate monohydrate, 50 mg / kg zinc sulfate monohydrate, 60 mg / kg manganese sulfate monohydrate, 1200 mg / kg magnesium sulfate heptahydrate, 3000 mg / kg calcium phosphate monohydrate, 100 mg / kg sodium chloride.

[0042] Preparation of experimental feed of Example 2

[0043] Experimental method: all ingredients except fish oil, soybean oil and lecithin are ground and filtered using a 200 μm mesh screen to collect the undersize; then the undersize is weighed and mixed according to the formula proportions to obtain mixture 1; then the formula proportion of fish oil, the formula proportion of soybean oil, the formula proportion of lecithin and water are added to mixture 1 in sequence and stirred vigorously until uniform to obtain mixture 2; then the obtained mixture 2 is made into puffed feed with a diameter of 2.5 mm and dried in a ventilated oven at 40°C until the moisture content of the feed is reduced to below 10%. Finally, the experimental feed is stored at -20°C.

[0044] Application Example 1

[0045] The 5 groups of experimental feed raw materials D1, D2, D3, D4 and D5 of Example 1 are respectively prepared into corresponding experimental feeds according to the preparation method of Example 2.

[0046] The juvenile Trachurus ovatus were obtained from a commercial hatchery (Yiyue Aquaculture Technology Co., Ltd.) in Ningde, Fujian Province, China, and were acclimated for two weeks in floating net cages (4.0 m x 3.0 m x 2.5 m) to adapt to the experimental conditions. Before the start of the feeding trial, all fish were subjected to a 24 h fasting period. Subsequently, 800 juvenile Trachurus ovatus with an initial average weight of 7.00 ± 0.03 g were randomly distributed among 20 smaller floating cages (1 m x 1 m x 1.5 m), with 40 fish per cage. The experimental diets were then randomly allocated to four cages per treatment. During the 8-week experiment, fish were fed twice daily at 7:00 a.m. and 6:00 p.m. to ensure their nutritional needs were met. During this period, water temperature was maintained within the optimal range of 28 ± 1 °C, and dissolved oxygen levels were always maintained above 6.0 mg / L.

[0047] After the 8-week feeding trial, the juvenile Trachurus ovatus of each group were subjected to a 24 h fasting period, followed by anesthesia with 100 mg / L eugenol solution. The body weight of the juvenile Trachurus ovatus in different treatment groups was measured, and the final average body weight (FBW), specific growth ratio (SGR), feed efficiency (FE), and survival rate (SR) were calculated. The specific results are shown in Table 2. The data are expressed as the mean and standard deviation.

[0048] Specific growth rate (%) = (ln final weight - ln initial weight) / experimental days x 100; Equation 1

[0049] Feed efficiency = (final weight - initial weight) / feed intake; Equation 2

[0050] Survival rate (%) = final fish number / initial fish number x 100. Equation 3

[0051] Table 3 Growth performance of juvenile Trachurus ovatus in different treatment groups

[0052] D1 D2 D3 D4 D5 IBW (g) 6.98±0.03 6.99±0.03 7.01±0.03 7.03±0.02 7.01±0.02 FBW (g) 31.00 ± 1.14 b ]] 28.17 ± 0.26 c ]] 33.41 ± 0.75 ab ]] 33.15 ± 1.09 ab ]] 33.73 ± 0.43 a ]] SGR (% / d) 3.55 ± 0.10 a ]] 3.32 ± 0.03 b ]] 3.72 ± 0.04 a ]] 3.69 ± 0.08 a ]] 3.74 ± 0.03 a ]] FE 0.75 ± 0.06 a ]] 0.63 ± 0.03 b ]] 0.77 ± 0.02 a ]] 0.78 ± 0.03 a ]] 0.79 ± 0.04 a ]] SR (%) 87.50±0.01 87.50±0.02 88.75±0.01 90.63±0.02 90.00±0.02

