Multifunctional anti-stress agent preparation for fish farming and application thereof

Abstract

The application belongs to the technical field of aquaculture, and discloses a multifunctional anti-stress agent preparation for fish culture and application thereof. The anti-stress agent preparation provided by the application contains the following components per 1 kg of the anti-stress agent preparation compound: 100 g of vitamin C, 25 g of curcumin, 120 g of taurine, 25 g of beta-glucan, 15 g of gamma-aminobutyric acid, 50 g of astragalus polysaccharide, 25 g of betaine, 10 g of chromium picolinate, 25 g of reduced glutathione, and 605 g of carrier material. The application takes California bass as the research object, and determines the blood biochemical indexes, tissue antioxidant capacity and survival rate of the fish under stress conditions such as high temperature, hypoxia, transportation and pathogenic bacteria infection after the anti-stress agent is applied. The anti-stress agent can effectively promote the growth and production performance of the fish, improve the immunity of the fish, reduce the damage of various stresses to the body, improve the survival rate of culture, and increase the economic benefits of the breeders.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, specifically relating to a multifunctional anti-stress agent preparation for fish farming and its application. Background Technology

[0002] Currently, aquaculture is increasingly characterized by large-scale farming, and modern farming techniques are constantly improving. However, the impact of stress factors on aquatic animals is becoming more and more prominent, and stress response has become one of the most troublesome problems for aquaculture farmers. During the farming process, fish are subjected to various factors such as low dissolved oxygen levels, water temperature changes, fright, high-density farming, pH changes, pond separation, and transportation, resulting in stress. This stress leads to decreased feed intake, reduced internal synthesis capacity, suppressed immune system, and decreased resistance, causing a series of changes in the fish's nutrition and physiological metabolism, resulting in the occurrence of various diseases, often accompanied by high mortality rates, causing incalculable losses to fishery production.

[0003] When fish are under prolonged stress, they require a large amount of energy to maintain physiological balance and resist stress responses, ensuring the normal function of various tissues and organs and the health of the fish. This necessitates the consumption of large amounts of vitamins, polysaccharides, amino acids, bioactive peptides, nucleotides, organic acids, trace elements, and other nutrients. Vitamins participate in redox reactions within the body and play a crucial role in the growth, development, survival, reproduction, and immunity of fish and shrimp. They are effective anti-stress agents and are widely used in intensive aquaculture to alleviate stress responses in fish, shrimp, and other aquatic animals. Polysaccharides are widely used in aquaculture in various ways. Bioactive polysaccharides can significantly improve the health of aquatic animals, resist pathogen invasion to alleviate animal stress, and increase the productivity of aquaculture water bodies. Organic acids and amino acids can regulate some key metabolic pathways in aquatic animals, remove excess reactive oxygen free radicals in the body, resist lipid peroxidation, and play a role in resisting environmental stress and preventing various diseases. Bioactive peptides can improve the immunity and antibacterial ability of fish and shrimp, enhance antioxidant defense function, and exert non-specific immunity on fish fry. Mineral elements are essential elements for the life activities of aquatic animals. They can buffer stress, improve body growth, enhance antioxidant performance and immune disease resistance, protect aquatic animals from pathogen infection, and improve the survival rate of aquatic animals during transportation. Some traditional Chinese medicines can also effectively improve the immune activity or stress resistance of aquatic animals.

[0004] Currently, vitamins, polysaccharides, amino acids, trace elements, taurine, and extracts of traditional Chinese medicine are widely added to fish feed to mitigate the negative effects of various stresses on fish. However, due to the complexity and diversity of factors causing fish stress, such as high temperature, hypoxia, transportation, pathogenic microorganisms, netting, and water quality, and the varying mechanisms of action and harms caused by different stressors, most anti-stress agents used in aquaculture are single-component or single-function products. Their anti-stress effects and application scope are limited, and the lack of market segmentation results in less than ideal practical application effects for existing anti-stress agents.

