Periodic feeding method of super-micro powder of vine tea in aquaculture and application thereof

Abstract

This application discloses a method for periodically feeding vine tea ultrafine powder in aquaculture and its application. The method includes the following steps: mixing vine tea ultrafine powder into aquatic animal feed at an addition rate of 1% to 8% of the total feed weight, and feeding it using a periodic feeding pattern; the periodic feeding pattern consists of continuous feeding for 7 days, followed by a 7-21 day break, which constitutes one cycle. This application, by pulverizing vine tea to an optimal particle size of 300 mesh, and by optimizing the optimal addition rate of 4% and the periodic feeding strategy of "7 days of continuous use, 14 days of break," fully utilizes the antioxidant and immune-enhancing effects of vine tea while effectively avoiding the potential liver and kidney burden and immunosuppression risks associated with long-term continuous use. Large-scale application trials show that this method achieves a protection rate of 52.91% to 65.14% in various aquatic animals, including crucian carp, largemouth bass, whiteleg shrimp, carp, and tilapia, demonstrating significant economic benefits and promising prospects for industrial application.

Description

Technical Field

[0001] This application relates to the field of aquaculture technology, specifically to a method for periodically feeding vine tea ultrafine powder in aquaculture and its application. Background Technology

[0002] In intensive aquaculture, high-density stocking and difficulties in water quality control lead to chronic stress in farmed animals, resulting in weakened immunity and frequent outbreaks of diseases such as bacterial septicemia and hepatopancreatic necrosis, causing significant economic losses to the industry. Traditionally, farmers have relied heavily on antibiotics and chemical drugs for disease control, but the drawbacks of these methods are becoming increasingly apparent: on the one hand, long-term use leads to increasing drug resistance in pathogens, making disease control more difficult year by year; on the other hand, drug residues pose a potential threat to the quality and safety of aquatic products and the ecological environment. Against this backdrop, developing green, safe, and efficient alternative technologies for disease control has become an urgent need for the sustainable development of the aquaculture industry.

[0003] Traditional Chinese medicine (TCM) is highly regarded in the industry due to its natural, environmentally friendly, and multifunctional characteristics. Modern research shows that the active ingredients in many TCM herbs, such as flavonoids, polysaccharides, and alkaloids, have biological functions such as antioxidation, immune regulation, and antibacterial and anti-inflammatory effects. Theoretically, these can enhance the animal's own immunity to achieve disease control, which aligns with the concept of green farming.

[0004] However, the actual application of traditional Chinese medicine in aquaculture has fallen far short of expectations, mainly due to the following two long-standing and unresolved technical problems:

[0005] Firstly, there is the issue of bioavailability of active ingredients in traditional Chinese medicine (TCM). In traditional applications, TCM is often added directly to feed in coarse powder form. The active ingredients in this coarse powder are encapsulated within the dense cell walls of the plant cells, resulting in extremely low dissolution rates and transmembrane absorption efficiency in the digestive tract of aquatic animals. This leads to the "active ingredients failing to reach the target site," resulting in slow onset of action and unstable effects. Although ultrafine grinding technology can theoretically improve dissolution rates by disrupting cell walls, for specific TCM herbs, optimizing the particle size for dissolution efficiency is not simply a matter of "the finer the better"—excessively fine particles may cause powder adhesion and agglomeration, hindering dissolution. Current technology lacks a systematic method for particle size selection based on the characteristics of different TCM herbs, preventing this technology from fully realizing its potential.

[0006] Secondly, there are safety risks associated with long-term continuous use. Existing research and applications largely focus on the short-term effects of traditional Chinese medicine (TCM) or continuous feeding patterns, but generally overlook a fundamental issue: TCM is essentially an exogenous active substance, and long-term continuous intake may accumulate metabolic burden, damage liver and kidney function, or even induce immunosuppression. This issue leaves the industrial application of TCM facing a strategic blind spot regarding "how long to use and how long to stop." The lack of a scientifically designed feeding rhythm not only may lead to resource waste but also potentially negatively impact animal health due to improper use, contradicting the original intention of disease control.

[0007] In summary, overcoming the two major technical bottlenecks mentioned above—ensuring efficient delivery of active ingredients through process optimization and mitigating long-term use risks through feeding strategy design—is a core technical problem urgently needing to be solved in this field. Establishing a method for applying traditional Chinese medicine that balances "efficient utilization" and "safety and sustainability" will provide crucial technical support for the green and healthy development of aquaculture. Summary of the Invention

[0008] In view of this, the purpose of this application is to provide a method for periodically feeding vine tea ultrafine powder in aquaculture and its application. This application involves pulverizing vine tea to 300 mesh to obtain the optimal dissolution efficiency of active ingredients, then adding it at a 4% dosage to the feed, and adopting a periodic feeding pattern of "7 consecutive days of feeding followed by 14 days of withdrawal." This allows the vine tea ultrafine powder to enhance the disease resistance of aquatic animals while providing the body with sufficient metabolic rest, avoiding the liver and kidney burden and immunosuppression risks that may result from long-term continuous use. Experimental results show that this method, in large-scale pond application to various aquatic animals such as crucian carp, largemouth bass, whiteleg shrimp, carp, and tilapia, significantly enhances the antioxidant capacity and non-specific immune levels of the animals, achieving a disease protection rate of 52.91%–65.14%, achieving a synergistic effect and demonstrating good prospects for industrial application.

