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

Abstract

The invention belongs to the technical field of fishery breeding, and provides a low-oxygen-resistant low-fish-meal compound feed as well as a preparation method and application thereof. The low-fish-meal compound feed is prepared from the following raw materials: fish meal, soybean meal, soybean protein isolate, flour, chicken meal, cassava starch, fish oil, soybean oil, lecithin, monocalcium phosphate, compound vitamins, compound minerals, choline, vitamin C, DL-Met, Lys-HCL, Thr, astaxanthin and chrysophyta laminarin. Firstly, the soybean meal, the soybean protein isolate and other composite plant proteins and the chicken meal are adopted to replace traditional fish meal, amino acid composition is balanced, the nutrition proportion of the compound feed is optimized, and the proportion of the fish meal is reduced to 20%; in addition, compound antioxidants such as astaxanthin and chrysophyta laminarin are added into the compound feed disclosed by the invention, so that the oxidation resistance of fish bodies can be remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of fishery breeding, and in particular to a low-oxygen-resistant low-fishmeal compound feed, a preparation method thereof, and an application thereof. Background Art

[0002] As an important marine economic fish, the scale of intensive farming of ovate pomfret has continued to expand in recent years due to its rapid growth, excellent meat quality and strong market demand. However, under the high-density farming model, the dissolved oxygen content in the water body fluctuates significantly, especially in the high temperature and peak feeding period in summer, which can easily lead to hypoxia stress (dissolved oxygen <3mg / L), resulting in decreased fish feeding rate, metabolic disorders, reduced immunity, and even large-scale mortality, causing serious economic losses. Existing conventional feeds are mostly designed for normal dissolved oxygen environments. Their high protein ratio relies on fish meal as the main protein source, which not only increases the burden of ammonia nitrogen excretion and accelerates the deterioration of water quality, but also due to insufficient antioxidant components, it cannot effectively remove hypoxia-induced reactive oxygen species, leading to tissue oxidative damage; at the same time, the feed lacks regulatory ingredients for intestinal health, and the risk of intestinal flora imbalance in a hypoxic environment increases, which directly affects the efficiency of nutrient absorption.

[0003] Furthermore, fishmeal accounts for as much as 30% to 50% of traditional feeds, and this overreliance has exacerbated issues such as marine resource depletion, ecological imbalance, and rising aquaculture costs. This heavy reliance on fishmeal in traditional aquaculture feeds has posed multiple challenges. Fishmeal resources are facing depletion due to overfishing, and drastic price fluctuations threaten the stability of the aquaculture industry. Fishmeal production further exacerbates marine ecological imbalances and disrupts the food chain. Fishmeal accounts for over 50% of feed costs, significantly compressing industry profit margins. Furthermore, high-fishmeal feeds emit high levels of nitrogen and phosphorus, exacerbating the risk of eutrophication. Against this backdrop, reducing fishmeal usage to below 20% has become an urgent industry need. However, existing feeds do not address both of these issues simultaneously. Therefore, there is an urgent need to develop new compound feeds that combine hypoxia tolerance with low fishmeal content. Summary of the Invention

[0004] The purpose of the present invention is to provide a hypoxia-resistant low-fish meal compound feed, a preparation method and application thereof. The compound feed prepared by the present invention can reduce dependence on fish meal while ensuring nutritional balance of fish by scientifically proportioning alternative protein sources such as plant protein and animal protein, promote the transformation of aquaculture towards resource conservation and environmental friendliness, and achieve the dual goals of ecological protection and economic benefits.

[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 low-fish meal compound feed that is resistant to hypoxia. The low-fish meal compound feed 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 isolated soy protein, 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 complex vitamins, 0.5-1.5 parts of complex minerals, 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 golden algae laminarin.

[0007] Preferably, the multivitamin contains the following raw materials in the following concentrations: 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.

[0008] Preferably, the composite mineral salt contains raw materials in the following concentrations: 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.

[0009] The present application also provides the use of the low-fishmeal compound feed in preparing oval pomfret feed.