[0005] To address the aforementioned issues, the applicant has developed a compound preparation that promotes fish growth and has anti-stress functions by combining the characteristics, complementary functions, and synergistic effects of components such as vitamins, active polysaccharides, amino acids, traditional Chinese medicine extracts, and mineral elements. This preparation can effectively alleviate various stress responses encountered during aquatic animal farming. Summary of the Invention

[0006] To address the shortcomings of existing technologies, a multifunctional anti-stress agent formulation for fish farming is provided. This compound formulation is made from natural ingredients, has a reasonable formula, and contains no other hormones. It can effectively alleviate various stresses in fish caused by high temperature, hypoxia, transportation, and pathogenic microorganisms, repair stress-induced bodily damage, promote fish production performance and improve fish quality, and reduce the feed conversion ratio.

[0007] Another object of the present invention is to provide the application of the anti-stress agent in fish farming production.

[0008] To achieve the above objectives, the present invention adopts the following technical measures:

[0009] A multifunctional anti-stress agent preparation for fish farming, each 1 kg of the anti-stress agent preparation includes: vitamin C 80-150g, curcumin 15-50g, taurine 100-180g, β-glucan 15-40g, γ-aminobutyric acid 10-30g, astragalus polysaccharide 30-80g, betaine 15-35g, glutamine 15-30g, chromium pyridinecarboxylate 6-18g, reduced glutathione 18-45g, and carrier substance 357-716g.

[0010] In the above-described scheme, the preferred composition of each 1 kg anti-stress compound preparation is as follows: Vitamin C 100g, curcumin 25g, taurine 120g, β-glucan 25g, γ-aminobutyric acid 15g, astragalus polysaccharide 50g, betaine 25g, chromium pyridinecarboxylate 10g, reduced glutathione 25g, and carrier substance 605g; the carrier substance is one or a mixture of two or more of glucose, maifanite, pine needle powder, zeolite powder, montmorillonite powder, sodium sulfate, and stone powder.

[0011] The application of anti-stress agents in fish farming production includes using the anti-stress agents of the present invention to prepare anti-stress feed or growth-promoting and stress-resistant feed additives.

[0012] In the above-described applications, the fish is preferably the California bass.

[0013] In the above applications, when used as a feed mixer, the following methods apply: During the fish fry stage, mix 1 kg of the anti-stress compound preparation with 100 kg of feed; during the fish rearing stage, mix 500 g of the anti-stress preparation with 100 kg of feed. Feed the fish containing the anti-stress preparation every other day using the normal feeding method for 40 consecutive days. Alternatively, apply the mixture to the entire pond at a rate of 250–500 g / mu•meter of water depth, once every other day for 40 consecutive days.

[0014] In the applications described above, the stress includes heat stress, hypoxia stress, transportation stress, or pathogen infection stress.

[0015] The scope of protection of this invention also includes the application of the above-mentioned anti-stress compound preparation in promoting the growth of California bass.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] (1) This invention creatively selects a certain effective concentration of curcumin, taurine, chromium pyridinecarboxylate, reduced glutathione, astragalus polysaccharide, vitamin C, γ-aminobutyric acid, and betaine, and mixes them to make an anti-stress agent. Then, 0.5% to 1% of the low-content anti-stress agent is added to the feed and fed to largemouth bass. The weight gain rate, survival rate, resistance to high temperature stress, resistance to hypoxia stress, resistance to transportation stress and resistance to pathogenic microorganism infection stress of largemouth bass are significantly improved. At the same time, the feed coefficient and breeding cost are significantly reduced.

