A feed additive for improving ammonia nitrogen tolerance of Litopenaeus vannamei and its application

Abstract

The present invention relates to a feed additive for improving ammonia nitrogen tolerance of Penaeus vannamei and its application, belonging to the technical field of fish farming, wherein the feed additive is composed of a nitrogen excretion-promoting amino acid complex, a TCA-promoting complex and a short-chain fatty acid; by weight, in the feed additive, the nitrogen excretion-promoting amino acid complex accounts for 45 to 55 parts, the TCA-promoting complex accounts for 18 to 22 parts, and the short-chain fatty acid accounts for 13 to 18 parts, with defatted rice bran as the remaining carrier; the nitrogen excretion-promoting amino acid complex can promote the metabolism of toxic ammonia to synthesize low-toxic compounds and excrete them from the body. The present invention also provides an application of the feed additive to improve the ammonia nitrogen tolerance of Penaeus vannamei, which can effectively slow down the nitrogen excretion and detoxification ability of Penaeus vannamei in a high ammonia nitrogen environment and improve its survival rate.

Description

Technical Field

[0001] The invention belongs to the technical field of fish breeding, and specifically relates to an additive for improving the ammonia nitrogen tolerance of Penaeus vannamei and an application thereof, that is, a nutritional solution that can effectively slow down the nitrogen excretion and detoxification ability of Penaeus vannamei in a high ammonia nitrogen environment and improve the ammonia nitrogen tolerance rate and survival rate of Penaeus vannamei. Background Art

[0002] Litopenaeus vannamei is an important aquaculture economic variety in my country. In 2022, the aquaculture output exceeded 1.34 million tons. It is currently the largest crustacean aquaculture variety in my country with broad market development prospects. However, while adopting a high-density aquaculture model to pursue aquaculture output, it also brings intensified aquaculture challenges. Among them, ammonia is the main environmental toxin in aquaculture water. Crustaceans are very sensitive to ammonia nitrogen. High ammonia nitrogen will inhibit their growth and even cause death, seriously affecting the efficiency of aquaculture. Therefore, it is urgent to develop a safe and efficient ammonia nitrogen tolerance additive for application in the aquaculture process of Litopenaeus vannamei. The published patent application CN 111642656A only improves the immune performance of shrimp by adjusting the feed formula to enhance its ammonia nitrogen tolerance. However, there are few reports on feed additives that can accurately regulate the tolerance of Litopenaeus vannamei in a high ammonia nitrogen environment based on its metabolic characteristics. Summary of the invention

[0003] In response to the above problems, our team found that non-ionic NH 3 It is easy to diffuse into the tissue through the cell membrane, increase the concentration of hemolymph ammonia nitrogen, and cause toxicity. In the vannamei shrimp, two detoxification pathways are retained to metabolize toxic ammonia into less toxic compounds. One is to consume glutamate under the catalysis of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) to generate non-toxic glutamine (Gln). The other is to convert toxic ammonia into urea through the urea cycle and excrete it from the body. Both detoxification processes require a large amount of energy. The tricarboxylic acid cycle (TCA) is the final metabolic pathway of the three major nutrients (sugars, lipids, and proteins), and it is also the fastest way to produce energy. Based on the above research content, the present invention provides a feed additive and its application for improving the ammonia nitrogen tolerance of vannamei shrimp.

[0004] The present invention is achieved through the following technical solutions:

[0005] A feed additive for improving ammonia nitrogen tolerance of Penaeus vannamei, the feed additive consisting of a nitrogen excretion-promoting amino acid complex, a TCA-promoting complex and short-chain fatty acids; in terms of weight, in the feed additive, the nitrogen excretion-promoting amino acid complex accounts for 45 to 55 parts, the TCA-promoting complex accounts for 18 to 22 parts, and the short-chain fatty acids account for 13 to 18 parts, with defatted rice bran serving as the remainder carrier; the nitrogen excretion-promoting amino acid complex can promote the metabolism of toxic ammonia to synthesize low-toxic compounds, which are then excreted from the body.

[0006] Furthermore, it is preferred that the nitrogen excretion promoting amino acid complex accounts for 50 parts, the TCA promoting complex accounts for 20 parts, and the short-chain fatty acids account for 15 parts;

[0007] The components of the nitrogen excretion promoting amino acid complex include L-glutamic acid and L-asparagine, and the compounding ratio of the two is (4-8): (3-5), preferably 6:4. L-glutamic acid is a synthetic substrate for glutamine, and its supplementation can increase the rate of glutamine synthesis. L-asparagine participates in the urea cycle, and citrulline carrying ammonia reacts with aspartic acid. The product is enzymatically hydrolyzed to generate ornithine and urea.