[0009] To achieve the above objectives, this application provides the following technical solution:

[0010] In a first aspect, this application provides a method for periodically feeding vine tea ultrafine powder into aquaculture, comprising the following steps:

[0011] Add vine tea ultrafine powder to aquatic animal feed at a dosage of 1% to 8% of the total feed weight, and feed it using a periodic feeding pattern. The periodic feeding pattern is to feed continuously for 7 days, followed by a 7 to 21-day withdrawal period, and repeat this cycle.

[0012] In some preferred embodiments, the amount of vine tea ultrafine powder added is 4% of the total weight of the feed.

[0013] In some embodiments, the particle size of the vine tea ultrafine powder is 200-400 mesh.

[0014] In some preferred embodiments, the particle size of the vine tea ultrafine powder is 300 mesh.

[0015] In some preferred embodiments, the drug withdrawal period is 14 days.

[0016] In some preferred embodiments, the periodic feeding pattern is repeated more than three times during the breeding cycle.

[0017] In some embodiments, the aquatic animal is a farmed species infected with or susceptible to Aeromonas hydrophila or Aeromonas vesiculosus. In some preferred embodiments, the aquatic animal is one of the following: crucian carp, largemouth bass, whiteleg shrimp, carp, and tilapia.

[0018] One or more species.

[0019] Secondly, this application provides a vine tea ultrafine powder for enhancing the disease resistance of aquatic animals. The vine tea ultrafine powder has a particle size of 200-400 mesh and is made from vine tea raw materials through ultrafine grinding. The vine tea contains ≥25% dihydromyricetin. Thirdly, this application provides the application of the method described in the first aspect in the preparation of feed additives for enhancing the disease resistance of aquatic animals.

[0020] Compared with the prior art, this application has at least the following advantages and beneficial effects:

[0021] First, particle size optimization significantly improves the dissolution efficiency of active ingredients. This application compared the cumulative dissolution rate of dihydromyricetin in vine tea powder with different particle sizes using an in vitro dissolution test system, determining that 300 mesh was the optimal particle size. This particle size can promote the dissolution of active ingredients by disrupting cell walls and increasing specific surface area, while avoiding powder adhesion and bridging caused by excessively fine particles, thereby achieving the best dissolution efficiency and laying the foundation for in vivo efficacy.

[0022] Second, the optimal dosage, balancing efficacy and cost, was determined through dosage screening. This application investigated the effects of dosages ranging from 0.5% to 8% on the antioxidant capacity and immune indicators of crucian carp through a dosage gradient experiment, determining 4% as the optimal dosage. The experiment showed that all immune indicators significantly improved starting from a 1% dosage, reaching a plateau at 4%, with a challenge protection rate of 58.83%; higher dosages (8%) did not show significant improvement. This dosage effectively activated the endogenous antioxidant defense system and immune function of aquatic animals while avoiding resource waste caused by excessive addition.

[0023] Third, the periodic feeding strategy mitigates the risks of long-term use and ensures the sustainability of immune enhancement. This application innovatively designs a periodic feeding pattern of "7 days of continuous feeding followed by 14 days of withdrawal." Comparative periodic experiments show that the key immune indicators such as SOD, CAT, and C3 in the crucian carp group with a 14-day withdrawal period are significantly better than those in the 7-day and 21-day withdrawal periods, with a challenge protection rate of 63.16%, far exceeding other periodic groups. This strategy provides the body with sufficient metabolic rest time, avoiding the liver and kidney burden and immunosuppression that may result from long-term continuous feeding, while effectively stimulating immune memory upon refeeding, forming a virtuous cycle of "activation-rest-reactivation." For the first time, it ensures both efficacy and safety for long-term use.

