Feed additive for improving hypoxia tolerance function of micropterus salmoides and feed composition thereof

Abstract

The invention provides a feed additive for improving the hypoxia tolerance function of micropterus salmoides. The feed additive is prepared from the following raw materials in percentage by mass: 20% of arachidonic acid and the balance of maltodextrin. The invention also provides a feed composition of the feed additive. The feed is prepared from the following raw materials in percentage by mass: 450 per mill of fish meal, 50 per mill of fermented hermetia illucens pulp, 69.5 per mill of enzymolysis chicken powder, 60 per mill of chicken liver powder, 60 per mill of enzymolysis soybean meal, 75 per mill of fermented soybean meal, 60 per mill of cottonseed meal, 29 per mill of dextrin, 40.5 per mill of cassava starch, 23 per mill of soybean oil, 23 per mill of linseed oil, 15 per mill of monocalcium phosphate, 5 per mill of choline chloride, 1 per mill of a micropterus salmoides premix, 1 per mill of zeolite powder and 20 per mill of a feed additive. The feed composition is used for improving the hypoxia tolerance of the micropterus salmoides. According to the invention, arachidonic acid is used as a functional active substance to prepare the hypoxia stress resistant functional feed, and the addition of arachidonic acid can significantly activate liver angiogenesis genes, promote the liver angiogenesis level and improve the hypoxia tolerance of largemouth bass.

Description

Technical Field

[0001] The invention belongs to the technical field of feed formulation, and particularly relates to a feed additive for improving the hypoxia tolerance function of largemouth bass, and a feed composition and application thereof. Background Art

[0002] With the intensive development of aquaculture and climate change, hypoxia stress in water bodies occurs frequently, leading to growth inhibition, decreased immunity and even mass death of fish.

[0003] At the same time, due to the environment such as high temperature, the hypoxia stress of the aquaculture water body often presents the characteristics of stage, long-term, and intermittent hypoxia stress. (Micropterus salmoides) As one of my country's most important farmed carnivorous fish, with an annual production exceeding 880,000 tons, it is highly sought after by consumers and farmers. Carnivorous fish generally exhibit low tolerance to hypoxia, and the largemouth bass's widespread farming and high-density culture make it particularly susceptible to hypoxic stress. Existing technologies primarily mitigate hypoxia through physical oxygenation (such as aeration equipment) or adjusting stocking density. Other approaches involve adjusting feed formulations (such as increasing starch levels or adding functional peptides). However, these methods are costly, complex, and unsuitable for carnivorous fish, resulting in limited success. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the deficiencies of the above-mentioned existing technologies and provide a feed additive and a feed composition and application thereof for improving the hypoxia tolerance function of largemouth seabass. The feed additive uses arachidonic acid as a functional active substance to prepare a hypoxia stress-resistant functional feed. The addition of arachidonic acid can significantly activate liver angiogenesis genes, promote the level of liver angiogenesis, and improve the hypoxia tolerance ability of largemouth seabass.

[0005] To solve the above technical problems, the technical solution adopted by the present invention is: a feed additive for improving the hypoxia tolerance of largemouth bass, the feed additive is composed of the following raw materials in mass fractions: 20% arachidonic acid and the balance maltodextrin.

[0006] The present invention also provides a feed composition of the above-mentioned feed additive, which is composed of the following raw materials in mass fractions: 450‰ fish meal, 50‰ fermented black soldier fly slurry, 69.5‰ enzymatic chicken meal, 60‰ chicken liver powder, 60‰ enzymatic soybean meal, 75‰ fermented soybean meal, 60‰ cottonseed meal, 29‰ dextrin, 40.5‰ cassava starch, 23‰ soybean oil, 23‰ linseed oil, 15‰ calcium dihydrogen phosphate, 5‰ choline chloride, 10‰ California sea bass premix, 10‰ zeolite powder, and 20‰ feed additives.

[0007] The present invention also provides an application of the feed composition, characterized in that the feed composition is used to improve the hypoxia tolerance of largemouth bass.

[0008] Compared with the prior art, the present invention has the following advantages: The feed additive of the present invention uses arachidonic acid as a functional active substance. The feed composition prepared by adding arachidonic acid can significantly activate liver angiogenesis genes, promote the level of liver angiogenesis, and improve the hypoxia tolerance of largemouth bass.