[0018] (2) Among the anti-stress agent components of this invention: curcumin has antioxidant, antibacterial and anti-inflammatory, antiviral, free radical scavenging, blood-activating and choleretic, and immune-enhancing effects; taurine has growth-promoting, immune-enhancing and antioxidant effects, and can also combine with free radicals and strong oxidizing substances in the body to enhance the body's antioxidant capacity. Together with γ-aminobutyric acid, it can significantly alleviate stress caused by transportation or high temperature during fish farming; the main functions of chromium pyridinecarboxylate are lowering blood sugar, lowering blood lipids, and... This invention enhances the body's immunity and improves the antioxidant and stress-resistant capabilities of animals. Reduced glutathione has the effect of eliminating free radicals and reducing oxidative stress, and is generally used in clinical drugs, human antioxidants, and food additives. This invention creatively formulates reduced glutathione into an anti-stress agent for farmed fish, which can promote fish growth and improve their stress resistance. Astragalus polysaccharide is a water-soluble heteropolysaccharide extracted, concentrated, and purified from the dried roots of Astragalus membranaceus or Astragalus mongholicus, and can be used as an immune booster for fish. These substances, including regulators, possess antiviral, antitumor, anti-aging, anti-radiation, anti-stress, and antioxidant properties, significantly improving fish survival rates during aquaculture. Vitamin C, characterized by its high content and fine particle size, exhibits high stability under high temperature and pressure, remaining completely free within the fish body and being effectively absorbed and utilized, directly improving survival and weight gain rates while reducing the feed conversion ratio. Furthermore, Vitamin C acts as an antioxidant, scavenging free radicals and reducing their attack on internal organs, significantly enhancing fish's stress resistance. Gamma-aminobutyric acid (GABA) has calming, anti-anxiety, and neuronal activity-reducing properties, preventing nerve cell overheating. GABA also promotes the secretion of gastric juice and growth hormone, and stimulates the animal's feeding center, thereby increasing growth rate and feed intake. Betaine enhances the tolerance of biological cells to high temperatures, high salt, and high osmotic environments, stabilizes enzyme activity (i.e., the function of biological macromolecules), regulates the osmotic pressure of gastrointestinal epithelial cells, prevents water loss, maintains intestinal ion balance, reduces energy requirements, and lowers stress.

[0019] (3) The main components and concentrations of the anti-stress agent of this invention were obtained through rigorous fish experiments. The effects of the compound anti-stress agent were also evaluated through fish transport stress test, fish hypoxia stress test, high temperature stress test and pathogenic microorganism stress test. The results showed that the compound anti-stress agent can significantly improve the antioxidant, hypoxia, high temperature and pathogenic microorganism stress resistance of largemouth bass, improve its survival rate under stress conditions, promote the growth of largemouth bass, and significantly reduce the feed coefficient.

[0020] (4) The raw materials for the anti-stress agent of this invention are common, readily available, and inexpensive. This invention achieves enhanced three-tiered antioxidant systems in fish tissues by scientifically combining and screening substances with different antioxidant and anti-stress mechanisms, preventing the formation of free radicals, simultaneously eliminating existing free radicals, limiting chain formation and proliferation, and repairing and eliminating cellular and molecular damage in stressed organisms. Furthermore, vitamins, as coenzymes in metabolism, promote the metabolism of glucose, protein, and lipids, enhancing immune response. This multifunctional anti-stress compound preparation effectively reduces the impact of stress on fish farming caused by decreased feed intake, weakened immune resistance, and reduced biosynthetic capacity. Using this product not only allows fish to quickly recover from stress but also improves liver detoxification, enhances immunity, increases feed intake, improves feeding efficiency, and improves production performance. Detailed Implementation

[0021] The following examples and experimental cases are used to illustrate the present invention, but are not intended to limit the scope of the invention. All materials involved in the invention are common commercially available products. Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the reagents and materials used in the following examples are commercially available unless otherwise specified.

[0022] Example 1:

[0023] A multifunctional anti-stress compound preparation for fish farming, calculated by weight, consists of the following components per 1 kg of anti-stress compound preparation: 80 g vitamin C, 15 g curcumin, 100 g taurine, 15 g β-glucan, 10 g γ-aminobutyric acid, 30 g astragalus polysaccharide, 15 g betaine, 6 g chromium pyridinecarboxylate, 18 g reduced glutathione, and 716 g carrier substance.

[0024] The carrier is glucose.

[0025] After preparing various powdered materials, the materials are stirred and mixed in a low-temperature drying environment to ensure that the mixture is fully dried. Then, the mixture is automatically and quantitatively packaged into 1kg bags, which are then vacuum-packed and sealed in bags made of aluminum-plastic composite film to obtain a multifunctional anti-stress preparation for fish.

