Application of resveratrol in culture of penaeus vannamei boone

Abstract

The invention belongs to the technical field of aquaculture, and particularly relates to application of resveratrol in culture of penaeus vannamei boone, which is characterized in that the application dosage and mode of the resveratrol are that the penaeus vannamei boone is fed according to the weight proportion of 0.033-0.067 per thousand of the total weight of the penaeus vannamei boone everyday. The resveratrol is used for improving the immune function of penaeus vannamei boone, improving the survival rate, increasing the body weight growth rate and counting immune cells. The resveratrol is also used for improving the anti-oxidative stress capability of the South America white, including improving the superoxide dismutase activity of the shrimp body, and / or improving the shrimp hepatopancreas lysozyme activity of the shrimp body, and / or improving the total anti-oxidative capability of the shrimp body. The resveratrol can enhance the immune epidemic prevention level of the penaeus vannamei population on stress sources such as vibrio infection, reduce the oxidative stress risk of the penaeus vannamei population and avoid the expansion of breeding loss caused by group stress.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and particularly relates to application of resveratrol in white shrimp farming. Background Art

[0002] The scientific name of the white shrimp Penaeus vannamei is Litopenaeus vannamei, which is the largest shrimp species farmed in my country. According to the "China Fisheries Statistical Yearbook" released by the Ministry of Agriculture and Rural Affairs in 2024, white shrimp accounts for more than 80% of the total shrimp farming volume (tons) in my country. During the farming of white shrimp, various stressors such as water pollution and pathogen infection will destroy the immune defense mechanism of the shrimp population. Even if water purification or antibiotic treatment is carried out, the shrimp population will still enter a state of oxidative stress due to stressors, resulting in collective acute hepatopancreatic necrosis, and aquaculture losses will increase sharply.

[0003] Based on general experience in animal husbandry, animals with stronger physiques usually have stronger immunity. Therefore, the prior art optimizes the nutritional ratio by adding a variety of nutrients to the feed, aiming to improve the appetite and nutrient absorption rate of white shrimp, thereby obtaining white shrimp with a larger meat yield, thereby indirectly strengthening the immune defense mechanism of the shrimp population. For example, Chinese patent CN 105309844 A proposes an efficient feed and preparation method that can improve the immunity of white shrimp. The feed includes more than 20 added components, which are fermented multiple times to improve the attractant effect, and improve the immunity of white shrimp by increasing the intestinal digestive enzyme activity. This empirical combination has achieved positive feedback in breeding practice and has been welcomed by the breeding industry. However, this addition scheme lacks clear scientific principle support and may contain ineffective or limited-effect substances. Summary of the invention

[0004] The inventors first studied the immune response mechanism of Penaeus vannamei after pathogen infection. As a basis, those skilled in the art have generally recognized the general process of shrimp responding to pathogen infection: after the invasion of pathogens, shrimp blood cells initiate an immune response, engulf, encapsulate, and nodule the pathogens and concentrate the pathogens in the stratum corneum, which are completely excreted during the next molting. In the process of shrimp blood cells removing pathogens, melanin with antibacterial effects is generated and deposited on the surface of the organism, so this immune response mechanism is called melanization. The most common pathogen in aquaculture is Vibrio. The inventors conducted a comprehensive analysis of the miRNA-mRNA regulatory network in shrimp blood cells after Vibrio infection to understand the immune response of Penaeus vannamei from the molecular mechanism level. The analysis results showed that the DNA damage of shrimp blood cells increased significantly after Vibrio infection, and the expression of messenger genes related to apoptosis and autophagy of shrimp blood cells was significantly upregulated. Autophagy is the process by which organisms remove cells with damaged genomes. The inventors have obtained preliminary inspiration that the activation of the autophagy pathway is the core immune mechanism of Penaeus vannamei to respond to pathogens. (See inventor's paper Wang, Feifei, et al. "Integrative analysis of the miRNA-mRNA regulation network in hemocytes of Penaeus vannamei following Vibrioalginolyticus infection." Developmental & Comparative Immunology 131(2022):104390.)