[0010] The present application also provides a method for preparing the low-fishmeal compound feed, comprising the following steps:

[0011] (1) grinding, sieving, and mixing fish meal, soybean meal, soy protein isolate, flour, chicken meal, tapioca starch, calcium dihydrogen phosphate, multivitamins, multiminerals, choline, vitamin C, DL-Met, Lys-HCL, Thr, astaxanthin, and kelp polysaccharide to obtain a mixture 1;

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

[0013] (3) Granulate the mixture 2 and dry it to obtain an expanded feed.

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

[0015] Preferably, the drying temperature in step (3) is 38-42° C., the diameter of the puffed feed is 2-3 mm, and the water content of the puffed feed is less than 10%.

[0016] The present application also provides the use of the low-fishmeal compound feed prepared by the preparation method in preparing oval pomfret feed.

[0017] The beneficial effects of the present invention are:

[0018] This application achieves synergistic benefits of low fish meal and low oxygen tolerance through multi-dimensional innovation. First, the nutritional ratio is optimized, the fish meal ratio is reduced to 20%, and composite plant proteins such as soybean meal and soy protein isolate and chicken meal are used to replace traditional fish meal to balance the amino acid composition; secondly, the functional ingredients are strengthened, and composite antioxidants such as astaxanthin and golden algae kelp polysaccharide are added to significantly improve the antioxidant capacity of the fish. The low oxygen survival rate in the experiment was increased to more than 85%. This application can alleviate the risk of eutrophication of aquaculture water bodies, while reducing dependence on oxygen enrichment equipment and saving energy consumption. Its low fish meal characteristics reduce feed costs, alleviating resource pressure while improving aquaculture benefits. This feed is particularly suitable for high-risk low-oxygen scenarios such as deep-sea cage aquaculture, providing technical support for the country's green and healthy aquaculture strategy, with both significant economic benefits and ecological value, injecting new impetus into the sustainable development of the aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Transcription levels of inflammation- and antioxidant-related genes in the liver. DETAILED DESCRIPTION

[0020] The invention provides a low-fish meal compound feed that is resistant to hypoxia. The low-fish meal compound feed 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 isolated soy protein, 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 complex vitamins, 0.5-1.5 parts of complex minerals, 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 golden algae laminarin.

[0021] In the present invention, the low-fishmeal 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 soy 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 complex vitamins, 1 part of complex minerals, 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 golden algae laminarin.

[0022] In the present invention, the multivitamin preferably contains the following raw materials in the following concentrations: 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, 245 0-2550 mg / kg choline chloride, 145-155 mg / kg ethoxyquinoline; further preferably containing the following raw materials in the following concentrations: 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 niacin, 20 mg / kg folic acid, 1.2 mg / kg biotin, 32 mg / kg retinyl acetate, 5 mg / kg cholecalciferol, 120 mg / kg α-tocopherol, 2000 mg / kg ascorbic acid, 2500 mg / kg choline chloride, and 150 mg / kg ethoxyquinoline.

[0023] In the present invention, the composite mineral salt preferably contains the following raw materials at the following concentrations: 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; further preferably containing raw materials in the following concentrations: 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 dihydrogen phosphate monohydrate and 100 mg / kg sodium chloride.

[0024] The present application also provides the use of the low-fishmeal compound feed in preparing oval pomfret feed.

[0025] The present application also provides a method for preparing the low-fishmeal compound feed, comprising the following steps:

[0026] (1) grinding, sieving, and mixing fish meal, soybean meal, soy protein isolate, flour, chicken meal, tapioca starch, calcium dihydrogen phosphate, multivitamins, multiminerals, choline, vitamin C, DL-Met, Lys-HCL, Thr, astaxanthin, and kelp polysaccharide to obtain a mixture 1;

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

[0028] (3) Granulate the mixture 2 and dry it to obtain an expanded feed.

[0029] In the present invention, the mesh size of the sieving in step (1) is preferably 190 to 210 μm, more preferably 200 μm.

