A method for post-spawning culture of blue crabs

Abstract

A method for breeding blue crab after spawning, characterized in that the method comprises a blue crab post-spawning microcirculation preparation, wherein the blue crab post-spawning microcirculation preparation contains at least one of the following components and contents: rosmarinic acid with a content of 300-400 mg / L, jasmolinic acid with a content of 250-350 mg / L, oleuropein with a content of 250-350 mg / L, and terpene alcohol with a content of 200-300 mg / L. The blue crab post-spawning microcirculation preparation can significantly improve the growth level, nutrient transport capacity, metabolic waste capacity, tissue repair capacity and fat and energy storage capacity of the post-spawning female crab, and promote the weight gain and rejuvenation of the post-spawning blue crab.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, specifically to a method for raising mud crabs after spawning. Background Technology

[0002] Mud crabs, as a high-value aquaculture species, occupy an important position in the global aquaculture industry. After spawning, female mud crabs often experience a decline in microcirculation, leading to problems such as obstructed nutrient transport, accumulation of metabolic waste, insufficient fat and energy reserves, and limited tissue repair capabilities. This microcirculatory disorder not only damages the hepatopancreas and gonads of female crabs but also causes insufficient fat reserves, physical weakness, and low efficiency in roe accumulation, thus seriously affecting their overall health, reproductive capacity, and market competitiveness. Furthermore, due to slow postpartum recovery, the survival rate and farming efficiency of female crabs are significantly reduced.

[0003] Currently, common postpartum recovery methods for female crabs in aquaculture mainly include increasing the amount of high-nutrient feed, improving aquaculture water quality, and using chemically synthesized additives. However, these measures have limited effectiveness in improving microcirculation function, and increasing the amount of high-nutrient feed and using chemically synthesized additives may cause environmental pollution, increase aquaculture costs, and pose food safety risks. Especially when addressing problems such as poor blood flow and obstructed oxygen and nutrient transport caused by microcirculatory disorders, existing technologies are still unable to effectively promote the production of roe and rejuvenate postpartum female crabs, limiting their overall recovery and improvement in health. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for raising mud crabs after spawning that can improve the microcirculation function, promote the increase of roe, and rejuvenate the crabs, in light of the above-mentioned existing technology.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: the method for raising mud crabs after spawning, characterized in that: it includes a mud crab post-spawning microcirculation preparation, wherein the mud crab post-spawning microcirculation preparation contains at least one of the following components and contents: rosmarinic acid at a content of 300-400 mg / L, caryopsisic acid at a content of 250-350 mg / L, oleuropein at a content of 250-350 mg / L, and terpene alcohol at a content of 200-300 mg / L.

[0006] Furthermore, the postpartum microcirculation preparation for mud crabs also includes the following components: rosmarinic acid, caryopsisic acid, oleuropein, and terpene alcohol, wherein the content of rosmarinic acid is 300-400 mg / L, the content of caryopsisic acid is 250-350 mg / L, the content of oleuropein is 250-350 mg / L, and the content of terpene alcohol is 200-300 mg / L.

[0007] Furthermore, the postpartum microcirculation preparation for mud crabs also includes the following components: catechin, quercetin, ursolic acid, and proanthocyanidins, wherein the content of catechin is 100-200 mg / L, the content of quercetin is 100-200 mg / L, the content of ursolic acid is 50-100 mg / L, and the content of proanthocyanidins is 50-100 mg / L.

[0008] Furthermore, the content of each component in the postpartum microcirculation preparation of mud crab is as follows: rosmarinic acid content is 350 mg / L, carrageenan content is 300 mg / L, oleuropein content is 300 mg / L, terpene alcohol content is 250 mg / L, catechin content is 150 mg / L, quercetin content is 150 mg / L, ursolic acid content is 75 mg / L, and proanthocyanidin content is 75 mg / L.

[0009] Furthermore, the preparation method of the postpartum microcirculation preparation for mud crabs includes the following steps:

[0010] 1) Mixing: Weigh 25-30 parts rosemary petals, 20-25 parts sage leaves, 20-25 parts olive leaves, 15-20 parts perilla leaves, 10-15 parts green tea leaves, 10-15 parts red onion, 5-10 parts blueberry leaves, and 5-10 parts grape seeds, and mix them to obtain the raw material mixture; wherein, 1 part = 1g;

[0011] 2) Ultrasonic extraction: After pulverizing 60-80 parts of the raw material mixture obtained in step 1), the pulverized powder is ultrasonically extracted using 70% ethanol solution as the extractant to obtain an extraction solution; wherein, 1 part = 1g;

[0012] 3) Filtration and centrifugation: The extraction solution obtained in step 2) is initially filtered to obtain a pre-filtered solution. After centrifuging the pre-filtered solution, the lower layer impurities are discarded to obtain the centrifuged solution.

