Additive and breeding method for improving cold resistance of large yellow croaker

Abstract

The present invention relates to an additive and a breeding method for improving the cold resistance of large yellow croaker, belonging to the technical field of fish breeding, wherein the additive is composed of the following components by mass per 100g: 2-4g of spermidine, 6-10g of resveratrol, 10-20g of pyruvate creatine, and the balance carrier defatted rice bran. The present invention also provides a breeding method for improving the cold resistance of large yellow croaker using the additive, wherein the additive is added to large yellow croaker feed, and the added amount accounts for 0.5-2% of the feed mass percentage. After the raw materials of the present invention are matched according to a certain ratio, the raw materials are effectively matched, synergistically enhanced, and the quality of cell mitochondria can be improved, lipid droplet mobilization can be enhanced, fat metabolism level can be improved, cell tricarboxylic acid cycle flux can be improved, nutrient metabolism energy supply can be promoted, cell energy supply can be improved, and large yellow croaker resistance to low temperature can be enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of fish breeding, and particularly relates to an additive and a breeding method for improving the cold resistance of large yellow croaker, that is, a nutritional solution that can increase the autophagy level of the fish body, mobilize nutrient metabolism, and thus improve the survival rate of large yellow croaker under low temperature stress. Background Art

[0002] Large yellow croaker (Larimichthys crocea), belonging to the order Perciformes, the family Sciaenidae, and the genus Larimichthys, is a warm-temperate offshore fish with delicious meat and high economic value. It is one of the main marine economic fish in my country. In 2022, the annual aquaculture output of large yellow croaker has reached 257,700 tons, making it the largest aquaculture species in my country. However, artificial breeding of large yellow croaker still faces some urgent problems. First of all, large yellow croaker is not resistant to low temperatures. The suitable temperature for breeding is 10-32°C, and feeding stops below 15°C. In a low temperature environment, those large yellow croakers with poor physique die due to malnutrition and lack of energy, causing huge economic losses to aquaculture practitioners. Therefore, the survival rate of large yellow croaker under low temperature stress is particularly important. Although the invention patent application with publication number CN103988798A discloses a method of transferring large yellow croaker to a winter breeding shed and conducting nutritional enhancement, it is limited to exogenous nutritional supplementation, and there are few reports on research on enhancing large yellow croaker's ability to withstand low temperatures by regulating its own nutrient utilization. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide an additive and a breeding method for improving the cold resistance of large yellow croaker. The additive is formulated into large yellow croaker feed for feeding, which can increase the autophagy level of the fish body and mobilize nutrient metabolism, thereby improving the survival rate of large yellow croaker under low temperature stress.

[0004] According to the results of many years of research in the applicant's laboratory, it was found that under stress conditions, fish consume a lot of energy to maintain homeostasis. The utilization of nutrients determines the energy level of cells. Mitochondria are important places for the metabolism of carbohydrates, lipids and amino acids in cells, and the quality of their state determines the efficiency of nutrient metabolism. Damaged or aged mitochondria are broken down into small molecule nutrients for cell reuse through autophagy. Mitochondrial autophagy is an important mechanism for maintaining the stability of the number and quality of mitochondria in cells. In addition, intracellular fat autophagy activates and promotes the process of lipolysis, which is an important link in lipid mobilization and metabolism. Autophagy degrades damaged cell structures and macromolecular cell components, and the small molecules produced by decomposition will participate in the tricarboxylic acid cycle for further metabolism to produce ATP, providing energy for resisting stress. It has been reported that the activation of the autophagy pathway caused by fasting can improve the cold resistance of fish. Therefore, in view of the above key points, a feed additive that can improve the cold resistance of large yellow croaker by enhancing autophagy is developed.

[0005] The present invention is achieved through the following technical solutions:

[0006] An additive for improving the cold resistance of large yellow croaker, characterized in that the additive comprises the following components per 100g: 2-4g of spermidine, 6-10g of resveratrol, 10-20g of creatine pyruvate, and the balance carrier defatted rice bran.