[0024] Fourth, the universality and reliability of the technology were verified through large-scale pond application across multiple species. This application conducted large-scale pond application trials on five aquatic animals: crucian carp, largemouth bass, whiteleg shrimp, carp, and tilapia. Results showed that the activities of antioxidant enzymes such as SOD and CAT in the serum or hepatopancreas of the experimental groups were significantly increased, while the MDA content was significantly decreased; immune indicators such as TP, ALB, GLB, and C3 were significantly increased; and the activities of LYZ and AKP in the hepatopancreas of whiteleg shrimp were also significantly increased. Throughout the entire rearing cycle, the cumulative mortality rate of each species in the experimental groups was significantly lower than that in the control group, with disease protection rates reaching: crucian carp 65.14%, largemouth bass 57.2%, whiteleg shrimp 52.91%, carp 60.75%, and tilapia 60.96%. In particular, even in the case of an outbreak of Aeromonas vera infection in the crucian carp pond, the protection rate of the experimental group still reached 65.14%, fully demonstrating the ability of the method applied in this application to control sudden bacterial diseases in actual production. Attached Figure Description

[0025] Figure 1 The cumulative dissolution rate of dihydromyricetin in vine tea powder of different particle sizes. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0027] The materials used in the following embodiments are not limited to those listed below, and other similar materials may be used instead. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0028] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may vary depending on the desired properties sought. At a minimum, each numerical parameter should be interpreted based at least on the reported significant figures and by applying common rounding techniques.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains.

[0030] The technical solution of this application and the technical effects achieved will be described in detail below through more specific embodiments.

[0031] Example 1: Comparison of in vitro dissolution rates of vine tea powders with different particle sizes

[0032] 1. Main test materials

[0033] The raw material for vine tea consists of broken and fragmented branches and leaves from the vine tea production process. It is a byproduct of vine tea production and is provided by vine tea producers in Enshi, Hubei Province. According to testing, the vine tea content is 25.6%.

[0034] 2. Test Methods

[0035] 2.1 Preparation of Vine Tea Powder with Different Particle Sizes

[0036] Take an appropriate amount of vine tea raw material and dry it in an oven at 60 ℃ until constant weight. Then, pulverize it using a universal pulverizer and an ultrafine pulverizer. After sieving, obtain vine tea powder with four particle sizes of 100 mesh, 200 mesh, 300 mesh and 400 mesh. Seal and store for later use.

[0037] 2.2 Dissolution test

[0038] The method was slightly modified from the "Dissolution Determination Method II" in the 2020 edition of the Chinese Pharmacopoeia. 300 mL of distilled water was added to a 250 mL beaker, followed by 10 g of vine tea powder of different particle sizes. The mixture was heated to 70 °C on a magnetic stirrer at 50 r / min. 5 mL samples were taken at 5, 10, 15, 30, 45, 60, and 90 min, and isothermal and equal volumes of distilled water were added simultaneously. The samples were filtered through a 0.45 μm microporous membrane, and the dihydromyricetin content of the filtrate was determined. The cumulative dissolution rate was calculated.

[0039] 2.3 Determination of dihydromyricetin content

[0040] High-performance liquid chromatography (HPLC) was used. This included:

[0041] (1) Chromatographic conditions: Octadecylsilane bonded silica gel was used as the packing material; methanol-0.05% phosphoric acid solution (55:45) was used as the mobile phase; detection wavelength was 291 nm; flow rate was 1.0 mL / min; column temperature was 30 °C; the theoretical plate number calculated based on the dihydromyricetin peak should not be less than 3000.

[0042] (2) Preparation of reference solution: Accurately weigh 15.0 mg of dihydromyricetin reference standard, place it in a 25 mL volumetric flask, dissolve and dilute to the mark with methanol, shake well, filter through a 0.45 μm filter membrane to prepare a 0.6 mg / mL stock solution. For the determination, pipette 1 mL from the stock solution into a 10 mL volumetric flask, dilute to the mark with methanol to prepare a 60 μg / mL DMY reference solution for later use.

[0043] (3) Preparation of test solution: The dissolution solution was left to stand overnight at 4 °C, filtered, and the crystals were dissolved in 20 mL of methanol and placed in a 25 mL volumetric flask. The solution was sonicated for 30 min, placed at room temperature, filtered, and the filtrate was diluted to 25 mL with methanol. The solution was then filtered through a 0.45 μm filter membrane for later use.

[0044] (4) Accurately pipette 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph for determination, calculate the content of dihydromyricetin in the test solution by peak area according to the external standard method, and calculate the cumulative dissolution rate.

[0045] Figure 1 The results show the cumulative dissolution rate of dihydromyricetin in vine tea powder of different particle sizes. The results indicate that the cumulative dissolution rate of vine tea powder of all particle sizes gradually increases with time, stabilizing after 90 minutes. Comparing different particle sizes, the cumulative dissolution rate is 100 mesh < 200 mesh < 400 mesh < 300 mesh. The 300 mesh vine tea powder has the highest cumulative dissolution rate at 90 minutes, reaching 83.5%; the dissolution rate of the 400 mesh vine tea powder is actually lower than that of the 300 mesh powder.

[0046] 4. Conclusion

[0047] The results of this embodiment indicate that 300 mesh is the optimal particle size for vine tea powder. Below 300 mesh, as the powder particle size decreases, the surface area increases dramatically, leading to a larger contact area with the dissolution medium and increased solubility of dihydromyricetin. However, 400 mesh vine tea powder is too small; upon contact with the dissolution medium, the powder surface absorbs moisture, easily causing adhesion, aggregation into irregular clumps, and significant bridging, thus reducing the dissolution of dihydromyricetin. Therefore, selecting 300 mesh as the preferred particle size for vine tea ultrafine powder ensures efficient dissolution while avoiding the adverse effects of excessively fine particle size.