[0013] 4) Concentration: The centrifuged solution obtained in step 3) is evaporated and concentrated to 1 / 2 volume at 45°C to obtain a concentrated solution;

[0014] 5) Dilution and Storage: The concentrated solution obtained in step 4) is diluted with water to obtain the postpartum microcirculation preparation of mud crabs. The volume ratio of concentrated solution to water during dilution is 1:20. The postpartum microcirculation preparation of mud crabs is placed in a sealed container and stored at 4°C. 70% ethanol solution as an extractant can dissolve the active ingredients of rosmarinic acid, caryopsisic acid, oleuropein, terpene alcohol, catechin, quercetin, ursolic acid, and proanthocyanidins; evaporation and concentration at 45°C can remove volatile impurities in the extract solution and also remove the above-mentioned active ingredients.

[0015] Furthermore, the postpartum microcirculation preparation for mud crabs is used in combination with an auxiliary solution. Each 4-6 mL of the auxiliary solution contains the following components and concentrations: 6-8 g of Clostridium butyricum, 4-6 g of L-arginine, 2-4 g of vitamin C, and 4-6 mL of 1× phosphate buffer. The postpartum microcirculation preparation for mud crabs is mixed with the auxiliary solution at a volume ratio of 5:1 to obtain the mixed preparation. L-arginine and vitamin C promote the effective absorption of the postpartum microcirculation preparation by the mud crabs, while the Clostridium butyricum component helps maintain water quality stability.

[0016] Furthermore, the method for preparing the auxiliary liquid includes the following steps:

[0017] 6) Raw material dissolution: In a sterile laminar flow hood, weigh 6-8 parts by weight of Clostridium butyricum powder, add 8-25 parts by weight of sterile water and stir thoroughly to obtain a Clostridium butyricum suspension; weigh 4-6 parts by weight of L-arginine powder and dissolve in 10-15 parts by weight of sterile water and stir thoroughly to obtain an L-arginine solution; weigh 2-4 parts by weight of vitamin C powder and dissolve in 8-12 parts by weight of sterile water and stir thoroughly to obtain a vitamin C solution; mix the obtained Clostridium butyricum suspension, L-arginine solution, vitamin C solution and 1×PBS buffer at a volume ratio of 8-25:10-15:8-12:4-6 to obtain a raw material mixed solution; wherein, 1 part by weight = 1g;

[0018] 7) Filtration and sterilization: The raw material mixture obtained in step 6) is aseptically filtered through a 0.22μm needle filter to obtain the auxiliary solution;

[0019] 8) Storage: After the auxiliary liquid obtained in step 8) is placed into a sealed container, it is stored in a light-proof environment at 4°C.

[0020] Furthermore, it includes a phased application process, specifically, three phases of application:

[0021] During the 3-7 days after the mud crabs spawn: apply once a day at 18:00, each time applying 10-15 ml of the mixed preparation per cubic meter of water.

[0022] During the 8-14 days postpartum period of mud crabs: apply the mixture once at 5:00 and once at 20:00 each day, with a dosage of 15-20 ml of the mixture per cubic meter of water each time;

[0023] During the 15-20 days after the mud crabs spawn: apply the mixture once at 5:00 and once at 20:00 every day, with each application being 20-30 ml of the mixture per cubic meter of water.

[0024] Furthermore, the application methods for the three stages in the phased application process are as follows:

[0025] During the 3-7 days after the mud crabs spawn: apply once a day at 18:00, each time applying 12.5ml of the mixed preparation per cubic meter of water;

[0026] During the 8-14 days postpartum period of mud crabs: apply the mixture once at 5:00 and once at 20:00 each day, with each application being 17.5 ml of the mixture per cubic meter of water.

[0027] During the 15-20 days after the mud crabs spawn: apply the mixture once at 5:00 and once at 20:00 every day, with each application being 25ml of the mixture per cubic meter of water.

[0028] Compared with the prior art, the advantages of the present invention are as follows:

[0029] 1. The ingredients in the postpartum microcirculation preparation for mud crabs can improve the microcirculation function of postpartum mud crabs, promote the production of roe, and restore their health. Specifically,

[0030] Rosmarinic acid and proanthocyanidins are mainly used to address the problem of obstructed nutrient transport in mud crabs after spawning: Rosmarinic acid can reduce the synthesis of triglycerides, thereby lowering triglyceride levels in the blood. Furthermore, by inhibiting the activity of HMG-CoA reductase (the rate-limiting enzyme in cholesterol synthesis), it reduces cholesterol synthesis and lowers cholesterol levels in the blood. It can also increase nitric oxide (NO) synthesis, dilate blood vessels, improve blood flow, and enhance the nutrient supply to organs such as the hepatopancreas and gonads. Proanthocyanidins, by improving blood circulation and gonadal nutrient supply, increase hepatopancreas and gonadal indices, and lower triglyceride and total cholesterol levels in the blood and lymph, thereby optimizing hepatopancreas function and lipid metabolism, and enhancing gonadal function, thus solving the problem of obstructed nutrient transport caused by lipid accumulation and metabolic disorders.