[0007] The present invention also provides a breeding method for improving the cold resistance of large yellow croaker by using the additive. The method comprises adding the additive to large yellow croaker feed, wherein the additive accounts for 0.5-2% of the mass percentage of the feed.

[0008] The principle of the additive of the present invention:

[0009] The spermidine can activate mitochondrial autophagy in cells, degrade damaged or aged mitochondria, promote cell repair, ensure the homeostasis and quality control of mitochondrial numbers, thereby improving mitochondrial function and enhancing the catabolism of lipids and amino acids in mitochondria.

[0010] The resveratrol can promote the autophagy of lipid droplets in cells, decompose lipid droplets into fatty acids, promote the lipolysis process, upregulate the gene expression of lipid decomposition and β-oxidation regulatory factors, and further mobilize fat metabolism.

[0011] The sodium pyruvate, as the starting metabolite of the tricarboxylic acid cycle, can increase the flux of the tricarboxylic acid cycle, promote the further metabolism of autophagy products amino acids and fatty acids, and provide energy for the body.

[0012] Among them, spermidine and resveratrol: synergistically promote cellular nutrient metabolism. Spermidine can promote autophagy of damaged mitochondria and improve mitochondrial quality, and resveratrol can promote lipophage. The two work synergistically to improve the decomposition and metabolism of macromolecular nutrients.

[0013] Among them, spermidine, resveratrol and sodium pyruvate: synergistically promote cell energy production, enhance the small molecule autophagy products through the tricarboxylic acid cycle to further metabolize and produce ATP energy supply.

[0014] The beneficial effects of the present invention compared with the prior art are as follows:

[0015] After the raw materials are mixed in a certain proportion, the raw materials work effectively together and synergize to improve the quality of cell mitochondria, enhance lipid droplet mobilization, increase fat metabolism levels, increase cellular tricarboxylic acid cycle flux, promote nutrient metabolism and energy supply, enhance cellular energy supply, and enhance the large yellow croaker's ability to resist low temperatures.

[0016] Adding the feed additive of the present invention into large yellow croaker feed can effectively improve the mobilization and utilization of nutrients by the fish body, increase the cell energy level, and improve the cold resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a statistical chart of the number of side floats in each group at 6.8℃;

[0018] Figure 2 The bar graph is the ATP content in the liver of each group of large yellow croaker;

[0019] Figure 3 The bar graphs are the expression levels of liver lipolysis (lpl, hsl) and β-oxidation genes (cpt1, acox1) in each group of large yellow croaker; each group of data columns from left to right are control group, experimental group 1, experimental group 2, experimental group 3, comparison group 1, comparison group 2, comparison group 3;

[0020] Figure 4 It is a bar graph of the expression levels of tricarboxylic acid cycle genes (cs, idh3) of each group of large yellow croaker. Each group of data columns is control group, experimental group 1, experimental group 2, experimental group 3, comparison group 1, comparison group 2, and comparison group 3 from left to right. DETAILED DESCRIPTION

[0021] Unless otherwise specified, the reagents used in the present invention can be purchased from commercial sources.

[0022] Control group: basic feed for large yellow croaker without adding compound additives.

[0023] Example 1

[0024] Test material 1: Large yellow croaker basic feed, with compound additives added, the amount of compound additives added is 1% of the total mass of the large yellow croaker basic feed.

[0025] Preparation of feed additives: 2 g of spermidine, 6 g of resveratrol, and 10 g of sodium pyruvate are mixed evenly by a step-by-step mixing method, and then the remaining 82 g of carrier defatted rice bran is mixed evenly.

[0026] Example 2

[0027] Test material 2: Large yellow croaker basic feed, with compound attractant added, the amount of compound additive added is 1% of the total mass of large yellow croaker basic feed.

[0028] Preparation of feed additives: 3 g of spermidine, 8 g of resveratrol, and 15 g of sodium pyruvate are mixed evenly by a step-by-step mixing method, and finally the remaining 74 g of carrier defatted rice bran is mixed evenly.