[0048] Example 2: Effects of different feeding dosages on the disease resistance of crucian carp

[0049] 1. Test materials

[0050] 1.1 Experimental Animals

[0051] Healthy crucian carp, with an average weight of 50±10 g, were purchased from a seedling farmer in Xinzhou District, Wuhan. They were temporarily raised for 7 days before the experiment, during which time they were fed a basic feed to acclimatize to the experimental environment.

[0052] 1.2 Experimental Feed

[0053] Basic feed: Commercial crucian carp compound feed, purchased from Tongwei Co., Ltd., batch number 20251008.

[0054] Vine tea ultrafine powder: 300 mesh vine tea ultrafine powder prepared according to the method in Example 1.

[0055] Experimental feeds: 0.5%, 1%, 2%, 4%, and 8% by mass of vine tea ultrafine powder (pregelatinized starch as binder, dried at low temperature) were added to the basal feed to prepare experimental feeds of different dosages. The control group was fed the basal feed (with an equal amount of pregelatinized starch added, also dried at low temperature).

[0056] 1.3 Infectious Bacterial Strains

[0057] Aeromonas hydrophila: Isolated from the liver of diseased crucian carp, identified as Aeromonas hydrophila, and stored at -80℃. Before use, it was revived and cultured in nutrient broth at 37℃ for 18 hours, and the bacterial concentration was adjusted to 1×10⁻⁶ with sterile physiological saline. 7 CFU / mL, for later use.

[0058] 1.4 Reagent Kit

[0059] Superoxide dismutase (SOD) assay kit, malondialdehyde (MDA) assay kit, catalase (CAT) assay kit: Nanjing Jiancheng Bioengineering Institute.

[0060] Total Protein (TP), Albumin (ALB), Globulin (GLB), Complement Protein 3 (C3) ELISA Kit: Wuhan Saipei Biotechnology Co., Ltd.

[0061] 2. Test Methods

[0062] 2.1 Experimental Grouping and Feed Management

[0063] Healthy crucian carp that were temporarily held were randomly divided into 6 groups: 0.5%, 1%, 2%, 4%, 8% and control group. Each group had 3 replicates, with 20 fish in each replicate, for a total of 360 fish.

[0064] The experiment was conducted in an indoor recirculating aquaculture system. Each 200L tank contained 20 fish. During the experiment, the water temperature was maintained at 25±2℃, dissolved oxygen ≥5.0 mg / L, ammonia nitrogen ≤0.2 mg / L, and pH 7.2–7.8. Fish were fed twice daily (9:00 AM and 4:00 PM) at 3%–5% of their body weight, with adjustments made based on feeding behavior to ensure satiation and no uneaten food. The experiment lasted 28 days.

[0065] 2.2 Sample Collection

[0066] After the feeding experiment, six fish from each group were randomly selected, and blood was collected from their tail veins. The blood samples were allowed to stand at 4 ℃ for 2 h, centrifuged at 3500 r / min for 10 min, and the serum was separated, aliquoted, and stored at -80 ℃ for later use in the determination of various indicators.

[0067] 2.3 Index Measurement

[0068] Serum SOD, CAT activity, and MDA levels were measured and calculated according to the kit instructions. Serum TP, ALB, GLB, and C3 levels were detected using an ELISA kit, following the instructions.

[0069] 2.4 Challenge Test

[0070] After the feeding trial, the remaining fish in each group (14 fish per replicate, 42 fish per group) were used for the challenge experiment. Each fish was injected intraperitoneally with 0.2 mL of a 1×10⁻⁶ solution. 7 CFU / mL Aeromonas hydrophila bacterial suspension. After challenge, the fish were observed continuously for 7 days, and the mortality rate of each group was recorded. The protection rate was calculated using the following formula:

[0071] Protection rate (%) = (mortality rate of control group - mortality rate of experimental group) / mortality rate of control group × 100%.

[0072] 3. Results

[0073] 3.1 Effects on antioxidant indices

[0074] Intensive, high-density aquaculture exposes aquatic animals to chronic environmental stress (such as water quality fluctuations and pathogen threats), leading to excessive production of reactive oxygen species (ROS) in their bodies. This triggers oxidative stress reactions, generating toxic metabolites such as malondialdehyde (MDA), which damage cell membrane integrity and function, ultimately resulting in cell damage, tissue lesions, and decreased immunity. This is also an intrinsic cause of outbreaks of various bacterial diseases in aquatic animals. Superoxide dismutase (SOD) is a key first-line enzyme that converts superoxide anion radicals (O2⁻) into hydrogen peroxide (H2O2); while catalase (CAT) is responsible for further decomposing H2O2 into harmless water and oxygen.