[0005] Another study by the inventor analyzed the response mechanism of white shrimp to nitrite pollutants in water bodies. The study found through experiments that the blood cell count of white shrimp in a nitrite-polluted environment decreased and respiratory metabolism was damaged, resulting in an increase in the mortality rate of shrimp; and the introduction of autophagy agonists can alleviate the decrease in shrimp blood cell counts and improve the survival rate of shrimp. In order to determine the signal molecules related to the autophagy activation process, the inventor further performed KEGG enrichment analysis on the transcriptome of white shrimp. The results showed that autophagy activation can significantly increase the expression of prophenoloxidase (proPO) family genes, and proPO is the main working enzyme of the aforementioned melanization. In other words, after the autophagy pathway is activated, the melanization effect will also increase. The inventor has gained further inspiration from the results of this study, that is, the activation of the autophagy pathway is a basic and universal process of the shrimp immune barrier, which can help the shrimp cope with the influence of various adverse factors. (See the inventor's paper Lin, Xinghao, et al. "Transcriptome analysis of hepatopancreas revealed the role of autophagy under nitrite stress in Pacific white shrimp (Penaeus vannamei)." Aquaculture International (2024): 1-22.)

[0006] In the above experiment, the activation process of the autophagy pathway occurred after the stressor, which was a passive reaction of the shrimp. If the shrimp can have a relatively active autophagy pathway response level before the adverse factors occur, the shrimp will have stronger immunity. Commonly used autophagy agonists in laboratory studies include rapamycin (antibiotics), lithium chloride, thapsigargin, etc. These compounds cannot be directly used in aquaculture due to economic costs, human safety and other factors. Therefore, the inventor's conception was further clarified to propose an autophagy agonist with reasonable economic cost, reliable safety, and effective against white shrimp.

[0007] Since many artificially designed new compound molecules lack human safety data, the inventors hope to dig out compounds that can activate autophagy in white shrimp from medicinal plants that have been used by humans or natural plant additives used empirically in the aquaculture industry. These natural plants may be mixed or used extensively because their active ingredients and mechanisms of action are unknown. The Ministry of Agriculture and Rural Affairs has currently approved a total of 52 Chinese medicinal materials and Chinese patent medicines for the prevention and control of aquaculture diseases, including the Chinese medicine Polygonum cuspidatum. Resveratrol, an extract of Polygonum cuspidatum, is a compound that has been widely discussed in the field of human health technology in recent years. On the one hand, many experimental studies based on in vitro cell models and mammalian models have found that resveratrol can achieve tumor cell clearance by activating the autophagy pathway (see the paper Alayev, Anya, et al. "The combination of rapamycin and resveratrol blocks autophagy and induces apoptosis in breast cancer cells." Journal of cellular biochemistry 116.3 (2015): 450-457. and the paper Thongchot, Suyanee, et al. "Preclinical evidence for preventive and curative effects of resveratrol on xenograftcholangiocarcinogenesis." Cancer Letters 582 (2024): 216589.). On the other hand, in related experiments on crustaceans, resveratrol alleviated oxidative stress damage induced by starvation in marine mussels (Mytilus coruscus).

[0008] In summary, the inventors studied the immune response mechanism of white shrimp to different stressors from the molecular biology level and found that passive activation of the autophagy pathway will lead to a decrease in the number of shrimp blood cells and damage the immune defense mechanism. Improving the response level of the autophagy pathway in the shrimp population can promptly remove damaged shrimp blood cells and quickly replenish new shrimp blood cells, ensuring that the number of shrimp blood cells is sufficient and maintaining the stability of the immune defense mechanism.