[0031] Olive glycosides and quercetin are mainly used to address the problem of metabolic waste accumulation: Olive glycosides enhance renal tubular cell function, promote uric acid excretion, and stimulate the immune system to increase the levels of antimicrobial peptides and lysozyme, reducing inflammatory responses. This, in turn, promotes the excretion of urea nitrogen, ammonia, and uric acid from the blood and lymph, thereby increasing the levels of antimicrobial peptides and lysozyme, reducing the accumulation of metabolic waste in the blood, alleviating the toxic burden caused by the accumulation of metabolic waste, and improving the liver's detoxification and immune protection functions. Quercetin, by activating urea cycle enzymes, enhances the liver's ability to convert ammonia into urea, accelerates renal urea excretion, and reduces the accumulation of metabolic waste in the blood.

[0032] Terpenols and catechins are primarily used to address the problem of insufficient fat and energy reserves in mud crabs after spawning. Terpenols maintain a dynamic balance of fat reserves by activating fatty acid synthases, while simultaneously increasing high-density lipoprotein levels and clearing low-density lipoproteins, optimizing lipid metabolism and energy supply, thereby improving fat metabolism efficiency and enhancing energy reserves, providing stable energy support for the recovery period of mud crabs. Catechins also enhance lipid and energy reserves. Their combined effect effectively solves the problem of insufficient fat and energy reserves in mud crabs after spawning.

[0033] Sagein and ursolic acid are mainly used to address the problem of limited tissue repair capacity: Sagein accelerates cell proliferation and damage repair by increasing the secretion level of repair factors (EGF, PDGF, TGF-β), thereby improving tissue regeneration capacity and accelerating the tissue repair process, enhancing cell proliferation and damage healing ability, and improving the efficiency of postpartum tissue repair in female mud crabs; ursolic acid activates fibroblasts, promotes collagen and hydroxyproline synthesis, and provides tissue structural support.

[0034] 2. The application of the above-mentioned microcirculation preparation for mollusc crabs can significantly improve the growth level, nutrient transport capacity, metabolic waste removal capacity, tissue repair capacity, and fat and energy storage capacity of female mollusc crabs after spawning. The survival rate is increased to 67.34-78.13%, the weight gain rate is increased to 28.30-36.03%, and the mollusc crabs are promoted to increase roe and recover after spawning. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to the embodiments.

[0036] The following is a preferred embodiment of the present invention. The post-spawning farming method for mud crabs in this embodiment is mainly used to promote the production and rejuvenation of mud crabs after spawning. Under natural conditions, female mud crabs often experience a decline in microcirculation function after spawning, leading to problems such as obstructed nutrient transport, accumulation of metabolic waste, insufficient fat and energy reserves, and limited tissue repair capabilities. Existing technologies mainly improve the growth status of mud crabs by increasing the amount of high-nutrient feed, improving aquaculture water quality, and using chemically synthesized additives. However, these methods do not specifically address the problem of decreased microcirculation function after spawning and are therefore less effective. Therefore, providing a post-spawning farming method for mud crabs that can improve microcirculation function, promote the production and rejuvenation of mud crabs would be beneficial. The following will provide a detailed description of this post-spawning farming method for mud crabs:

[0037] Example 1: Rosmarinic Acid Content Experiment

[0038] The specific steps of the preparation method for the postpartum microcirculation preparation of mud crabs in this embodiment are as follows:

[0039] I. Ultrasonic Extraction of Rosmarinic Acid

[0040] Step 1: Preparation of plant materials

[0041] (1) Material preparation: Weigh out 22.5, 25, 27.5, 30, and 32.5 grams of rosemary petals respectively. (The plant materials can be purchased from the market.)

[0042] (2) Grinding: The above 5 plant materials were coarsely ground using a grinder (model: LD-1300A high-speed universal grinder, Zhejiang Wenling Linda Machinery Co., Ltd.) to control the particle size to 0.5-1 mm. The ground materials were then placed into dry 100ml sealed glass beakers (100ml round-bottom glass beaker, Shanghai Heqi Glass Instrument Co., Ltd.) for later use.

[0043] Step 2: Ultrasonic-assisted ethanol extraction

[0044] (3) Solvent preparation: Use 70% ethanol solution (commercially available) as the extraction solvent. Dispense 100ml of 70% ethanol solution into four 500ml round-bottom glass flasks (500ml round-bottom glass flasks, Shanghai Heqi Glass Instrument Co., Ltd.).

[0045] (4) Plant material preparation: Take 5 portions of crushed rosemary petals and add them to 5 500ml round-bottom flasks containing 100ml of 70% ethanol solution.