[0030] In the present invention, the drying temperature in step (3) is preferably 38-42°C, more preferably 40°C, the diameter of the puffed feed is preferably 2-3 mm, more preferably 2.5 mm, and the water content of the puffed feed is preferably <10%, more preferably 8%.

[0031] The present application also provides the use of the low-fishmeal compound feed prepared by the preparation method in preparing oval pomfret feed.

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

[0033] Example 1 Raw material composition of experimental feed

[0034] Five experimental diets with equal nitrogen and lipid content were prepared: D1, D2, D3, D4, and D5. D1 served as the control group, containing a normal fishmeal diet; D2 served as the low-fishmeal diet; D3 served as the low-fishmeal diet supplemented with 0.05% astaxanthin; D4 served as the low-fishmeal diet supplemented with 0.05% chrysophyll laminarin; and D5 served as the low-fishmeal diet supplemented with both 0.05% astaxanthin and 0.05% chrysophyll laminarin. The composition of each experimental diet is shown in Table 1, and the nutrient content of each experimental diet is shown in Table 2.

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

[0036]

[0037]

[0038] Table 2 Nutrient content of experimental diets 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 content 10.26 10.43 10.19 10.28 10.35

[0040] Note: The multivitamins 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 niacin, 20 mg / kg folic acid, 1.20 mg / kg biotin, 32 mg / kg retinyl acetate, 5 mg / kg cholecalciferol, 120 mg / kg α-tocopherol, 2000 mg / kg ascorbic acid, 2500 mg / kg choline chloride, and 150 mg / kg ethoxyquin.

[0041] The complex 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 dihydrogen phosphate monohydrate, and 100 mg / kg sodium chloride.

[0042] Example 2 Preparation of experimental feed

[0043] Experimental Method: All ingredients except fish oil, soybean oil, and lecithin were pulverized and filtered through a 200μm sieve, collecting the undersize. The undersize was then weighed and mixed according to the formula ratio to produce Mixture 1. The formula ratios of fish oil, soybean oil, lecithin, and water were then added to Mixture 1 in that order and stirred vigorously until uniform, producing Mixture 2. Mixture 2 was then formed into extruded feed with a diameter of 2.5mm and dried in a ventilated oven at 40°C until the moisture content dropped below 10%. Finally, the experimental feed was stored at -20°C.

[0044] Application Example 1

[0045] The five groups of experimental feed raw materials D1, D2, D3, D4, and D5 in Example 1 were prepared according to the preparation method of Example 2 to obtain corresponding experimental feeds.

[0046] Juvenile silver pomfret (Scutellaria ovata) were obtained from a commercial seedling farm in Ningde, Fujian Province, China (Ningde Lingling Fishery Technology Co., Ltd.). They were housed in 4.0 m × 3.0 m × 2.5 m seawater floating cages for a two-week acclimation period to acclimate the fish to the experimental conditions. All fish were fasted for 24 hours before the start of the feeding experiment. Subsequently, 800 silver pomfret juveniles, with an average initial weight of 7.00 ± 0.03 g, were randomly distributed into 20 smaller floating cages (1 m × 1 m × 1.5 m each), with 40 silver pomfret juveniles per cage. The experimental diets were then randomly distributed into four of these cages. During the eight-week experiment, the fish were fed twice daily at 7:00 AM and 6:00 PM 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 consistently above 6.0 mg / L.

[0047] At the end of the 8-week feeding experiment, each group of juvenile pomfret was fasted for 24 hours and then anesthetized with a 100 mg / L eugenol solution. The body weights of the juvenile pomfret in the different treatment groups were measured, and the final average body weight (FBW), specific growth ratio (SGR), feed efficiency (FE), and survival rate (SR) were calculated. Detailed results are shown in Table 2. Data are presented as means and standard deviations.