[0075] The effects of different doses of vine tea ultrafine powder on the antioxidant index of crucian carp serum are shown in Table 1.

[0076] Table 1. Effects of different addition amounts on antioxidant indices in crucian carp serum.

[0077]

[0078] Note: Different letters on the right side of the same column indicate significant differences (P < 0.05).

[0079] Table 1 shows that with the increase of the amount of vine tea ultrafine powder added, the activities of SOD and CAT gradually increased, while the content of MDA gradually decreased, indicating that feeding vine tea ultrafine powder effectively activated the aquatic animals' own endogenous antioxidant defense system. Starting from a 1% addition level, all indicators showed significant differences compared to the control group (P < 0.05); the 4% and 8% groups had significantly higher indicators than other groups (P < 0.05), but there was no significant difference between the 4% and 8% groups (P > 0.05).

[0080] 3.2 Effects on non-specific immune indicators

[0081] TP and ALB reflect the overall nutritional and metabolic status of animals. Significant increases in both levels in crucian carp serum indicate that feeding with ultrafine vine tea powder can improve the body's nutritional status. GLB and C3 (especially C3 as a core initiating protein of the complement system) are directly related to immune levels, indicating that feeding with ultrafine vine tea powder can improve the body's immune status. The effects of different doses of ultrafine vine tea powder on non-specific immune indicators in crucian carp serum are shown in Table 2.

[0082] Table 2. Effects of different additive amounts on non-specific immune indicators in crucian carp serum.

[0083]

[0084] Note: Different letters on the right side of the same column indicate significant differences (P < 0.05).

[0085] The results showed that with the increase of the amount of vine tea ultrafine powder added, the levels of TP, ALB, GLB and C3 gradually increased. The levels of all indicators in the 4% and 8% groups were significantly higher than those in the other groups (P < 0.05), but there was no significant difference between the two groups (P > 0.05).

[0086] 3.3 Impact on virus challenge protection rate

[0087] The effects of different doses of vine tea ultrafine powder on the mortality rate and protection rate of crucian carp after viral challenge are shown in Table 3.

[0088] Table 3 Results of the challenge test

[0089]

[0090] Table 3 shows that as the amount of vine tea ultrafine powder added increases, the protection rate gradually increases, reaching the highest in the 4% group (58.83%), while the 8% group shows a slight decrease (52.94%).

[0091] 4. Conclusion

[0092] Based on the combined results of antioxidant indicators, immune indicators, and virus challenge protection rates, the addition of vine tea ultrafine powder at least 1% significantly improves the disease resistance of crucian carp, with 4% achieving the best effect. Increasing the content to 8% does not significantly enhance the effect. Therefore, this application determines the addition range of vine tea ultrafine powder to be 1%~8%, preferably 4%.

[0093] Example 3: Effects of different feeding strategies on the disease resistance of crucian carp

[0094] 1. Test materials

[0095] Same as Example 2.

[0096] 2. Test Methods

[0097] 2.1 Experimental grouping and feeding management

[0098] Healthy crucian carp were randomly divided into three groups, with 20 fish per replicate. All experimental groups were fed experimental feed supplemented with 4% vine tea ultrafine powder (300 mesh), while the control group was fed a basal diet.

[0099] Experiment 1 (Drug Withdrawal Period Study): Three experimental groups were set up: ① 7 days of feeding, 7 days of withdrawal; ② 7 days of feeding, 14 days of withdrawal; ③ 7 days of feeding, 21 days of withdrawal. Each group was fed according to the corresponding cycle, and the experiment lasted for 84 days.

[0100] Experiment 2 (Feeding Time Study): Two experimental groups were set up: ① fed for 7 days, then stopped for 14 days; ② fed for 14 days, then stopped for 14 days. Each group was fed according to the corresponding cycle, and the experiment lasted for 84 days.

[0101] The feeding and management conditions are the same as in Example 2.

[0102] 2.2 Sample Collection and Index Measurement

[0103] Same as Example 2.

[0104] 2.3 Challenge Test

[0105] Same as Example 2.

[0106] 3. Test Results

[0107] 3.1 Effects of drug withdrawal period on antioxidant and immune indicators

[0108] The effects of different drug withdrawal periods on the serum indicators of crucian carp are shown in Table 4.

[0109] Table 4. Effects of different drug withdrawal periods on serum antioxidant and non-specific immune indicators in crucian carp.

[0110]

[0111] Note: Different lowercase letters in the superscript of data in the same column indicate significant differences (P < 0.05).

[0112] The results showed that the SOD, CAT, ALB, GLB, and C3 levels in crucian carp in the 14-day withdrawal group were significantly higher than those in the 7-day and 21-day withdrawal groups (P < 0.05), while the MDA content was significantly lower than in the other groups (P < 0.05). The 7-day withdrawal group showed the second highest levels, while the 21-day withdrawal group showed the worst results.