[0053] As shown in Table 3, compared with the control group D1, the final body weight, specific growth rate and feed efficiency of the low fish meal group D2 were significantly reduced (P<0.05). Notably, after adding astaxanthin (group D3), chrysolaminarin (group D4) or both (group D5) in the low fish meal feed, the above growth indicators were significantly improved compared with group D2 (P<0.05), and restored to the level comparable to the control group (P>0.05). There was no significant difference in survival rate among the experimental groups (P>0.05). This finding indicates that although low fish meal feed can significantly inhibit the growth performance and feed utilization efficiency of the Trachinotus ovatus, the addition of functional additives such as astaxanthin or chrysolaminarin can effectively alleviate this negative impact and maintain the growth performance at a normal level.

[0054] Application Example 2

[0055] After the determination of growth performance indicators, 10 Trachinotus ovatus juveniles were randomly selected from each experimental net cage for tissue sample collection. The liver tissue was quickly frozen in liquid nitrogen for analysis of antioxidant markers and gene expression levels, and the intestinal tissue was also quickly frozen in liquid nitrogen for subsequent digestive enzyme activity detection.

[0056] 1. Detection of antioxidant capacity and digestive enzyme activity

[0057] The collected intestinal or liver tissue was added to pre-cooled physiological saline at a ratio of 1:10 (w / v), homogenized under ice bath conditions, and then centrifuged at 4°C and 2000 rpm for 30 minutes to obtain tissue supernatant, which was used to determine liver antioxidant parameters, including catalase CAT (A007-1), superoxide dismutase SOD activity (A001-1), total antioxidant capacity T-AOC (A015-2-1) and malondialdehyde MDA (A003-1) content, as well as intestinal digestive enzyme activities: pepsin, lipase and alpha-amylase. All tests were performed according to the operating procedures provided in the kit instructions from Nanjing Jiancheng Biological Engineering Institute. The specific test results are shown in Tables 4 and 5.

[0058] Table 4 Antioxidant capacity of Trachinotus ovatus juveniles in different treatment groups

[0059]

[0060] According to the results of Table 4, compared with the control group D1, the activities of superoxide dismutase (SOD) and catalase (CAT) and the total antioxidant capacity (T-AOC) of the liver in the low fish meal group D2 were significantly reduced, while the content of malondialdehyde (MDA) was significantly increased (P<0.05). After adding astaxanthin (D3 group), chrysolaminarin (D4 group) or both (D5 group) in the low fish meal, the antioxidant indexes were significantly improved compared with D2 group (P<0.05), and the activities of SOD, CAT and T-AOC in D5 group were better than those in other experimental groups. The results showed that low fish meal diet could weaken the antioxidant capacity of fish and induce oxidative damage, but the addition of astaxanthin or chrysolaminarin could effectively alleviate this negative effect, and the combined use of the two showed a synergistic effect in improving the antioxidant capacity, which provided an important basis for the functional optimization of low fish meal diet.

[0061] Table 5 Digestive capacity of juvenile Trachinotus ovatus in different treatment groups

[0062]

[0063] As shown in Table 5, compared with the control group D1, the activities of intestinal lipase and pepsin in the low fish meal group D2 were significantly reduced (P<0.05). After adding astaxanthin (D3 group), chrysolaminarin (D4 group) or both (D5 group) in the low fish meal, the activities of lipase and pepsin were significantly improved compared with D2 group (P<0.05), and the activities of lipase in D3, D4 and D5 groups were significantly higher than those in the control group (P<0.05). There was no significant difference in the activities of a-amylase among the experimental groups (P>0.05). This finding showed that low fish meal diet could inhibit the digestion efficiency of fat and protein, but the addition of astaxanthin or chrysolaminarin could effectively reverse this inhibition, and the enhancement effect of lipase activity was particularly significant, which provided an important basis for the functional improvement of low fish meal diet.