[0009] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The figure shows the effects of different feeds on the asphyxiation point (A) and oxygen consumption rate (B) of largemouth bass in Example 1 of the present invention (* indicates P < 0.05).

[0011] Figure 2 The effects of different feeds on liver angiogenesis and related gene expression in largemouth bass (Black bass) in Example 1 of the present invention are shown in Figure 1. (A. Largemouth bass liver tissue pathology section, light green fill indicates blood vessels, scale bar is 400 μm; B. Percentage of liver vascular area; C. mRNA expression levels of liver angiogenesis-related genes. * indicates P <0.05; ** indicates P <0.01;*** indicates P <0.001.) Figure 3 The levels of PGE2 and EETs in the liver of largemouth bass and their synthesis-related gene expression levels in different feed groups under intermittent hypoxia stress in Example 1 of the present invention. (* indicates p <0.05; ** indicates p <0.01; *** indicates p <0.001.) DETAILED DESCRIPTION

[0012] Example 1 The feed additive of this embodiment is composed of the following raw materials in mass fractions: 20% arachidonic acid (AA) and the balance maltodextrin.

[0013] The feed composition containing the above-mentioned feed additives in this embodiment is composed of the following raw materials in mass fractions: 450‰ fish meal, 50‰ fermented black soldier fly slurry, 69.5‰ enzymatic chicken meal, 60‰ chicken liver powder, 60‰ enzymatic soybean meal, 75‰ fermented soybean meal, 60‰ cottonseed meal, 29‰ dextrin, 40.5‰ tapioca starch, 23‰ soybean oil, 23‰ linseed oil, 15‰ calcium dihydrogen phosphate, 5‰ choline chloride, 10‰ California sea bass premix, 10‰ zeolite powder, and 20‰ the above-mentioned feed additives.

[0014] That is, in the formula of this embodiment, the level of pure arachidonic acid added is 0.4%.

[0015] The feed composition of this embodiment is used to improve the hypoxia tolerance of largemouth bass.

[0016] 1 Materials and Methods 1.1 Experimental fish and daily management The largemouth bass used in the experiment in this example were all from the Balidian Comprehensive Experimental Base of the Zhejiang Freshwater Fisheries Research Institute. The experimental fish were temporarily raised in a circulating water system for 2 weeks. The experimental fish were starved for 24 hours before the formal experiment.

[0017] 1.2 Breeding experiment and sample collection Feed preparation: Largemouth bass basic feed ingredients were purchased from Hanbei Aquatic Feed Company. All ingredients were ground, passed through a 60-mesh sieve, weighed, and mixed uniformly. A fat source was added, along with 15% water. Pellets were pelleted using a pelletizer, dried, and stored at -20°C until use. The fat source consisted of a 1:1 mixture of soybean oil and linseed oil. A control feed and an AA feed were prepared with equal nitrogen and lipid content. The feed formula and composition are shown in Table 1.

[0018] Table 1 Feed ingredients and nutritional components Feeding Experiment: The experiment was conducted in a recirculating aquaculture system at the Comprehensive Experimental Base of the Zhejiang Freshwater Fisheries Research Institute. The tanks had a radius of 90 cm and a water depth of 50 cm. Two hundred and forty healthy juvenile largemouth bass (12.52 ± 0.02 g) were randomly divided into two groups, each containing three parallel tanks, with 40 fish per tank. During the experiment, the fish were fed a diet at 3% of their body weight twice daily, with weights adjusted every two weeks for an eight-week period. The water temperature was maintained at 26-29°C, dissolved oxygen >7 mg / L, pH 6.8-7.3, ammonia nitrogen <0.3 mg / L, and nitrite <0.15 mg / L. Following a 24-hour fast, each tank was counted and weighed. Final mean body weight (FBW), weight gain rate (WGR), feed conversion ratio (FCR), and survival rate (SR) were calculated for each group. Six experimental fish were randomly selected from each tank, and their body weight and length were measured. The weights of the internal organs and liver were weighed, and the fatness fraction (CF), viscera-to-body ratio (VSI) and liver-to-body ratio (HSI) of each group of experimental fish were calculated.