[0113] 3.2 The impact of drug withdrawal period on the protection rate against viral challenge

[0114] The effects of different withdrawal periods on the protection rate of crucian carp against viral infection are shown in Table 5.

[0115] Table 5. Effects of different drug withdrawal periods on the protection rate of crucian carp against viral challenge.

[0116]

[0117] The results showed that the protection rate was highest in the 14-day withdrawal group (63.16%), significantly higher than that in the 7-day withdrawal group (42.11%) and the 21-day withdrawal group (10.53%). This result is likely due to the fact that long-term administration may have caused immunosuppression or adverse effects on the liver and kidneys, and that discontinuing administration for a certain period can avoid these risks. The results indicate that 14 days is the optimal withdrawal period because the body's immune level is still relatively high at 7 days, while it is extremely low at 21 days, and is at an intermediate level at 14 days. Continuing administration at 14 days can then stimulate the body's immunity to reach a higher level again.

[0118] 3.3 Effects of feeding time on antioxidant and immune indicators

[0119] The effects of different feeding times on the serum parameters of crucian carp are shown in Table 6.

[0120] Table 6. Effects of different feeding times on serum antioxidant and nonspecific immune indicators in crucian carp.

[0121]

[0122] Note: Different lowercase letters in the superscript of data in the same column indicate significant differences (P < 0.05).

[0123] Table 6 shows that there were significant differences between the experimental groups and the control group (P < 0.05). The levels of T-SOD, CAT, TP, ALB, GLB, and C3 in the crucian carp fed for 14 days were higher than those in the group fed for 7 days, while the MDA content was lower, but the differences were not significant (P > 0.05). Therefore, although the effect of feeding for 14 days was better than that of feeding for 7 days, the difference in effect was not significant, and considering the cost of medication, feeding for 7 days was chosen as the optimal feeding method.

[0124] 3.4 The effect of feeding time on the protection rate against viral infection

[0125] The effects of different feeding times on the protection rate of crucian carp against viral infection are shown in Table 7.

[0126] Table 7. Effects of different feeding times on the virus challenge protection rate of crucian carp.

[0127]

[0128] As shown in Table 7, the protection rate of the 14-day feeding group (58.82%) was slightly higher than that of the 7-day feeding group (52.94%), but the difference was not significant.

[0129] 4. Conclusion

[0130] The results of this embodiment show that:

[0131] (1) The withdrawal period has a significant impact on the disease resistance of crucian carp. The effect is best when the medication is withdrawn for 14 days, followed by 7 days, and the effect is worst when the medication is withdrawn for 21 days. Therefore, the preferred withdrawal time is 14 days.

[0132] (2) The feeding time had no significant effect on the disease resistance of crucian carp; there was no significant difference in the effect between feeding for 7 days and feeding for 14 days. Considering the cost of medication, the optimal feeding time was 7 days.

[0133] (3) The experiment lasted for 84 days and consisted of 4 complete cycles (21 days / cycle), verifying that the periodic feeding pattern could be repeated more than 3 times. The optimal feeding strategy was determined to be: continuous feeding for 7 days, followed by a 14-day withdrawal period, with this as one cycle, and repeated cyclically.

[0134] Example 4: Datang Application Test

[0135] 1. Test materials

[0136] 1.1 Test Location and Time

[0137] The trials will be conducted simultaneously at the following five locations from June to September 2025:

[0138] Huangpi District, Wuhan: California bass raised in enclosures and barrels;

[0139] Yiyang, Hunan: Pond and cage culture of crucian carp;

[0140] Wuhan Caidian: Greenhouse farming of Litopenaeus vannamei;

[0141] Huangshi Yangxin: Pond-raised carp;

[0142] Huangshi Daye: Tilapia farming in ponds.

[0143] 1.2 Experimental Animals

[0144] The species and scale of aquaculture at each experimental site are shown in Table 8.

[0145] Table 8. Breeding species and scale at each experimental site

[0146]

[0147] 1.3 Experimental Feed

[0148] Experimental group: 4% of 300-mesh vine tea ultrafine powder (same as in Example 2) was added to the commercial feed, and pregelatinized starch was used as a binder. The feed was dried at low temperature and then stored for later use. The feeding method was to feed continuously for 7 days, stop for 14 days, and repeat the cycle for 21 days. The experiment lasted for 84 days.

[0149] Control group: fed commercial feed without added vine tea ultrafine powder, and other management was the same as the experimental group.

[0150] 2. Test Methods

[0151] 2.1 Feeding and Management

[0152] Each experimental site was managed according to local conventional breeding practices, maintaining identical conditions between the experimental and control groups except for feed. Feeding and mortality data were recorded daily.