[0064] 2. Transcription levels of inflammation and antioxidant-related genes in the liver

[0065] The liver samples of juvenile Trachinotus ovatus in each net cage were taken, and the Trizol reagent (TaKaRa, Dalian, China) was used for RNA extraction according to the instructions. Then, the concentration and purity of the extracted RNA were quantified using Nanodrop 2000 spectrophotometer (Thermo, Waltham, USA), and further confirmed by 1% agarose gel electrophoresis. Then the extracted RNA was reversely transcribed into cDNA using primescrisript RT reagent kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. TMTotal RNA was reverse-transcribed into cDNA by RT kit (TaKaRa, Dalian, China) for qRT-PCR analysis. The qRT-PCR procedure and specific primer sequences used are referred to "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).

[0066] The specific qRT-PCR conditions of the present application are as follows:

[0067] The experimental instruments are LightCycler 480 Real-Time Fluorescent Quantitative PCR Instrument (Roche Applied Science, Switzerland) and Premix ExTaq TM II (Takara, Dalian, China) kit;

[0068] The primer sequences used for fluorescent quantitative PCR are shown in Table 6;

[0069] Table 6 Primer sequences used for fluorescent quantitative PCR

[0070]

[0071] The reaction system of 20 μL is as follows: 10 μL Premix ExTaq TM II, 0.8 μL Forward Primer, 0.8 μL Reverse Primer, 6.4 μL RNase Free dH2O, 2 μL cDNA;

[0072] The reaction conditions were set to 95℃ for 1 min, followed by 40 cycles (95℃, 5 s; 60℃, 15 s; 72℃, 20 s).

[0073] 2 -ΔΔCt The expression levels of the target genes were calculated, and all results were normalized to the expression of the housekeeping gene β-actin to ensure consistency and accuracy, as shown in Figure 1 .

[0074] As Figure 1 shown, compared with the control group (D1), the mRNA expression levels of NF-E2-related nuclear factor 2 (Nrf2), manganese superoxide dismutase (Mn-SOD), and heme oxygenase-1 (HO-1) in the low fish meal group (D2) were significantly reduced (P<0.05), while the expression of Kelch-like ECH-associated protein 1 (Keap1) was significantly increased (P<0.05). After supplementing astaxanthin (D3 group), laminarin (D4 group), or both (D5 group) in the low fish meal diet, the gene expression levels of Nrf2, Mn-SOD, and HO-1 were significantly restored compared with the D2 group (P<0.05), and the levels in the relevant treatment groups even significantly exceeded those in the control group (P<0.05). Notably, the gene expression levels of Nrf2, Mn-SOD, and HO-1 reached the highest in the D5 group, showing a trend of better performance than the single additive group. These results suggest that low fish meal diet may damage the fish antioxidant defense system by activating the negative feedback regulation mechanism of the Keap1-Nrf2 signaling pathway, while astaxanthin and laminarin may more effectively activate the pathway through a synergistic mechanism, thereby enhancing the antioxidant capacity of the body.

[0075] In terms of pro-inflammatory cytokine expression, the low fish meal group (D2) showed significant activation of inflammatory response compared with the control group (D1), specifically manifested as significantly increased mRNA expression levels of pro-inflammatory factors IL-1β and IL-8 (P<0.05). The feed additive intervention experiment showed that astaxanthin (D3 group), laminarin (D4 group), and their combined addition (D5 group) could significantly inhibit the up-regulation of pro-inflammatory factor expression induced by low fish meal diet (P<0.05). Notably, the expression levels of IL-1β and IL-8 in each additive treatment group were not only significantly lower than those in the D2 group (P<0.05), but the relevant treatment groups even showed a trend of being lower than the control group (P<0.05). These results suggest that low fish meal diet may induce inflammatory response by activating the pro-inflammatory signaling pathway, while astaxanthin, laminarin, or their combination can effectively alleviate the inflammatory state and may enhance the anti-inflammatory defense capacity of the body.