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

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

[0050] Survival rate (%) = final number of fish / initial number of fish × 100. Formula 3

[0051] Table 3 Growth performance of juvenile pomfret 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) <![CDATA[31.00±1.14 b ]]> <![CDATA[28.17±0.26 c ]]> <![CDATA[33.41±0.75 ab ]]> <![CDATA[33.15±1.09 ab ]]> <![CDATA[33.73±0.43 a ]]> SGR (% / d) <![CDATA[3.55±0.10 a ]]> <![CDATA[3.32±0.03 b ]]> <![CDATA[3.72±0.04 a ]]> <![CDATA[3.69±0.08 a ]]> <![CDATA[3.74±0.03 a ]]> FE <![CDATA[0.75±0.06 a ]]> <![CDATA[0.63±0.03 b ]]> <![CDATA[0.77±0.02 a ]]> <![CDATA[0.78±0.03 a ]]> <![CDATA[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 terminal body weight, specific growth rate, and feed efficiency of the low-fishmeal group D2 were significantly reduced (P < 0.05). Notably, when astaxanthin (group D3), kelp (group D4), or both were added to the low-fishmeal diet (group D5), the above growth indicators were significantly improved compared with the D2 group (P < 0.05) and returned to levels comparable to the control group (P > 0.05). There were no significant differences in survival rates among the experimental groups (P > 0.05). This finding suggests that although a low-fishmeal diet significantly inhibits the growth performance and feed utilization efficiency of scad, the addition of functional additives such as astaxanthin or kelp can effectively mitigate this negative impact, maintaining growth performance at normal levels.

[0054] Application Example 2

[0055] After completing the growth performance index determination, this study randomly selected 10 egg-shaped pomfret juveniles from each experimental cage for tissue sample collection. The liver tissue was quickly frozen in liquid nitrogen and used for analysis of antioxidant markers and gene expression levels. The intestinal tissue was also quickly frozen in liquid nitrogen and preserved for subsequent digestive enzyme activity detection.

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

[0057] The collected intestinal or liver tissues were added to pre-cooled physiological saline at a ratio of 1:10 (w / v) and homogenized in an ice bath. The tissue supernatant was obtained by centrifugation at 2000 rpm for 30 minutes at 4°C. The supernatant was used to measure 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 α-amylase. All tests were performed strictly in accordance with the operating procedures of the kit instructions provided by Nanjing Jiancheng Bioengineering Research Institute. The specific test results are shown in Tables 4 and 5.

[0058] Table 4 Antioxidant capacity of juvenile pomfret in different treatment groups

[0059]

[0060] The results in Table 4 show that compared with the control group D1, liver superoxide dismutase (SOD) and catalase (CAT) activities and total antioxidant capacity (T-AOC) in the low-fishmeal group D2 were significantly reduced, while malondialdehyde (MDA) content was significantly increased (P < 0.05). Adding astaxanthin (group D3), chrysophyll laminarin (group D4), or both (group D5) to the low-fishmeal diet significantly improved antioxidant indices compared with group D2 (P < 0.05), with SOD, CAT activities, and T-AOC in group D5 exceeding those in the other experimental groups. These results suggest that a low-fishmeal diet can weaken fish antioxidant capacity and induce oxidative damage, but the addition of astaxanthin or chrysophyll laminarin can effectively mitigate this negative effect. The combined use of astaxanthin and chrysophyll laminarin exhibits a synergistic effect in enhancing antioxidant capacity, providing important insights for optimizing the functionality of low-fishmeal diets.

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

[0062]

[0063] As shown in Table 5, compared with the control group D1, the intestinal lipase and pepsin activities in the low-fishmeal group D2 were significantly reduced (P < 0.05). When astaxanthin (group D3), chrysocolla polysaccharide (group D4), or both were added to the low-fishmeal diet (group D5), lipase and pepsin activities were significantly increased compared with those in the D2 group (P < 0.05). The lipase activity in the D3, D4, and D5 groups was significantly higher than that in the control group (P < 0.05). There were no significant differences in α-amylase activity among the experimental groups (P > 0.05). This finding suggests that a low-fishmeal diet inhibits the digestion efficiency of fat and protein, but that the addition of astaxanthin or chrysocolla polysaccharide can effectively reverse this inhibitory effect, with the enhanced lipase activity being particularly significant. This provides important evidence for improving the functionality of low-fishmeal diets.