[0008] Its supplementation can increase the urea cycle flow. The nitrogen excretion-promoting amino acid complex can promote the metabolism of toxic ammonia to synthesize low-toxic compounds and excrete them from the body.

[0009] The components of the TCA-promoting complex include citric acid, ferrous fumarate and succinic acid, and the mass ratio thereof is (3-5):(2-4):(2-4), preferably 4:3:3. Citric acid, ferrous fumarate and succinic acid are intermediate metabolites of the TCA cycle, and their supplementation can promote the high-flux TCA cycle. The TCA complex can promote the TCA cycle and provide energy for nitrogen excretion metabolism.

[0010] The components of the short-chain fatty acids include acetic acid, propionic acid, butyric acid and isobutyric acid, and the mass ratio thereof is (2-4):(2-4):(1-3):(1-3), preferably 3:3:2:2. The short-chain fatty acids can be used as metabolic raw materials to provide energy and maintain the energy homeostasis of the shrimp under ammonia nitrogen stress.

[0011] The present invention also provides an application of the feed additive to improve the ammonia nitrogen tolerance of Penaeus vannamei. The application method is to add the additive into Penaeus vannamei feed, and the amount of the additive accounts for 1.5%-3.5% of the weight of the basic feed formula of Penaeus vannamei.

[0012] Furthermore, the dosage of the additive accounts for 2.5% of the feed of Penaeus vannamei.

[0013] The beneficial effects of the present invention compared with the prior art are as follows:

[0014] The feed additive for improving ammonia nitrogen tolerance of Penaeus vannamei of the present invention can effectively slow down the nitrogen excretion and detoxification ability of Penaeus vannamei in a high ammonia nitrogen environment, and improve its survival rate. The present invention is based on the latest research theory, has clear regulatory targets, is safe and efficient, has a reasonable formula, has synergistic effects between the components, and the combined use has better and more stable effects than single use. The preparation method is simple, the conditions are well controllable, the feed stability is good, and it has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The bar graphs are for the ATP content in the hepatopancreas of Litopenaeus vannamei in each experimental group;

[0016] Figure 2 This is a bar graph of the effects of each experimental group on TCA in the hepatopancreas of Penaeus vannamei. 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, and comparison group 3. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below through specific implementation cases. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art are all within the scope of the claims attached to this application.

[0018] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents from the conventional market.

[0019] Control group 1: basic feed for Penaeus vannamei without compound additives. Formula of basic feed for Penaeus vannamei: fish meal: 20 parts; soybean meal: 25 parts; peanut meal: 15 parts; wheat flour: 22 parts; gluten: 3 parts; squid paste: 3 parts; fish oil: 2.6 parts; lecithin oil: 1.5 parts; compound vitamins: 1.5 parts; compound minerals: 1.5 parts.

[0020] Example 1: Add compound additives to the basic feed of Penaeus vannamei, with an addition amount of 2.5%, including 50 parts of nitrogen excretion-promoting amino acid compound, 20 parts of TCA compound, and 15 parts of short-chain fatty acids.

[0021] The components of the nitrogen excretion-promoting amino acid complex include L-glutamic acid and L-asparagine, with a compound ratio of 6:4; the components of the TCA-promoting complex include citric acid, ferrous fumarate and succinic acid, with a mass ratio of 4:3:3; the components of the short-chain fatty acids include acetic acid, propionic acid, butyric acid and isobutyric acid, with a mass ratio of 3:3:2:2.

[0022] Example 2: Add compound additives to the basic feed of Penaeus vannamei, with an addition amount of 1.5%, 45 parts of nitrogen excretion-promoting amino acid complex, 18 parts of TCA complex, and 13 parts of short-chain fatty acids.

[0023] The components of the nitrogen excretion-promoting amino acid complex include L-glutamic acid and L-asparagine, with a compound ratio of 6:4; the components of the TCA-promoting complex include citric acid, ferrous fumarate and succinic acid, with a mass ratio of 5:2:2; the components of the short-chain fatty acids include acetic acid, propionic acid, butyric acid and isobutyric acid, with a mass ratio of 4:4:1:1.