[0009] In view of this, the inventors used resveratrol, which has the effect of autophagy agonist, as feed or immune enhancer for white shrimp. The inventors further found in the experiment that resveratrol can not only effectively enhance the immunity of shrimp, but also enhance the ability of shrimp to resist oxidative stress, improve the health of shrimp hepatopancreas, and avoid the chain reaction of stress and hepatopancreas necrosis that leads to the expansion of breeding losses.

[0010] The present invention is achieved through the following technical solutions:

[0011] The application of resveratrol in the cultivation of whiteleg shrimp is characterized in that the application dosage and method of the resveratrol are to feed the whiteleg shrimp daily at a weight ratio of 0.033‰ to 0.067‰ of the total weight of the shrimp.

[0012] Preferably, the resveratrol is used to enhance the immune function of Penaeus vannamei.

[0013] Preferably, the resveratrol is used to improve the survival rate of Penaeus vannamei.

[0014] Preferably, the resveratrol is used to increase the weight growth rate of Penaeus vannamei.

[0015] Preferably, the resveratrol is used to increase the immune cell count of Penaeus vannamei.

[0016] Preferably, the resveratrol is used to increase the blood cell count of Penaeus vannamei.

[0017] Preferably, the resveratrol is used to improve the anti-oxidative stress capacity of Penaeus vannamei.

[0018] Preferably, the anti-oxidative stress ability includes:

[0019] (i) increasing the superoxide dismutase activity of shrimp; and / or

[0020] (ii) increasing the activity of shrimp hepatopancreas lysozyme; and / or

[0021] (iii) Enhance the total antioxidant capacity of shrimp.

[0022] Preferably, the use comprises treating Vibrio vannamei infection in white shrimp.

[0023] Preferably, the treatment of Vibrio vannamei infection in white shrimp comprises:

[0024] (i) increasing the superoxide dismutase activity of shrimp after Vibrio infection; and / or

[0025] (ii) increasing the hepatopancreas lysozyme activity of shrimp after Vibrio infection; and / or

[0026] (iii) Enhance the total antioxidant capacity of shrimp after Vibrio infection.

[0027] The present invention has the following technical effects:

[0028] Compared with the prior art, the present invention proposes the application and dosage range of resveratrol in the cultivation of white shrimp based on the study of the immune mechanism of white shrimp. Feeding white shrimp with resveratrol at a weight ratio of 0.033‰ to 0.067‰ of the total weight of shrimp every day can improve the basic immune level of the shrimp; when stressors exist, resveratrol can also improve the anti-oxidative stress level of the shrimp, avoiding the expansion of cultivation losses caused by oxidative stress of the shrimp. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a comparison chart of the effects of resveratrol on the blood cell count of white shrimp.

[0030] Figure 2 This is a comparison chart of the effects of resveratrol on superoxide dismutase activity in Nanmeibai;

[0031] Figure 3 This is a comparison chart of the effects of resveratrol on the activity of hepatopancreas lysozyme in white shrimp Penaeus vannamei;

[0032] Figure 4 This is a comparison chart of the effects of resveratrol on the total antioxidant capacity of white shrimp;

[0033] Figure 5 This is a comparison chart of the effect of resveratrol on the blood cell count of Penaeus vannamei when Vibrio infection occurs;

[0034] Figure 6 This is a comparison chart of the effect of resveratrol on superoxide dismutase activity of Nanmeibai when Vibrio infection occurs;

[0035] Figure 7 This is a comparison chart of the effect of resveratrol on the hepatopancreas lysozyme activity of Penaeus vannamei when Vibrio infection occurs;

[0036] Figure 8 This is a comparison chart of the effects of resveratrol on the total antioxidant capacity of white shrimp when Vibrio infection occurs. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the specification. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0038] Example 1

[0039] This example is a control group, which is used to observe the baseline immune level of Penaeus vannamei when it is raised without resveratrol.