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

[0065] Liver samples of juvenile pomfret in each cage were collected and RNA was extracted using Trizol reagent (TaKaRa, Dalian, China) according to the manufacturer's instructions. The concentration and purity of the extracted RNA were then quantified using a Nanodrop 2000 spectrophotometer (Thermo, Wasserhaus, USA) and further confirmed by 1% agarose gel electrophoresis. TMTotal RNA was reverse transcribed into cDNA using a RT assay kit (TaKaRa, Dalian, China) and prepared for qRT-PCR analysis. The qRT-PCR procedure and specific primer sequences used were referred to “Dietary Tribonema sp. supplementation increased growth performance, antioxidant capacity, immunity and improved hepatic health in goldenpompano (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 goldenpompano (Trachinotus ovatus) [J]. Aquaculture, 2020, 529(1)).

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

[0067] The experimental instruments were: LightCycler 480 real-time fluorescence quantitative PCR instrument (Roche Applied Science, Switzerland) and Premix ExTaq TM II (Takara, Dalian, China) kit;

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

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

[0070]

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

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

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

[0074] like Figure 1 As shown, in terms of antioxidant gene expression, compared with the control group (D1), the low-fishmeal group (D2) showed significantly decreased mRNA expression levels of nuclear factor of NF-E2-related 2 (Nrf2), manganese superoxide dismutase (Mn-SOD), and heme oxygenase-1 (HO-1), while the expression of Kelch-like ECH-associated protein 1 (Keap1) was significantly increased (P<0.05). When the low-fishmeal diet was supplemented with astaxanthin (D3), kelp (D4), or both (D5), the gene expression levels of Nrf2, Mn-SOD, and HO-1 were significantly restored compared with those in the D2 group (P<0.05), and the levels in these 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 their highest levels in the D5 group, showing a trend of improvement over the single-additive group. These results suggest that low fishmeal diets may damage the antioxidant defense system of fish by activating the negative feedback regulatory mechanism of the Keap1-Nrf2 signaling pathway, while astaxanthin and kelp polysaccharides may more effectively activate this pathway through a synergistic mechanism, thereby enhancing the body's antioxidant capacity.

[0075] In terms of pro-inflammatory cytokine expression, the low-fishmeal group (D2) exhibited significant activation of the inflammatory response compared to the control group (D1), as evidenced by significantly increased mRNA expression levels of the pro-inflammatory cytokines IL-1β and IL-8 (P < 0.05). Feed additive intervention experiments demonstrated that astaxanthin (D3), kelp (D4), and their combined addition (D5) significantly inhibited the upregulation of pro-inflammatory cytokines induced by the low-fishmeal diet (P < 0.05). Notably, IL-1β and IL-8 expression levels in each additive-treated group were not only significantly lower than those in the D2 group (P < 0.05), but also showed a trend toward lower expression than in the control group (P < 0.05). These results suggest that the low-fishmeal diet may induce inflammatory responses by activating pro-inflammatory signaling pathways, and that astaxanthin, kelp, or their combination can effectively alleviate the inflammatory state and potentially enhance the body's anti-inflammatory defenses.

[0076] Among anti-inflammatory cytokines, the mRNA expression levels of transforming growth factor β1 (TGF-β1) and interleukin 10 (IL-10) were significantly downregulated in the low-fishmeal group (D2) compared with the control group (D1) (P<0.05). Notably, the addition of astaxanthin (D3), kelp (D4), or a combination of the two (D5) to the low-fishmeal diet significantly reversed this inhibitory effect (P<0.05). The combined addition group (D5) had the most significant effect on increasing the expression of TGF-β1 and IL-10, which was not only significantly higher than that of the D2 group (P<0.05), but also showed an enhanced trend that was superior to that of each single additive group. These results suggest that astaxanthin and kelp may more effectively activate anti-inflammatory signaling pathways through a synergistic mechanism, thereby improving the problem of reduced anti-inflammatory ability caused by low-fishmeal diets.