[0029] Example 3: Test material 3: Large yellow croaker basic feed, with compound additives added, the amount of compound additives added being 1% of the total mass of the large yellow croaker basic feed.

[0030] Preparation of feed additives: 4 g of spermidine, 10 g of resveratrol and 20 g of sodium pyruvate are mixed evenly by a step-by-step mixing method, and finally the remaining 66 g of carrier defatted rice bran is mixed evenly.

[0031] Example 4: Comparative feed 1: a basic feed for large yellow croaker, with a single additive added, and the amount of the additive added is 1% of the total mass of the basic feed for large yellow croaker.

[0032] Preparation of feed additive: 4 g of spermidine and 96 g of carrier defatted rice bran are mixed evenly by a step-by-step mixing method.

[0033] Example 5: Comparative material 2

[0034] A single additive is added to the basic feed for large yellow croaker, and the amount of the additive added is 1% of the total mass of the basic feed for large yellow croaker.

[0035] Preparation of feed additive: 10g of resveratrol and 90g of carrier defatted rice bran are mixed evenly by a step-by-step mixing method.

[0036] Example 6: Comparative feed 3: a basic feed for large yellow croaker, with a single additive added, and the amount of the additive added is 1% of the total mass of the basic feed for large yellow croaker.

[0037] Preparation of feed additive: 20g of sodium pyruvate and 80g of carrier defatted rice bran are mixed evenly by a step-by-step mixing method.

[0038] Embodiment 7,

[0039] 1. Experimental Grouping and Preparation

[0040] 840 large yellow croakers of uniform size and robust physique were selected, with an initial body weight of 15.2±0.03g, and randomly divided into 7 groups (control group, experimental group 1-3, comparison group 1-3), with three replicates in each group and 40 fish in each replicate. The feed formulas of each group are shown in Tables 1 and 2.

[0041] The experiment was conducted in a temperature-controlled circulating water system. During the experiment, the water temperature was (20±0.5)℃, the salinity was controlled at 29-30‰, the dissolved oxygen was higher than 7mg / l, and the ammonia nitrogen and nitrite content was lower than 0.1mg / l. Artificial apparent satiation was fed twice a day for 14 days. After 14 days, a low temperature experiment was conducted, with the scheme of dropping the temperature by 2℃ every 4 hours.

[0042] 2. Experimental Plan

[0043] 2.1 Sample collection

[0044] At the end of the breeding experiment, six large yellow croakers were randomly selected from each treatment, and liver samples were collected in cryopreservation tubes after being anesthetized with MS-222. After being quickly frozen with liquid nitrogen, they were stored at -80°C for analysis.

[0045] 2.2 Behavioral Observation

[0046] During the low-temperature experiment, the activity status of the fish in the water was observed, and the number of large yellow croakers that lost their balance and floated on their backs on the water surface was recorded.

[0047] 2.3 The ATP level in liver tissue was detected using a commercial kit (Nanjing Jiancheng Bioengineering Institute).

[0048] 2.4 The expression levels of genes related to lipid metabolism and tricarboxylic acid cycle were detected by real-time fluorescence quantitative PCR.

[0049] 2.5. The experimental data are expressed as mean ± standard error (X ± SD). SPSS18.0 software was used for data analysis. ANOVA was used to analyze the significant differences between the data. The values ​​with significant differences (P < 0.05) were marked with superscript letters.

[0050] 3. Results

[0051] 3.1 Effects of the additive of the present invention on the behavior of large yellow croaker.

[0052] During the cooling process, when the temperature is above 18°C, the behavior of the large yellow croaker is not affected; at 13-15°C, the swimming behavior is significantly reduced; at 10-13°C, the fish swim slowly and some lie still. When the temperature drops to 7.4°C, some large yellow croakers begin to lose balance and float sideways on the water. When the temperature drops to 5.2°C, all large yellow croakers float sideways. Half of the control group floated sideways at 6.8°C. At this time, the number of groups 1-3 floating sideways in the test group was significantly lower than that in the control group, and there was no significant difference between groups 1-3 and the control group. This shows that this additive can effectively improve the cold resistance of large yellow croaker. The statistics of the number of groups floating sideways are shown in the following table. Figure 1 shown.