[0026] Example 2:

[0027] A multifunctional anti-stress compound preparation for fish farming, calculated by weight, consists of the following components per 1 kg of anti-stress compound preparation: 100 g vitamin C, 25 g curcumin, 120 g taurine, 25 g β-glucan, 15 g γ-aminobutyric acid, 50 g astragalus polysaccharide, 25 g betaine, 10 g chromium pyridinecarboxylate, 25 g reduced glutathione, and 605 g carrier substance.

[0028] The carrier is glucose.

[0029] After preparing various powdered materials, the materials are stirred and mixed in a low-temperature drying environment to ensure that the mixture is fully dried. Then, the mixture is automatically and quantitatively packaged into 1kg bags, which are then vacuum-packed and sealed in bags made of aluminum-plastic composite film to obtain a multifunctional anti-stress preparation for fish.

[0030] Example 3:

[0031] A multifunctional anti-stress compound preparation for fish farming, calculated by weight, consists of the following components per 1 kg: Vitamin C 130g, Curcumin 35g, Taurine 150g, β-glucan 30g, γ-aminobutyric acid 20g, Astragalus polysaccharide 65g, Betaine 25g, Chromium pyridinecarboxylate 15g, Reduced glutathione 35g, and Carrier substance 485g.

[0032] The carrier is glucose.

[0033] After preparing various powdered materials, the materials are stirred and mixed in a low-temperature drying environment to ensure that the mixture is fully dried. Then, the mixture is automatically and quantitatively packaged into 1kg bags, which are then vacuum-packed and sealed in bags made of aluminum-plastic composite film to obtain a multifunctional anti-stress preparation for fish.

[0034] Example 4:

[0035] A multifunctional anti-stress compound preparation for fish farming, calculated by weight, consists of the following components per 1 kg of anti-stress compound preparation: Vitamin C 150g, Curcumin 50g, Taurine 180g, β-glucan 40g, γ-aminobutyric acid 30g, Astragalus polysaccharide 80g, Betaine 35g, Chromium pyridinecarboxylate 18g, Reduced glutathione 45g, and Carrier substance 357g.

[0036] The carrier is glucose.

[0037] After preparing various powdered materials, the materials are stirred and mixed in a low-temperature drying environment to ensure that the mixture is fully dried. Then, the mixture is automatically and quantitatively packaged into 1kg bags, which are then vacuum-packed and sealed in bags made of aluminum-plastic composite film to obtain a multifunctional anti-stress preparation for fish.

[0038] Example 5:

[0039] Application effects of the multifunctional anti-stress agent formulations for fish farming prepared in Examples 1-4:

[0040] Feeding experiment:

[0041] Five hundred California bass fry with an average weight of 12g ± 0.5g from a local California bass fry breeding company in Foshan City, Guangdong Province, were randomly divided into five groups: a normal temperature group, a control group, and experimental groups A, B, C, and D, with 100 fish in each group.

[0042] The control group consisted of commercially available California bass basal diets, which were the standard feeds used for the daily feeding of California bass.

[0043] Experimental Group A: The control group received conventional feed plus the anti-stress agent from Example 1, added at a ratio of 4.0 kg per 1.0 ton of feed;

[0044] Experimental Group B: The control group received standard feed plus the anti-stress agent from Example 2, added at a ratio of 4.0 kg per 1.0 ton of feed.

[0045] Experimental Group C: The control group received standard feed plus the anti-stress agent from Example 3, at a rate of 4.0 kg per 1.0 ton of feed.

[0046] Experimental group D: The control group used conventional feed plus the anti-stress preparation from Example 4, which was added at a ratio of 4.0 kg per 1.0 ton of feed.

[0047] During the experiment, water quality and environmental hygiene were carefully managed. Fish were fed twice daily, at 8:00 AM and 6:00 PM, ensuring they were fully fed. After feeding, to maintain water quality, feed on the water surface and feces at the bottom of the buckets were promptly cleaned. The anti-stress agent was mixed with feed and fed for 40 days. After the experiment, the number of fish in each group was counted to calculate the survival rate. Then, six fish were randomly selected from each group and weighed, with each group weighed three times. Growth performance indicators for each group were calculated, including specific growth rate (SGR), weight gain (WG), average daily weight gain (ADG, g·d⁻¹), feed conversion ratio, and survival rate. The final results are expressed as mean + standard error (see Table 1). SPSS 20.0 data analysis software was used to perform significance analysis on the experimental data.