[0153] 2.2 Sample Collection and Index Measurement

[0154] At the end of the experiment, 10 largemouth bass, 10 crucian carp, 10 common carp, and 10 tilapia were randomly collected from each pond. Blood was collected from the tail vein, serum was separated, and antioxidant and immune indicators were measured as in Example 2. Twenty whiteleg shrimp were collected, and hepatopancreas was collected. After homogenization, the activities of SOD, CAT, MDA, lysozyme (LYZ), and alkaline phosphatase (AKP) were measured, all according to the kit instructions.

[0155] 2.3 Protection Rate Calculation

[0156] Record the cumulative number of deaths in each group throughout the entire trial period, and calculate the protection rate using the following formula:

[0157] Protection rate (%) = (mortality rate of control group - mortality rate of experimental group) / mortality rate of control group × 100%.

[0158] 3. Test Results

[0159] 3.1 Effects on serum parameters of crucian carp

[0160] The effects of vine tea ultrafine powder on the antioxidant and immune indicators of crucian carp serum are shown in Table 9.

[0161] Table 9. Antioxidant and non-specific immune indicators in crucian carp serum.

[0162]

[0163] Note: "*" indicates a significant difference compared to the control group (P < 0.05).

[0164] 3.2 Effects on serum markers of California bass

[0165] The effects of vine tea ultrafine powder on serum antioxidant and immune indicators of California bass are shown in Table 10.

[0166] Table 10 Antioxidant and non-specific immune indicators in the serum of California bass

[0167]

[0168] Note: "*" indicates a significant difference compared to the control group (P < 0.05).

[0169] 3.3 Effects on serum markers in carp

[0170] The effects of vine tea ultrafine powder on the antioxidant and immune indicators of carp serum are shown in 11.

[0171] Table 11 Antioxidant and non-specific immune indicators in carp serum

[0172]

[0173] Note: "*" indicates a significant difference compared to the control group (P < 0.05).

[0174] 3.4 Effects on serum markers in tilapia

[0175] The effects of vine tea ultrafine powder on the antioxidant and immune indicators of tilapia serum are shown in Table 12.

[0176] Table 12 Antioxidant and non-specific immune indicators in tilapia serum

[0177]

[0178] Note: "*" indicates a significant difference compared to the control group (P < 0.05).

[0179] 3.5 Effects on hepatopancreatic parameters of Litopenaeus vannamei

[0180] The effects of vine tea ultrafine powder on antioxidant and immune indicators of the hepatopancreas of Litopenaeus vannamei are shown in Table 13.

[0181] Table 13 Antioxidant and non-specific immune indicators in the hepatopancreas of Litopenaeus vannamei.

[0182]

[0183] Note: "*" indicates a significant difference compared to the control group (P < 0.05).

[0184] 3.6 Impact on cumulative mortality rate and protection rate

[0185] Table 14 shows the cumulative mortality and protection rate of each variety during the entire trial period.

[0186] Table 14. Animal mortality during the experiment.

[0187]

[0188] According to fish farmers, during the trial period, the experimental animals fed with vine tea ultrafine powder were generally more active than the control group in the later stages. A mass mortality occurred in the crucian carp pond, and bacterial isolation upon dissection identified it as Aeromonas verrucosa infection. During this period, the control group suffered a total mortality of 218 fish, while the experimental group suffered only 76, resulting in a protection rate of 65.14%, demonstrating the significant control effect of the proposed method against Aeromonas verrucosa infection. In the early stages of the trials with California bass and Litopenaeus vannamei, mortality was higher due to their smaller size, but decreased later. The California bass control group suffered a total mortality of 98 fish, while the experimental group suffered 42, resulting in a protection rate of 57.2%; the Litopenaeus vannamei control group suffered a total mortality of 327 fish, while the experimental group suffered 154, resulting in a protection rate of 52.91%; the carp control group suffered a total mortality of 107 fish, while the experimental group suffered 42, resulting in a protection rate of 60.75%; and the tilapia control group suffered a total mortality of 146 fish, while the experimental group suffered 57, resulting in a protection rate of 60.96%.

[0189] 4. Conclusion

[0190] This embodiment verifies the effectiveness of the method of this application in pond culture of five different aquatic animals. The results show that:

[0191] (1) The method of this application can significantly increase the levels of SOD, CAT, TP, ALB, GLB and C3 in the serum of crucian carp, largemouth bass, carp and tilapia, and significantly reduce the content of MDA (P<0.05).

[0192] (2) The method of this application can significantly increase the activity of SOD, CAT, LYZ and AKP in the hepatopancreas of Litopenaeus vannamei and significantly reduce the content of MDA (P<0.05).

[0193] (3) Throughout the entire breeding cycle, the cumulative mortality rate of all species in the experimental group was significantly lower than that in the control group, with a protection rate of 52.91%~

[0194] The protection rate was between 65.14% and 65.14% against Aeromonas verrucosa infection.