[0053] 3.2 Effect of the additive of the present invention on the ATP content in the liver tissue of large yellow croaker.

[0054] Depend on Figure 2 It can be seen that after adding the additive of the present invention, the liver ATP content of the test groups 1-3 was significantly higher than that of the control group (P<0.05), and there was no significant difference between the comparison groups 1-3 and the control group (P>0.05). It shows that adding the feed additive of the present invention can effectively increase the ATP content of tissues.

[0055] 3.3 Effects of the additive of the present invention on lipid metabolism and tricarboxylic acid cycle in the liver of large yellow croaker.

[0056] Depend on Figure 3It can be seen that after adding the feed additive, the expression levels of liver lipolysis (lpl, hsl) and β-oxidation genes (cpt1, acox1) in the test groups 1-3 were significantly higher than those in the control group (P<0.05), and there was no significant difference between the comparison groups 1-3 and the control group (P>0.05). This shows that adding the feed additive of the present invention can effectively improve fat decomposition and promote fat mobilization.

[0057] Depend on Figure 4 It can be seen that after adding the feed additive, the expression levels of tricarboxylic acid cycle genes (cs, idh3) in the test groups 1-3 were significantly higher than those in the control group (P<0.05), and there was no significant difference between the comparison groups 1-3 and the control group (P>0.05). This shows that adding the feed additive of the present invention can effectively enhance fat decomposition and promote fat mobilization.

[0058] In summary, by adding the additive of the present invention to large yellow croaker feed, the utilization of nutrients by large yellow croaker can be effectively improved and its cold resistance can be enhanced.

[0059] Table 1 Feed formula for each group (% dry diet)

[0060]

[0061] Note:

[0062] Vitamin premix (mg / kg diet): Vitamin B1, 25; Vitamin B2, 45; Vitamin B6, 20; Vitamin K, 10; Vitamin B12, 10; Inositol, 800; Niacin, 200; Folic acid, 20; Biotin, 60; Vitamin A, 32; Vitamin D, 5; Vitamin E, 240; Pantothenic acid phosphate, 2000; Microcrystalline cellulose, 11533.

[0063] Mineral premix (mg / kg diet): CoCl 2 (1%), 50; CuSO 4 ·5H 2 O, 10; FeSO 4 7H 2 O, 80; ZnSO4·H 2 O, 50; MnSO 4 ·H 2 O, 45; MgSO 4 7H 2 O, 1,200; H 2 NaOSe (1%), 20; zeolite powder, 3485.

[0064] Others: monocalcium phosphate 0.50%, choline chloride 0.25%, attractant 1% (betaine: sulfobetaine: threonine: glycine: 5-phosphoinosine = 4:2:2:1:1); calcium propionate 0.05%; ethoxyquinoline 0.05%; sodium alginate 1%; yttrium trioxide 0.1%.

[0065] Table 2 Feed additive formula for the experimental group

[0066]

[0067] Example 8

[0068] In this embodiment, when the additive of test group 1 is added to the basic feed of large yellow croaker at an amount of 0.5% and the additive of test group 3 is added to the basic feed of large yellow croaker at an amount of 2%, both can play a good role in resisting cold.

Claims

1. An additive for improving the cold resistance of large yellow croaker, characterized in that: The additive is composed of the following components per 100 g: 2-4 g of spermidine, 6-10 g of resveratrol, 10-20 g of sodium pyruvate, and the balance is defatted rice bran as a carrier.

2. A method for cultivating large yellow croaker by using the additive according to claim 1 to improve the cold resistance of large yellow croaker, characterized in that: The method comprises the steps of adding the additive to large yellow croaker feed, wherein the additive amount accounts for 0.5-2% of the mass percentage of the feed.

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