[0076] Among the anti-inflammatory cytokines, the mRNA expression levels of transforming growth factor beta 1 (TGF-β1) and interleukin 10 (IL-10) in the low fish meal group (D2) were significantly down-regulated compared with the control group (D1) (P<0.05). Notably, when astaxanthin (D3 group), chrysolaminarin (D4 group), or both (D5 group) were added to the low fish meal feed, the inhibitory effect was significantly reversed (P<0.05), among which the combined addition group (D5) had the most significant effect on the expression of TGF-β1 and IL-10, not only significantly higher than the D2 group (P<0.05), but also showing an enhanced trend better than each single additive group. These results suggest that astaxanthin and chrysolaminarin may more effectively activate the anti-inflammatory signaling pathway through a synergistic mechanism, thereby improving the problem of decreased anti-inflammatory capacity caused by low fish meal feed.

[0077] Application Example 3

[0078] After 8 weeks of feeding experiment, the off-water acute hypoxia stress experiment of juvenile Trachinotus ovatus was carried out. During the experiment, 20 healthy juvenile fish were randomly selected from each experimental group and placed in a water-free foam box for 15 minutes of off-water exposure treatment, and then they were put back into the original net cage for recovery. The standard for determining death was the cessation of gill cover movement within 1 hour, and the number of deaths in each group was accurately recorded. The experimental results showed that compared with the control group (D1), the survival rate of the low fish meal group (D2) after acute hypoxia stress was significantly reduced (P<0.05), indicating that low fish meal feed significantly affected the fish's ability to tolerate low oxygen. Notably, after supplementing astaxanthin (D3 group), chrysolaminarin (D4 group), or both (D5 group) in the low fish meal feed, the survival rate of each additive group was significantly higher than that of D1 and D2 groups (P<0.05), among which the survival rate of the combined addition group (D5) was the best (P<0.05) (as shown in Table 7). These results fully demonstrate that astaxanthin and chrysolaminarin can effectively enhance the tolerance of Trachinotus ovatus to acute hypoxia stress, and the two additives have a significant synergistic effect in improving the fish's ability to resist hypoxia.

[0079] Table 7 Survival rate of juvenile Trachinotus ovatus after off-water stress

[0080] Survival rate (%) D1 55 ± 2.04 c ]] D2 38.75 ± 2.39 d ]] D3 62.5 ± 1.44 b ]] D4 62.5 ± 3.23 b ]] D5 70 ± 2.04 a ]]

[0081] In summary, low fish meal feed can significantly inhibit fish growth performance (final body weight, specific growth rate and feed efficiency), reduce liver antioxidant enzyme (SOD, CAT) activity and total antioxidant capacity (T-AOC), weaken intestinal lipase and pepsin activity, inhibit Nrf2-Keap1 signaling pathway and down-regulate antioxidant gene (Nrf2, Mn-SOD, HO-1) expression, destroy the balance of pro-inflammatory factors (IL-1β, IL-8) and anti-inflammatory factors (TGF-β1, IL-10), and ultimately lead to decreased antioxidant capacity, exacerbated inflammation and weakened hypoxia tolerance in fish. It is worth noting that the addition of astaxanthin or dulse polysaccharide in low fish meal feed can not only effectively reverse the above negative effects and restore all indicators to normal levels, but also show significant synergistic effects in improving antioxidant capacity, improving inflammation and enhancing hypoxia tolerance when used in combination, providing important theoretical basis and practical guidance for the functional optimization of low fish meal feed.

[0082] From the above examples, the present application provides a low fish meal compound feed with hypoxia tolerance and its preparation method and application. The low fish meal compound feed contains the following raw materials: fish meal, soybean meal, soybean protein isolate, flour, chicken meal, cassava starch, fish oil, soybean oil, lecithin, calcium dihydrogen phosphate, complex vitamins, complex minerals, choline, vitamin C, DL-Met, Lys-HCL, Thr, astaxanthin and dulse polysaccharide. First, the present application uses compound plant protein such as soybean meal and soybean protein isolate to replace traditional fish meal with chicken meal to balance the amino acid composition and optimize the nutritional ratio of the compound feed, reducing the fish meal proportion to 20%. Second, the compound feed of the present application adds compound antioxidants such as astaxanthin and dulse polysaccharide, which can significantly improve the antioxidant capacity of fish.