[0048] Table 1. Growth performance of fish in each group after 40 days of feeding with anti-stress agents mixed into feed.

[0049] .

[0050] Note: Different numbers of superscript "*" indicate significant differences between the data.

[0051] Table 1 shows the experimental results of the effect of a multifunctional anti-stress agent formulation for fish farming on the growth performance of largemouth bass. It can be seen that the anti-stress agent prepared in this invention, when mixed with feed and fed to largemouth bass, improved the growth rate and daily weight gain, reduced the feed conversion ratio, and increased the feed conversion rate compared to the conventional feed control group. Furthermore, the experimental indicators of Example 2 (B) were superior to those of Examples 1 (A), 3 (C), and 4 (D). Although the survival rate of the experimental groups was higher than that of the control group, there were no significant differences among them. This indicates that the anti-stress feed additive provided by this invention has a significant effect on improving the growth performance of fish.

[0052] Heat stress test

[0053] After the anti-stress preparation was mixed with feed and the fish were cultured, 20 fish from each of the control and experimental groups were randomly selected for heat treatment. The water temperature was raised to 35±0.5℃ and maintained for 24 hours. During this period, the condition and survival of the fish in each group were recorded. After 24 hours of heat stress, serum was collected from 5 fish in each heat stress experimental group. The levels of superoxide dismutase (SOD), alkaline phosphatase (AKP), lysozyme (LZM), malondialdehyde (MDA), acetylcholinesterase (AcH-E), and cortisol (COR) in the serum were measured using a commercially available kit (Nanjing Jiancheng Biotechnology Research Institute, China). The final results are expressed as mean + standard error, and SPSS 20.0 data analysis software was used to perform significance analysis on the experimental data.

[0054] Table 2. Changes in antioxidant and immune-related indicators in fish serum and fish survival status after heat stress.

[0055] .

[0056] Note: Different numbers of superscript "*" indicate significant differences between the data.

[0057] Table 2 shows that a multifunctional anti-stress agent for fish farming, when mixed with feed and fed to largemouth bass, significantly improves the antioxidant capacity, immunity, and survival rate of largemouth bass under high-temperature conditions. It also significantly increases the levels of SOD, AKP, LZM, and AcH-E enzymes in the fish serum, while reducing MAD and COR levels, thus minimizing the damage caused by high-temperature stress and improving aquaculture efficiency. Furthermore, the experimental indicators in Example 2 (B) are superior to those in Example 1 (A), Example 3 (C), and Example 4 (D).

[0058] Simulated transportation stress test

[0059] After the anti-stress preparation feeding experiment concluded, 20 fish from each of the control and experimental groups were randomly selected for a simulated transport stress experiment. The 20 fish from each group were placed in transport bags and oxygenated. All transport bags containing fish were placed in a constant temperature (30℃) shaker with a shaking frequency of 50 rpm / min to simulate transport for 12 hours. During this period, the condition and survival rate of the fish in each group were recorded. After 12 hours, serum was collected from 5 fish in each of the simulated transport experimental groups. The levels of superoxide dismutase (SOD), alkaline phosphatase (AKP), lysozyme (LZM), malondialdehyde (MDA), acetylcholinesterase (AcH-E), and cortisol (COR) in the serum were measured using a commercially available kit (Nanjing Jiancheng Biotechnology Research Institute, China). The final results are expressed as mean + standard error, and SPSS 20.0 data analysis software was used for statistical significance analysis of the experimental data.

[0060] Table 3. Changes in antioxidant and immune-related indicators in fish serum and fish survival status after transport stress.

[0061] .

[0062] Note: Different numbers of superscript "*" indicate significant differences between the data.

[0063] Table 3 shows that a multifunctional anti-stress agent for fish farming, when mixed with feed and administered to largemouth bass, significantly improves the antioxidant capacity and immunity of largemouth bass during transportation. It also significantly increases the levels of SOD, AKP, LZM, and AcH-E enzymes in the fish serum, while reducing MAD and COR levels, thus minimizing damage caused by transportation stress and improving aquaculture efficiency. Furthermore, the experimental indicators in Example 2 (B) are superior to those in Example 1 (A), Example 3 (C), and Example 4 (D).