[0195] The above results demonstrate that the method described in this application effectively activates the endogenous antioxidant defense system of aquatic animals through periodic feeding of vine tea ultrafine powder. Its mechanism of action lies in the fact that in intensive aquaculture environments, aquatic animals produce excessive reactive oxygen species (ROS), triggering oxidative stress and generating toxic metabolites such as malondialdehyde (MDA), which damage cell membrane integrity, leading to cell damage and decreased immunity. The method described in this application significantly enhances the activities of superoxide dismutase (SOD) and catalase (CAT). SOD can convert superoxide anion free radicals (O2·⁻) into hydrogen peroxide, while CAT further decomposes hydrogen peroxide into harmless water and oxygen, thereby effectively scavenging free radicals and reducing oxidative damage.

[0196] In terms of immune regulation, the method described in this application significantly increased the levels of total protein (TP), albumin (ALB), globulin (GLB), and complement protein 3 (C3). Increased TP and ALB levels reflect an improvement in the body's overall nutritional and metabolic status; increased GLB and C3 (as core initiating proteins of the complement system) directly indicate an enhanced level of non-specific immunity.

[0197] For Litopenaeus vannamei, the method described in this application also significantly enhances the activities of lysozyme (LYZ) and alkaline phosphatase (AKP). LYZ hydrolyzes peptidoglycan in bacterial cell walls, directly killing Gram-positive bacteria and also inhibiting some Gram-negative bacteria; AKP not only participates in metabolism but is also related to phagocytosis, phosphate group transfer, and immune regulation. The enhancement of the activities of these two key enzymes directly strengthens the organism's antibacterial ability.

[0198] In summary, the method described in this application demonstrates significant disease resistance enhancement in various aquatic animals through multiple mechanisms, including activating the antioxidant system, improving immunity, and enhancing antibacterial capabilities, exhibiting good universality and reliability. The best results were achieved by consistently feeding the animal at a 4% feed additive rate for 7 days, followed by a 14-day rest period, with each cycle lasting 21 days.

[0199] Based on the above embodiments, this application also provides a vine tea ultrafine powder for enhancing the disease resistance of aquatic animals, which can be prepared by the following method: take vine tea raw material (dihydromyricetin content not less than 25%), dry it at 60°C, and pulverize it to 300 mesh using an ultrafine pulverizer to obtain the powder.

[0200] Based on the above embodiments, this application provides the application of the aforementioned method in the preparation of feed additives for enhancing the disease resistance of aquatic animals. The 300-mesh vine tea ultrafine powder prepared above is mixed into commercial feed at a ratio of 4% of the total feed weight, using pregelatinized starch as a binder. After low-temperature drying, the vine tea ultrafine powder feed additive is obtained. The method of using this additive is as described in this application: continuous feeding for 7 days, followed by a 14-day withdrawal period, with this as one cycle. As shown in Example 4, the feed additive prepared using this method achieved a protection rate of 52.91%~65.14% in pond culture of crucian carp, largemouth bass, whiteleg shrimp, carp, and tilapia, significantly enhancing the disease resistance of aquatic animals. Therefore, the periodic feeding method of vine tea ultrafine powder in aquaculture described in this application can be used to prepare feed additives that enhance the disease resistance of aquatic animals.

[0201] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A method for periodically feeding vine tea ultrafine powder into aquaculture, characterized in that, Includes the following steps: Add vine tea ultrafine powder to aquatic animal feed at a dosage of 1% to 8% of the total feed weight, and feed it using a periodic feeding pattern. The periodic feeding pattern is to feed continuously for 7 days, followed by a 7 to 21-day withdrawal period, and repeat this cycle.

2. The periodic feeding method according to claim 1, characterized in that, The amount of vine tea ultrafine powder added is 4% of the total weight of the feed.

3. The periodic feeding method according to claim 1, characterized in that, The particle size of the vine tea ultrafine powder is 200~400 mesh.

4. The periodic feeding method according to claim 3, characterized in that, The particle size of the vine tea ultrafine powder is 300 mesh.

5. The periodic feeding method according to claim 1, characterized in that, The drug withdrawal period is 14 days.

6. The periodic feeding method according to claim 1, characterized in that, The periodic feeding pattern is repeated more than three times during the breeding cycle.

7. The periodic feeding method according to claim 1, characterized in that, The aquatic animals mentioned are farmed species that are infected with or susceptible to Aeromonas hydrophila and Aeromonas vernix.

8. The periodic feeding method according to claim 1, characterized in that, The aquatic animals mentioned are one or more of the following: crucian carp, largemouth bass, whiteleg shrimp, carp, and tilapia.

9. A type of vine tea ultrafine powder for enhancing the disease resistance of aquatic animals, characterized in that, The vine tea ultrafine powder has a particle size of 200-400 mesh, and is made from vine tea raw materials through ultrafine grinding, and the vine tea contains ≥25% dihydromyricetin.

10. The use of the periodic feeding method according to any one of claims 1 to 8 in the preparation of feed additives for enhancing the disease resistance of aquatic animals.

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