[0077] Application Example 3

[0078] After an 8-week feeding experiment, an acute hypoxic stress experiment was conducted on juvenile scads (Oval pomfret) exposed to water out of water. During the experiment, 20 healthy juveniles were randomly selected from each experimental group and placed in a waterless foam tank for 15 minutes. They were then returned to their original cages for recovery. Mortality was determined by the cessation of operculum movement within 1 hour, and the number of fish killed in each group was accurately recorded. The results showed that compared with the control group (D1), the survival rate of the low-fishmeal group (D2) after acute hypoxia was significantly reduced (P < 0.05), indicating that the low-fishmeal diet significantly affected the fish's tolerance to hypoxia. Notably, supplementing the low-fishmeal diet with astaxanthin (D3), chrysophyte laminarin (D4), or both (D5) significantly improved the survival rate of each supplement compared to the D1 and D2 groups (P < 0.05), with the combined supplementation group (D5) achieving the highest survival rate (P < 0.05) (Table 7). These results fully demonstrate that astaxanthin and golden algae laminarin can effectively enhance the tolerance of oval pomfret 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 Pomfret after out-of-water stress

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

[0081] In summary, low-fishmeal diets significantly inhibit fish growth performance (terminal 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 the Nrf2-Keap1 signaling pathway, downregulate antioxidant gene expression (Nrf2, Mn-SOD, HO-1), and disrupt the balance between pro-inflammatory cytokines (IL-1β, IL-8) and anti-inflammatory cytokines (TGF-β1, IL-10), ultimately leading to decreased antioxidant capacity, aggravated inflammatory responses, and weakened hypoxia tolerance in fish. Notably, the addition of astaxanthin or kelp polysaccharide to low-fishmeal diets not only effectively reversed these negative effects and restored various indicators to normal levels, but their combined use also demonstrated significant synergistic effects in enhancing antioxidant capacity, improving inflammatory status, and enhancing hypoxia tolerance, providing important theoretical basis and practical guidance for the functional optimization of low-fishmeal diets.

[0082] As can be seen from the above examples, the present invention provides a hypoxia-resistant low-fishmeal compound feed, its preparation method, and application. The low-fishmeal compound feed contains the following raw materials: fish meal, soybean meal, soy 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 golden algae laminarin. First, the present application uses composite plant proteins such as soybean meal and soy protein isolate and chicken meal to replace traditional fish meal, balancing the amino acid composition, optimizing the nutritional ratio of the compound feed, and reducing the fish meal ratio to 20%. Second, the compound feed of the present application adds composite antioxidants such as astaxanthin and golden algae laminarin, which can significantly enhance the antioxidant capacity of fish.

[0083] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A hypoxia-resistant low-fishmeal compound feed, characterized in that: The low-fishmeal compound feed 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 isolated soy protein, 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 monocalcium phosphate, 0.5-1.5 parts of complex vitamins, 0.5-1.5 parts of complex minerals, 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 golden algae laminarin.

2. The low fish meal compound feed according to claim 1, characterized in that The multivitamin contains the following raw materials in the following concentrations: 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 fish meal compound feed according to claim 2, characterized in that The composite mineral salt contains raw materials in the following concentrations: 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. Use of the low-fishmeal compound feed according to any one of claims 1 to 3 in preparing oval pomfret feed.

5. The method for preparing the low-fishmeal compound feed according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) grinding, sieving, and mixing fish meal, soybean meal, soy protein isolate, flour, chicken meal, tapioca starch, calcium dihydrogen phosphate, multivitamins, multiminerals, choline, vitamin C, DL-Met, Lys-HCL, Thr, astaxanthin, and kelp polysaccharide to obtain a mixture 1; (2) mixing fish oil, soybean oil, lecithin, water, and mixture 1 to obtain mixture 2; (3) Granulate the mixture 2 and dry it to obtain an expanded feed.

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

7. The preparation method according to claim 6, characterized in that The drying temperature in step (3) is 38-42° C., the diameter of the puffed feed is 2-3 mm, and the water content of the puffed feed is less than 10%.

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

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