[0019] The indicator calculation formula is as follows: Weight gain rate (WGR, %) = (Wt-W0) / W0×100 Specific growth rate (GR, % / d) = (LnWt-LnW0) / t×100 Feed coefficient ratio (FCR) = F / (Wt-W0) Survival rate (SR, %) = Nt / N0 × 100 Visceral somatic index (VSI) = (VW / W) × 100 Hepatosomatic index (HSI) = (HW / W) × 100 Fullness (CF, g / cm 3 )=W / L 3 Where W0 and Wt represent the initial and final average weights (g), respectively; t represents the number of days in culture (d); F represents the average feed intake (g); Nt and N0 represent the number of largemouth bass at the end and start of the experiment, respectively; VW, HW, and W represent the weights of viscera, liver, and whole fish, respectively (g); and L represents body length (cm).

[0020] Sample Collection: Ten largemouth bass were randomly selected from each tank for apnea point and oxygen consumption rate testing; three fish were randomly selected for crude body composition analysis. Sixty fish were randomly assigned to three aquariums in each group for a two-week intermittent hypoxia stress experiment. Following the hypoxia stress experiment, blood and liver samples were collected from each group for testing of liver enzyme activities, hemoglobin levels, pathological sections, gene expression, and fatty acid composition. In addition, liver tissues from six fish in each group were randomly selected for measurement of prostaglandin E2 (PGE2) and epoxyeicosatrienoic acid (EETs) levels.

[0021] 1.2 Hypoxia stress experiment and sample collection Hypoxia Stress Experiment: To simulate the hypoxic exposure pattern experienced in aquaculture, this study subjected two groups of largemouth bass to intermittent hypoxia: a control diet hypoxia stress group (CON-HS) and an AA diet hypoxia stress group (AA-HS). Intermittent hypoxia stress was controlled by injecting nitrogen or air into the aquarium. At 5:00 PM daily, nitrogen was injected into the water to reduce dissolved oxygen levels, reducing them to 1.5 mg / L within approximately one hour. The fish were then kept in this hypoxic environment for three hours. Following the hypoxic exposure, aeration was immediately performed to restore oxygen levels to above 7.0 mg / L. The experimental group was fed equal amounts of the CON diet and the AA diet (Table 1) for a two-week period.

[0022] Sample collection: After the experiment, the experimental fish were anesthetized with MS-222 (60 mg / L, Sigma). Three experimental fish were randomly selected from each group, and blood was drawn from the tail vein using a 1-mL sterile syringe soaked in sodium heparin anticoagulant. Liver tissues of nine fish in each group were used for enzyme activity detection and for gene expression analysis. The liver tissues of three fish were mixed into one replicate, for a total of three replicates, frozen in liquid nitrogen and stored at -80°C. In addition, liver tissues of three fish in each group were stored in 4% paraformaldehyde for pathological section analysis. Liver tissues of three fish in each group were frozen in liquid nitrogen and stored at -80°C for gene expression and fatty acid composition analysis.

[0023] 1.4 Index detection Crude component analysis: The basic components (crude protein, crude fat, moisture, and ash) of the experimental feed and fish were determined according to AOAC standard methods. The atmospheric pressure drying method was used, in accordance with GB6435-86. Ash was determined by the muffle furnace ignition method, in accordance with GB / T 6438-2007. Crude protein was determined by the Kjeldahl method, in accordance with GB / T 6432-94. Crude fat was determined by the Soxhlet extraction method, in accordance with GB / T 6433-2006.

[0024] Liver enzyme activity assays: Malondialdehyde (MDA; Biyuntian, S0131M), superoxide dismutase (SOD; Biyuntian, S0101M), catalase (CAT; Nanjing Jiancheng, A007-2-1), and total antioxidant capacity (T-AOC; Biyuntian, S0119) in the liver tissues of largemouth bass were determined according to the manufacturer's instructions.

[0025] Pathological sections: Largemouth bass liver tissue was fixed in 4% paraformaldehyde solution for 24 hours, dehydrated, paraffin-permeabilized, embedded, sectioned, and stained to prepare liver tissue sections. The level of intrahepatic angiogenesis was observed by H&E staining, and the proportion of liver vascular area was calculated using Image J.

[0026] Gene expression analysis: Liver RNA was extracted and reverse transcribed into cDNA, followed by real-time fluorescence quantitative PCR. β- actin and 18s RNA For double internal reference, use 2 -ΔΔCT Methods: Quantitative analysis was performed to determine the mRNA expression of genes related to angiogenesis, PGE2, and EETs synthesis in liver tissue of largemouth bass. The primers for each target gene are shown in Table 2.