[0040] 1.1 Experimental Materials

[0041] Vannamei shrimp, purchased from the local breeding base in Zhoushan City, Zhejiang Province;

[0042] Vibrio parahaemolyticus, from the intestinal bacteria screening of whiteleg shrimp with acute hepatopancreatic necrosis in Guangzhou, cultured and preserved in the laboratory;

[0043] Total antioxidant capacity detection kit (FRAP method) was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0044] Total SOD activity detection kit (WST-8 method) was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0045] Lysozyme (LZM) test kit (with standard) (turbidimetric method) was purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.;

[0046] The basic diet was purchased from Tongwei Agricultural Development Co., Ltd. The main raw materials were fish meal, soybean meal, squid paste, flour, fish oil, amino acids, vitamins, trace elements, etc. The nutritional components are shown in Table 1.

[0047] Table 1 Nutrient composition of basic diet

[0048]

[0049] 1.2 Experimental procedures

[0050] S1. Prepare sufficient amount of whiteleg shrimps of the same growth batch, take 100 of them as the experimental group, and the rest of the whiteleg shrimps as the alternative group;

[0051] S2. Both the experimental group and the alternative group were fasted for three days, and 100% of the water was changed every day; if there were individuals in the experimental group that died, they were supplemented from the alternative group to maintain the number of white shrimp in the experimental group at 100;

[0052] S3. Feed the experimental group with the basic diet for 14 days; the total daily feeding amount is 1% of the total shrimp weight, divided into three equal feedings, with an interval of 6 hours each; if any individual dies, remove it in time and recalculate the feeding amount;

[0053] S4. After 7 days of feeding, the survival rate, body length, body weight, superoxide dismutase activity, shrimp hepatopancreas lysozyme activity, and total antioxidant capacity of Penaeus vannamei were measured;

[0054] S5, 100% water change;

[0055] S6. After 14 days of feeding, the survival rate, body length, body weight, superoxide dismutase activity, shrimp hepatopancreas lysozyme activity, and total antioxidant capacity of Penaeus vannamei were measured again;

[0056] S7, 100% water change;

[0057] S8. Continue fasting for 48 hours. After 24 hours of fasting, add 1×10 CFU Vibrio parahaemolyticus into the aquaculture water. After 48 hours of fasting, measure the superoxide dismutase activity, hepatopancreas lysozyme activity and total antioxidant capacity of white shrimp.

[0058] 1.3 Determination methods of various indicators

[0059] Shrimp blood cell counting method: Use a syringe containing sodium citrate anticoagulant to draw blood from white shrimp in a 1:1 volume ratio of blood to anticoagulant, and use a hemocytometer to count blood cells.

[0060] Determination of superoxide dismutase activity, shrimp hepatopancreas lysozyme activity, and total antioxidant capacity: Follow the instructions of the corresponding detection kits.

[0061] Example 2

[0062] This example is an experimental group, and the effect of feeding resveratrol at 0.033‰ of the total shrimp weight on Penaeus vannamei was observed.

[0063] 1.1 Experimental Materials

[0064] Resveratrol was purchased from Beijing Puxitang Biotechnology Co., Ltd., product number R10010, and the structural formula is as follows;

[0065]

[0066] The other materials are the same as those in Example 1.

[0067] 1.2 Experimental procedures

[0068] S1. Prepare sufficient amount of whiteleg shrimps of the same growth batch, take 100 of them as the control group, and the rest of the whiteleg shrimps as the alternative group;

[0069] S2, the control group and the alternative group were all fasted for three days, and 100% of the water was changed every day; if there were individuals in the control group who died, they were supplemented from the alternative group to keep the number of white shrimp in the control group at 100;

[0070] S3. The control group was fed with the basic diet for 14 days; the total daily feeding amount was 1% of the total shrimp weight, and it was fed in three equal amounts, with an interval of 6 hours each time; during the second feeding of the day, resveratrol was added at a ratio of 0.033‰ of the total shrimp weight (i.e. 0.01% of the total feed weight); if any individual died, it was removed in time and the feeding amount was recalculated;