[0024] Example 3: A composite additive was added to the basic feed of Penaeus vannamei, with the addition amount being 3.5%, wherein the nitrogen excretion-promoting amino acid complex accounted for 55 parts, the TCA-promoting complex accounted for 22 parts, and the short-chain fatty acid accounted for 18 parts.

[0025] The components of the nitrogen excretion-promoting amino acid complex include L-glutamic acid and L-asparagine, with a compound ratio of 6:4; the components of the TCA-promoting complex include citric acid, ferrous fumarate and succinic acid, with a mass ratio of 5:3:2; the components of the short-chain fatty acids include acetic acid, propionic acid, butyric acid and isobutyric acid, with a mass ratio of 4:3:2:1.

[0026] Example 4 Comparative Example 1: In the control group of Example 1, only the nitrogen excretion-promoting amino acid complex was added to the feed, and other conditions remained unchanged.

[0027] Example 5 Comparative Example 2: In the control group of Example 1, only the TCA-promoting complex was added to the feed, and other conditions remained unchanged.

[0028] Example 6 Comparative Example 3: In the control group of Example 1, only short-chain fatty acids were added to the feed, and other conditions remained unchanged.

[0029] Example 7 Selected Penaeus vannamei with good vitality and neat specifications (average specifications: 10.0 ± 0.30g), randomly divided into 7 treatments, fed the control group and the feed described in Examples 1-6, respectively, 3 repetitions were set for each treatment, 40 tails for each repetition, and four feedings per day (7 am, 11 am, 3 pm, 7 pm, and 7 pm) were implemented, and the feeding was continued for 14 days. After the breeding stage, a toxicity test was carried out. The water environment with different ammonia nitrogen concentrations (5.0, 7.0, 10.0, 15.0, 17.0, and 20.0 mg / L) was configured with ammonium chloride for preliminary testing to determine the half-lethal concentration of ammonia nitrogen in the test shrimp under the breeding conditions in 24h. The uniform configuration pre-test obtained a half-lethal concentration of ammonia nitrogen water, and 20 tail shrimps were taken for each repetition for 12h, 24h, and 36h toxicity test, and the mortality rate of the test shrimp at each time point was determined.

[0030] After the experiment, 6 test shrimps were randomly selected from each pool to take hemolymph and hepatopancreas samples, and stored in a -80℃ refrigerator for analysis of hemolymph ammonia concentration, glutamine (Gln), and urea (Urea) content; hepatopancreas glutamate dehydrogenase (GDH) and glutamine synthetase (GS) activities; hepatopancreas ATP content; hepatopancreas TCA key enzyme gene expression levels. The determination of biochemical indicators used commercial kits from Nanjing Jiancheng Biotechnology Co., Ltd. The gene expression level was detected by real-time fluorescence quantitative PCR. Data statistics: SPSS17.0 software was used to analyze the data results, and the significant differences between the data were analyzed by ANOVA. The values ​​with significant differences (P<0.05) were marked with English superscript letters.

[0031] Experimental results: The effects of each experimental group on the survival rate of Penaeus vannamei under ammonia nitrogen stress are shown in Table 1. After adding the feed additive, the survival rate of Example 1-3 groups under ammonia nitrogen stress was significantly higher than that of the control group (P<0.05), indicating that adding the feed additive of the present invention can effectively improve the ammonia nitrogen tolerance of shrimp.

[0032] The effects of each experimental group on the nitrogen excretion capacity of Penaeus vannamei are shown in Table 2. After adding the feed additive, the blood ammonia concentration, glutamine content and urea content in the hemolymph of the Example 1-3 groups were significantly higher than those of the control group (P<0.05). This shows that adding the feed additive of the present invention can effectively improve the nitrogen excretion capacity of shrimp. The effects of each experimental group on the ATP content in the hepatopancreas of Penaeus vannamei are shown in Table 2. Figure 1 After adding the feed additive, the ATP content in the hepatopancreas tissue of the Example 1-3 group was significantly higher than that of the control group (P<0.05). This shows that adding the feed additive of the present invention can effectively improve the energy homeostasis of shrimp under ammonia nitrogen stress.

[0033] Effects of each experimental group on TCA in the hepatopancreas of Penaeus vannamei Figure 2 After adding the feed additive, the gene expression levels of TCA key metabolic regulatory factors in the hepatopancreas tissue of Example 1-3 groups were significantly higher than those of the control group (P<0.05). This shows that adding the feed additive of the present invention can effectively improve the TCA flux of shrimp under ammonia nitrogen stress.