[0027] Table 2 Primer list Fatty acid composition analysis: Total lipids from largemouth bass liver were extracted using the chloroform / methanol method and methyl-esterified with boron trifluoride. Fatty acid composition was determined by GC-MS. A 1 μL sample was injected; the injection temperature was 260°C, and the detection temperature was 270°C. The column temperature program was: initial temperature 150°C, hold for 5 min, then increase to 230°C at a rate of 5°C / min, hold for 20 min. Hydrogen and air flow rates were 30 and 40 mL / min, respectively, and nitrogen carrier gas flow rate was 1.5 mL / min. Chromatographic peaks were calibrated with fatty acid standards, and fatty acid content and composition were calculated. Data were processed and analyzed using SPSS 22.0 software. All data were compared using t-tests using SPSS 22.0, with a significance level set at P < 0.05.

[0028] Detection of arachidonic acid derivatives: The PGE2 level in the liver tissue of largemouth bass was determined using a fish PGE2 assay kit according to the manufacturer's instructions; the EETs level in the liver tissue of largemouth bass was determined by liquid chromatography-mass spectrometry.

[0029] 1.5 Asphyxia Point and Oxygen Consumption Rate Testing: In a 250L confined water body, record the initial dissolved oxygen level in the water and record the dissolved oxygen level every 0.5h until the end of the first breeding experiment. Randomly select 10 largemouth bass from each group to determine their asphyxia point. The dissolved oxygen level at which the largemouth bass fainted after being placed in the water was considered the asphyxia point. The oxygen consumption rate of each experimental fish group was calculated according to the following formula: Oxygen consumption rate (mg / g / h) = (DO0-DO4) × V / W Where DO0 and DO4 are the initial dissolved oxygen in the water and the dissolved oxygen after 4 hours (mg / L), respectively; V represents the volume of the water (L); and W is the total weight of the experimental fish (g).

[0030] 1.6 Data Processing and Statistics The test results were expressed as mean ± standard deviation (mean ± SD). t -test was used to compare the two groups of data. Statistical analysis was performed using IBM SPSS Statistics 22. P A value <0.05 was considered significant.

[0031] 2. Results 2.1 Effects of arachidonic acid supplementation in feed on growth performance of largemouth bass The growth performance and feed utilization ability of largemouth bass fed with two groups of diets are shown in Table 3. The results showed that there were no significant differences in the final weight, weight gain rate, feed conversion rate, survival rate, and fatness of largemouth bass between the control group and the AA group (P>0.05); however, the liver-to-body ratio and organ-to-body ratio of largemouth bass in the AA group were significantly lower than those in the control group ( P <0.05). In terms of fish body composition, there was no significant difference in crude protein, ash, and water content between the AA group and the control group ( P >0.05), the fat-promoting level of fish in the AA group was significantly lower than that in the control group ( P <0.05). We also tested the fatty acid composition of the liver tissues of the two experimental fish groups. The results showed that AA (arachidonic acid), C20:3n-6 (AA desaturated substrate), and C22:4n-6 (AA elongation product) in the liver tissues of the largemouth bass in the AA group showed different degrees of accumulation ( P <0.05; Table 4).

[0032] Table 3 Effects of arachidonic acid supplementation on growth performance of largemouth bass Note: * in the table indicates P Significant difference at the <0.05 level, ** indicates P The difference was significant at the <0.01 level.

[0033] Table 4 Fatty acid composition of liver tissue of largemouth bass in each group after 8 weeks of feeding experiment Note: * in the table indicates P Significant difference at the <0.05 level, ** indicates P The difference was significant at the <0.01 level.

[0034] 2.2 Effects of exogenous AA supplementation on the asphyxiation point and oxygen consumption rate of largemouth bass The test content of this part is the low oxygen treatment (referred to as treatment a) to measure the asphyxiation point and oxygen consumption rate. The treatment method is: place the fish in a closed water space, and determine its asphyxiation point by testing the dissolved oxygen in the water and the status of the experimental fish. Treatment a is acute hypoxia stress, and generally only half a day is needed to obtain the results. Figure 1 The result in is the result of processing a.

[0035] like Figure 1 As shown in A, the largemouth bass in the AA group showed significantly enhanced hypoxia tolerance after being fed with the AA feed. The asphyxiation point was significantly lower at 0.5 mg / L than that of the control group (CON) at 1.08 mg / L, and the syncope time was extended from 8 hours to 10 hours. At the same time, the oxygen consumption rate of the largemouth bass in the AA group was significantly higher than that of the control group ( P <0.05; Figure 1 B).