[0071] S4. After 7 days of feeding, the survival rate, body length, body weight, superoxide dismutase activity, shrimp hepatopancreas lysozyme activity, and total antioxidant capacity of Penaeus vannamei were measured;

[0072] S5, 100% water change;

[0073] S6. After 14 days of feeding, the survival rate, body length, body weight, superoxide dismutase activity, shrimp hepatopancreas lysozyme activity, and total antioxidant capacity of Penaeus vannamei were measured;

[0074] S7, 100% water change;

[0075] S8. Continue fasting for 48 hours. After 24 hours of fasting, add 1×10 CFU Vibrio parahaemolyticus into the aquaculture water. After 48 hours of fasting, measure the superoxide dismutase activity, hepatopancreas lysozyme activity and total antioxidant capacity of white shrimp.

[0076] 1.3 Determination methods of various indicators

[0077] Same as Example 1.

[0078] Example 3

[0079] This example is an experimental group, and the effect of feeding resveratrol at 0.067‰ of the total shrimp weight on Penaeus vannamei was observed.

[0080] 1.1 Experimental Materials

[0081] Same as Example 2.

[0082] 1.2 Experimental procedures

[0083] S1. Prepare sufficient amount of whiteleg shrimps of the same growth batch, take 100 of them as the control group, and the rest of the whiteleg shrimps as the alternative group;

[0084] S2, the control group and the alternative group were all fasted for three days, and 100% of the water was changed every day; if there were individuals in the control group who died, they were supplemented from the alternative group to keep the number of white shrimp in the control group at 100;

[0085] S3. The control group was fed with the basic diet for 14 days; the total daily feeding amount was 1% of the total shrimp weight, and it was fed in three equal amounts, with an interval of 6 hours each time; during the second feeding of the day, resveratrol was added at a ratio of 0.067‰ of the total shrimp weight (i.e. 0.02% of the total feed weight); if any individual died, it was removed in time and the feeding amount was recalculated;

[0086] S4. After 7 days of feeding, the survival rate, body length, body weight, superoxide dismutase activity, shrimp hepatopancreas lysozyme activity, and total antioxidant capacity of Penaeus vannamei were measured;

[0087] S5, 100% water change;

[0088] S6. After 14 days of feeding, the survival rate, body length, body weight, superoxide dismutase activity, shrimp hepatopancreas lysozyme activity, and total antioxidant capacity of Penaeus vannamei were measured;

[0089] S7, 100% water change;

[0090] S8. Continue fasting for 48 hours. After 24 hours of fasting, add 1×10 CFU Vibrio parahaemolyticus into the aquaculture water. After 48 hours of fasting, measure the superoxide dismutase activity, hepatopancreas lysozyme activity and total antioxidant capacity of white shrimp.

[0091] 1.3 Determination methods of various indicators

[0092] Same as Example 1.

[0093] The following experimental results show the effect of resveratrol on white shrimp before Vibrio infection.

[0094] The effects of resveratrol on the survival rate, body length and weight of white shrimp are shown in Table 2. When the resveratrol feeding amount was 0.033‰ of the total shrimp weight, the survival rate, body length and weight growth rate of white shrimp on the 14th day were improved. When the feeding amount was further increased to 0.067‰ of the total shrimp weight, resveratrol still improved these indicators, but the effect was not as good as the previous group.

[0095] Table 2 Effects of resveratrol on survival rate, body length and body weight of white shrimp

[0096]

[0097] Effect of resveratrol on the blood cell count of Penaeus vannamei (unit: 10 7 / ml) Figure 1 After 7 days of feeding, the effect of resveratrol did not appear for the time being; after 14 days of feeding, the experimental group fed with resveratrol had a higher blood cell count, and this gain effect was positively correlated with the dose of resveratrol.