[0083] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. A low-fishmeal compound feed tolerant to low oxygen levels, characterized in that, The low-fishmeal compound feed contains the following ingredients in parts by weight: 19-21 parts fishmeal, 20-22 parts soybean meal, 4-6 parts soy protein isolate, 22-24 parts wheat flour, 13-15 parts chicken meal, 0.5-1.5 parts tapioca starch, 2-3 parts fish oil, 5-7 parts soybean oil, 1-3 parts lecithin, 0.5-1.5 parts calcium dihydrogen phosphate, 0.5-1.5 parts compound vitamins, 0.5-1.5 parts compound minerals, 0.3-0.7 parts choline, 0.3-0.7 parts vitamin C, 0.3-0.6 parts DL-Met, 0.7-0.9 parts Lys-HCl, 0.3-0.5 parts Thr, 0.03-0.07 parts astaxanthin, and 0.03-0.07 parts golden algae kelp polysaccharide.

2. The low-fishmeal compound feed according to claim 1, characterized in that, The compound vitamin contains the following concentrations of raw materials: 23-27 mg / kg vitamin B1, 43-47 mg / kg vitamin B2, 19-21 mg / kg pyridoxine hydrochloride, 0.05-0.15 mg / kg vitamin B12, 9-11 mg / kg vitamin K3, 790-810 mg / kg inositol, 58-62 mg / kg pantothenic acid, 195-205 mg / kg niacin, 19-21 mg / kg folic acid, 1.1-1.3 mg / kg biotin, 30-34 mg / kg retinyl acetate, 4-6 mg / kg cholecalciferol, 115-125 mg / kg α-tocopherol, 1950-2050 mg / kg ascorbic acid, 2450-2550 mg / kg choline chloride, and 145-155 mg / kg ethoxyquin.

3. The low-fishmeal compound feed according to claim 2, characterized in that, The composite mineral salt contains the following concentrations of raw materials: 1.5-2.5 mg / kg sodium fluoride, 0.6-1.0 mg / kg potassium iodide, 48-52 mg / kg cobalt chloride hexahydrate, 9-11 mg / kg copper sulfate pentahydrate, 78-82 mg / kg ferrous sulfate monohydrate, 48-52 mg / kg zinc sulfate monohydrate, 58-62 mg / kg manganese sulfate monohydrate, 1190-1210 mg / kg magnesium sulfate heptahydrate, 2990-3010 mg / kg calcium dihydrogen phosphate monohydrate, and 95-105 mg / kg sodium chloride.

4. The application of the low-fishmeal compound feed according to any one of claims 1 to 3 in the preparation of oval pomfret feed.

5. The method for preparing low-fishmeal compound feed according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Fish meal, soybean meal, soy protein isolate, flour, chicken meal, tapioca starch, calcium dihydrogen phosphate, compound vitamins, compound minerals, choline, vitamin C, DL-Met, Lys-HCl, Thr, astaxanthin and kelp polysaccharide are pulverized, sieved and mixed to obtain mixture 1; (2) Mix fish oil, soybean oil, lecithin, water and mixture 1 to obtain mixture 2; (3) Granulate the mixture 2 and dry it to obtain extruded feed.

6. The preparation method according to claim 5, characterized in that, The sieve size in step (1) is 190~210μm.

7. The preparation method according to claim 6, characterized in that, The drying temperature in step (3) is 38~42℃, the diameter of the extruded feed is 2~3mm, and the moisture content of the extruded feed is <10%.

8. The application of the low-fishmeal compound feed prepared by the preparation method according to any one of claims 5 to 7 in the preparation of oval pomfret feed.

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