[0036] 2.3 Effects of intermittent hypoxia stress on the antioxidant capacity of largemouth bass liver in different diet groups This part is an intermittent hypoxia stress experiment (in line with actual production, recorded as treatment b), which lasts for 2 weeks. The experimental fish are treated with hypoxia once a day, and then various indicators are tested to determine the effects of different feed groups on the hypoxia tolerance of the experimental fish.

[0037] The antioxidant activities of liver tissue of largemouth bass in different diet groups under intermittent hypoxia stress are shown in Table 5. Compared with the intermittent hypoxia stress control group (control-hypoxia, i.e. CON-HS), the T-AOC level, CAT activity and SOD activity of liver tissue of largemouth bass in the intermittent hypoxia stress AA group (AA-hypoxia, i.e. AA-HS) were significantly increased ( P <0.05, while there was no significant difference in MDA levels in liver tissues of largemouth bass between the two groups ( P> 0.05), indicating that AA feeding improved the liver antioxidant capacity of largemouth bass under intermittent hypoxia stress to a certain extent.

[0038] Table 5 Effects of intermittent hypoxic stress on the antioxidant capacity of liver of largemouth bass in the control group and AA group Note: * in the table indicates P Significant difference at the <0.05 level, ** indicates P The difference was significant at the <0.01 level.

[0039] 2.4 Effects of intermittent hypoxia stress on liver angiogenesis in largemouth bass (Black bass) fed different diets Under intermittent hypoxia stress, AA (AA-hypoxia) feeding significantly increased the level of liver angiogenesis in largemouth bass under intermittent hypoxia stress, as reflected by a significant increase in the proportion of liver vascular area in liver tissue (P <0.05; Figure 2 A-2B). At the same time, compared with the control group (control-hypoxia), AA feeding significantly increased the expression of angiogenesis-related genes in the liver tissue of largemouth bass under intermittent hypoxia stress ( vegfa 、 vegfr2 、 mmp2 、 jagged and notch1 ) expression level ( P <0.05; Figure 2 C).

[0040] 2.5 Effects of intermittent hypoxia stress on the content of arachidonic acid derivatives in the liver of largemouth bass fed different diets To explore the physiological mechanism of high angiogenesis in the AA group (AA-hypoxia), we detected the contents of arachidonic acid derivatives in the liver tissue of largemouth bass fed different diets under intermittent hypoxia stress, especially the levels of PGE2 and EETs with pro-angiogenic activity. The results showed that the levels of PGE2 and EETs in the liver tissue of the AA group were significantly higher than those in the control group (control-HS) ( P <0.05; Figure 3 AB), and its synthesis-related genes also showed significantly higher expression levels than the control group ( P <0.05; Figure 3 C).

[0041] In summary, the feed composition of the present invention uses arachidonic acid as the functional active substance to prepare a functional feed resistant to hypoxia stress. The addition of arachidonic acid can significantly activate liver angiogenesis genes, promote the level of liver angiogenesis, and improve hypoxia tolerance. The functional active substance arachidonic acid in the feed composition of the present invention improves the hypoxia tolerance of largemouth bass by promoting liver angiogenesis.

[0042] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent variation made to the above embodiment based on the essence of the invention technology shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A feed additive for improving the hypoxia tolerance of largemouth bass, characterized in that: The feed additive is composed of the following raw materials in mass fractions: 20% arachidonic acid and the balance maltodextrin.

2. A feed composition containing the feed additive according to claim 1, characterized in that: The feed composition is composed of the following raw materials in different mass fractions: 450‰ fish meal, 50‰ fermented black soldier fly slurry, 69.5‰ enzymatically hydrolyzed chicken meal, 60‰ chicken liver powder, 60‰ enzymatically hydrolyzed soybean meal, 75‰ fermented soybean meal, 60‰ cottonseed meal, 29‰ dextrin, 40.5‰ cassava starch, 23‰ soybean oil, 23‰ linseed oil, 15‰ calcium dihydrogen phosphate, 5‰ choline chloride, 10‰ California sea bass premix, 10‰ zeolite powder, and 20‰ feed additives.

3. A use of the feed composition according to claim 2, characterized in that: The feed composition is used for improving the hypoxia tolerance of largemouth bass.

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