[0064] Hypoxia stress test

[0065] After the anti-stress preparation feeding experiment concluded, 20 fish from each of the control and experimental groups were randomly selected for hypoxia stress testing. Each group's 20 fish were placed in a 10L water tank and kept statically for 6 hours without any aeration. The condition and survival rate of the fish were recorded during this period. Six hours later, serum samples were collected from 5 fish in each of the simulated transport experimental groups. A commercially available kit (Nanjing Jiancheng Biotechnology Research Institute, China) was used to measure the levels of superoxide dismutase (SOD), antioxidant capacity (AOC), alkaline phosphatase (AKP), lysozyme (LZM), malondialdehyde (MDA), acetylcholinesterase (AcH-E), and cortisol (COR) in the serum. The final results are expressed as mean + standard error, and SPSS 20.0 data analysis software was used for significance analysis of the experimental data.

[0066] Table 4. Changes in antioxidant and immune-related indicators in fish serum and fish survival status after hypoxic stress.

[0067] .

[0068] Note: Different numbers of superscript "*" indicate significant differences between the data.

[0069] Table 4 shows that a multifunctional anti-stress agent for fish farming, when mixed with feed and administered to largemouth bass, significantly improves their antioxidant capacity, immunity, and survival rate under hypoxic conditions. It also significantly increases the levels of SOD, AKP, LZM, and AcH-E enzymes in the fish serum, while reducing MAD levels, thus mitigating the damage caused by hypoxic stress. After 6 hours of hypoxia, the survival rate of fish fed the anti-stress agent was 75% or higher, significantly higher than the 30% of the control group. Furthermore, no fish fed with anti-stress agent B showed mortality. In addition, the experimental indicators of Example 2 (B) are superior to those of Examples 1 (A), 3 (C), and 4 (D).

[0070] Pathogenic microorganism infection stress test

[0071] After the feed feeding and rearing trial concluded, 20 fish from each of the control and experimental groups were randomly selected for a pathogenic microorganism infection stress test. Each fish was intraperitoneally injected with 0.2 mL of bacterial solution at a concentration of 5*10⁻⁶. 8 A suspension of Aeromonas vesiculosus at CFU / ml was administered, and the water temperature was maintained at 28±0.5℃. The condition and survival of fish in each group were continuously observed and recorded within 24 hours after infection with the pathogen. The number of surviving fish was counted every 2 hours after injection of Aeromonas vesiculosus.

[0072] Table 5. Survival status of fish in each group within 24 hours after infection with Aeromonas versicolor following pathogen stress.

[0073] .

[0074] Table 5 shows that a multifunctional anti-stress agent for fish farming, when mixed with feed and fed to largemouth bass, can effectively improve the largemouth bass's ability to resist pathogenic microorganisms. Although there was no significant difference in the final mortality rate between the experimental groups and the control group, the mortality process of the fish fed with the anti-stress agent was significantly slowed down.

Claims

1. The application of a multifunctional anti-stress agent formulation for fish farming in the preparation of a growth-promoting and stress-resistant feed additive for California bass, wherein each 1 kg of the anti-stress agent formulation comprises the following components: Vitamin C 100g, curcumin 25g, taurine 120g, β-glucan 25g, γ-aminobutyric acid 15g, astragalus polysaccharide 50g, betaine 25g, chromium pyridinecarboxylate 10g, reduced glutathione 25g, carrier substance 605g; the carrier substance is glucose, and the stress is heat stress, hypoxia stress, transportation stress, or pathogenic microorganism infection stress.

2. According to claim 1, the feed additive is used as follows: during the fish fry stage, mix 1 kg of anti-stress agent with 100 kg of feed; during the fish growth stage, mix 500 g of anti-stress agent with 100 kg of feed, and feed the fish containing the anti-stress agent once every other day according to the normal feeding method for 40 consecutive days; or sprinkle the feed throughout the pond at a rate of 250-500 g / mu•meter water depth, sprinkle once every other day for 40 consecutive days.