[0034] In summary, the composite feed additive of the present invention is added into feed, which can effectively improve the ability of Penaeus vannamei to excrete nitrogen and detoxify, and enhance its tolerance to ammonia nitrogen.

[0035] Table 1: Effects of each experimental group on the survival rate of Penaeus vannamei under ammonia nitrogen stress (%)

[0036] project Control group Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Mortality 12h <![CDATA[18.16±2.24 c ]]> <![CDATA[10.16±1.19 a ]]> <![CDATA[12.17±2.51 a ]]> <![CDATA[11.72±1.96 a ]]> <![CDATA[14.35±1.73 b ]]> <![CDATA[15.84±1.36 bc ]]> <![CDATA[17.02±2.38 c <!-- 3 -->]]> Mortality rate 24h <![CDATA[35.27±3.38 c ]]> <![CDATA[24.27±2.93 a ]]> <![CDATA[26.76±3.56 a ]]> <![CDATA[27.13±3.14 a ]]> <![CDATA[30.61±1.89 b ]]> <![CDATA[32.50±4.15 bc ]]> <![CDATA[34.02±4.07 c ]]> Mortality rate 36h <![CDATA[56.13±3.02 b ]]> <![CDATA[41.13±3.68 a ]]> <![CDATA[42.79±4.11 a ]]> <![CDATA[44.10±3.08 a ]]> <![CDATA[51.86±2.43 b ]]> <![CDATA[54.69±3.28 b ]]> <![CDATA[53.12±5.41 b ]]>

[0037] Table 2: Effects of each experimental group on the nitrogen excretion capacity of Penaeus vannamei

[0038]

Claims

1. A feed additive for improving ammonia nitrogen tolerance of Litopenaeus vannamei, characterized in that: The feed additive is composed of a nitrogen excretion-promoting amino acid complex, a TCA-promoting complex, and short-chain fatty acids; in terms of weight, the nitrogen excretion-promoting amino acid complex accounts for 45 to 55 parts, the TCA-promoting complex accounts for 18 to 22 parts, and the short-chain fatty acids account for 13 to 18 parts, with defatted rice bran serving as the remaining carrier; the nitrogen excretion-promoting amino acid complex can promote the metabolism of toxic ammonia to synthesize low-toxic compounds and excrete them from the body; The components of the nitrogen excretion-promoting amino acid complex are composed of L-glutamic acid and L-asparagine, and the composite mass ratio of the two is (4-8):(3-5); The components of the TCA-promoting complex are composed of citric acid, ferrous fumarate and succinic acid, and the mass ratio thereof is (3-5):(2-4):(2-4); The components of the short-chain fatty acids are composed of acetic acid, propionic acid, butyric acid and isobutyric acid, and the mass ratio thereof is (2-4):(2-4):(1-3):(1-3).

2. A feed additive for improving ammonia nitrogen tolerance of Litopenaeus vannamei according to claim 1, characterized in that: The nitrogen excretion promoting amino acid complex accounts for 50 parts, the TCA promoting complex accounts for 20 parts, and the short-chain fatty acids account for 15 parts.

3. A feed additive for improving ammonia nitrogen tolerance of Litopenaeus vannamei according to claim 1, characterized in that: The mass ratio of the L-glutamic acid to the L-asparagine is 6:

4.

4. A feed additive for improving ammonia nitrogen tolerance of Litopenaeus vannamei according to claim 1, characterized in that: The mass ratio of citric acid, ferrous fumarate and succinic acid in the components of the TCA-promoting complex is 4:3:

3.

5. A feed additive for improving ammonia nitrogen tolerance of Litopenaeus vannamei according to claim 1, characterized in that: The mass ratio of the acetic acid, propionic acid, butyric acid and isobutyric acid is 3:3:2:

2.

6. An application of the feed additive according to any one of claims 1 to 5 to improve the ammonia nitrogen tolerance of Penaeus vannamei, the application method comprising adding the feed additive to a basic feed of Penaeus vannamei, wherein the amount of the additive accounts for 1.5% to 3.5% of the weight of the basic feed of Penaeus vannamei.

7. The use according to claim 6, characterized in that: The amount of the feed additive used accounts for 2.5% of the weight of the basic feed formula of Penaeus vannamei.

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