[0098] Effects of resveratrol on the antioxidant capacity of Penaeus vannamei Figure 2 , Figure 3 , Figure 4 shown. Figure 2 , Figure 3 , Figure 4 The performance of the shrimp groups in the three examples was compared in terms of superoxide dismutase activity (unit: umo / mgprot), shrimp hepatopancreas lysozyme activity (unit: U / mgprot), and total antioxidant capacity (unit: mmol / mg). For each index, the experimental group with a resveratrol feeding amount of 0.033‰ of the total shrimp body weight showed the highest numerical level and the best health status; however, there was no positive correlation between the index and the feeding amount.

[0099] The following experimental results show the effect of resveratrol on white shrimp when infected with Vibrio parahaemolyticus.

[0100] The changes in shrimp blood cell counts (unit of measurement: 107 / ml) before and after infection with Vibrio parahaemolyticus in each embodiment are as follows: Figure 5 As shown. After infection with Vibrio parahaemolyticus, the blood cell counts of shrimps in the three examples all decreased, indicating that the white shrimps cleared the cells destroyed by Vibrio parahaemolyticus through autophagy. After the infection, the experimental group fed with 0.033‰ resveratrol by total shrimp weight retained more shrimp blood cells and had stronger immunity.

[0101] The changes in the antioxidant capacity of each embodiment before and after Vibrio infection are as follows: Figure 6 , Figure 7 , Figure 8 shown. Figure 6 , Figure 7 , Figure 8 They correspond to changes in three indicators: superoxide dismutase activity (unit: umo / mgprot), shrimp hepatopancreatic lysozyme activity (unit: U / mgprot), and total antioxidant capacity (unit: mmol / mg). For superoxide dismutase activity, the numerical level of the experimental group fed with 0.033‰ of resveratrol by total shrimp body weight remained almost unchanged, while the other two groups showed a decrease in enzyme activity and an increase in the risk of oxidative stress; for shrimp hepatopancreatic lysozyme activity, the experimental group fed with resveratrol did not show a significant improvement; but for the total antioxidant capacity of shrimp, the experimental group fed with 0.033‰ of resveratrol by total shrimp body weight showed the highest numerical level. A higher numerical level indicates that the shrimp group has a stronger ability to resist oxidative stress, which can reduce the risk of loss expansion due to oxidative stress.

Claims

1. The application of resveratrol in the cultivation of white shrimp, characterized in that: The dosage and method of application of resveratrol is to feed the shrimp daily at a weight ratio of 0.033‰ to 0.067‰ of the total weight of the shrimp.

2. The use according to claim 1, characterized in that: The resveratrol is used for enhancing the immune function of white shrimp.

3. The use according to claim 2, characterized in that: The resveratrol is used to improve the survival rate of white shrimp.

4. The use according to claim 2, characterized in that: The resveratrol is used to increase the weight growth rate of white shrimp.

5. The use according to claim 2, characterized in that: The resveratrol is used for increasing the immune cell count of white shrimp.

6. The use according to claim 5, characterized in that: The resveratrol is used for increasing the blood cell count of Penaeus vannamei.

7. The use according to claim 1, characterized in that: The resveratrol is used for improving the anti-oxidative stress capability of white shrimp.

8. The use according to claim 7, characterized in that: The anti-oxidative stress ability includes: (i) increasing the superoxide dismutase activity of shrimp; and / or (ii) increasing the activity of shrimp hepatopancreas lysozyme; and / or (iii) Enhance the total antioxidant capacity of the shrimp hepatopancreas.

9. The use according to claim 2, characterized in that: The application includes treating Vibrio vannamei infection in white shrimp.

10. The use according to claim 9, characterized in that: The treatment of Vibrio vannamei infection in white shrimp comprises: (i) increasing the superoxide dismutase activity of shrimp after Vibrio infection; and / or (ii) increasing the hepatopancreas lysozyme activity of shrimp after Vibrio infection; and / or (iii) Enhance the total antioxidant capacity of shrimp hepatopancreas after Vibrio infection.

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