[0046] (5) Ultrasonic extraction: Place 5 flasks into an ultrasonic extractor (model: YM-T2000CT multi-purpose constant temperature ultrasonic extractor, Shanghai Yuming Instrument Co., Ltd.), and set the extraction conditions according to the following parameters: ultrasonic power: 300 watts, ultrasonic frequency: 40 kHz, temperature: 50℃, extraction time: 40 minutes. After extraction, cool the extract to room temperature.

[0047] Step 3: Filtration and centrifugation

[0048] (6) Coarse filtration: The extract was initially filtered using a filter (model: MS-0.45μm polyethersulfone (PES) membrane filter, Shanghai Mosu Scientific Instruments Co., Ltd.) to remove larger particles.

[0049] (7) Centrifugation: Transfer the coarsely filtered extract to a centrifuge (model: TG16-WS benchtop high-speed centrifuge, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.), and centrifuge using the following parameters: speed: 10000 rpm, time: 15 minutes. After centrifugation, collect the supernatant and discard any remaining impurities.

[0050] Step 4: Rotary evaporation concentration

[0051] (8) Concentration: Transfer the supernatant after centrifugation to a rotary evaporator (model: RE-52AA rotary evaporator, Shanghai Yarong Biochemical Instrument Factory) for concentration. Set the following parameters: water bath temperature: 45℃, rotation speed: 80rpm. Evaporate and concentrate to approximately 50ml of concentrate to ensure that the active ingredients in the liquid are retained.

[0052] Step 5: Preparation of liquid formulation

[0053] (9) Dilution: Dilute 50ml of concentrate with purified water to 1000ml, stir thoroughly to make a postpartum microcirculation preparation for blue crabs.

[0054] (10) Storage: Dispense the prepared mud crab postpartum microcirculation preparation into 2L transparent glass containers and seal them for storage. The containers should be kept away from light and placed in a refrigerator at 4°C to prevent degradation of the active ingredients.

[0055] II. Preparation of Auxiliary Fluid

[0056] The postpartum microcirculation preparation for mud crabs needs to be used simultaneously with an auxiliary solution. This auxiliary solution contains 6–8 g of Clostridium butyricum, 4–6 g of L-arginine, 2–4 g of vitamin C, and 4–6 ml of 1×phosphate buffer (1×PBS), which aims to promote the effective absorption of the postpartum microcirculation preparation by mud crabs and maintain water quality stability.

[0057] Experimental materials: Clostridium butyricum: 6-8g, L-arginine: 4-6g, vitamin C: 2-4g, phosphate buffered saline (PBS): 4-6ml; 500mL glass beaker (model: GG-17, Shanghai Guanghua Glass Instrument Co., Ltd.). The glass beaker was placed in an autoclave (DXL-100S automatic high-pressure steam sterilizer, Shandong Dexiang Instrument Co., Ltd.) for high-temperature and high-pressure sterilization, and then set aside for later use.

[0058] Step 5: Preparation of auxiliary solution

[0059] (11) Dissolving Clostridium butyricum: In a sterile laminar flow hood (model: BSC-1100IIA2-X, Guangdong Keyipu Laboratory Equipment Research and Development Co., Ltd.), take 7g of Clostridium butyricum powder and add 16.5ml of sterile purified water; use an electric stir bar (model: IKA RW20 Digital Dissolver, Beijing Labtech Instrument Co., Ltd.) to stir at medium speed for 4 minutes to ensure that Clostridium butyricum is completely dissolved and forms a uniform suspension.

[0060] (12) Add L-arginine and vitamin C: Dissolve 5g of L-arginine in 12.5ml of sterile water and stir for 4 minutes until completely dissolved. Then dissolve 2-4g of vitamin C in 10ml of sterile water and stir for 3 minutes to ensure that the solution is transparent and free of precipitate. Finally, add the two solutions to the butyric acid clostridium suspension while stirring slowly until well mixed.

[0061] (13) Add PBS buffer: Take 5 ml of PBS buffer and slowly add it to the above mixture. Continue stirring for 4 minutes to ensure that all components are fully dissolved and mixed evenly to form a stable solution.

[0062] Step 6: Filtration and Aseptic Processing

[0063] (14) Filtration: The mixed solution was aseptically filtered through a 0.22μm needle filter (model: Syringe Filters 0.22μm, Zhejiang Saifen Technology Co., Ltd.) to remove bacteria, fungal spores and other microbial contaminants.

[0064] (15) Dispensing and storage: Dispense the filtered auxiliary solution into 500mL sterile transparent glass cups (model: GG-17, Shanghai Guanghua Glass Instrument Co., Ltd.), seal and store in a refrigerator at 4℃, away from light to prevent degradation of active ingredients.

[0065] III. Mixed Formulations

[0066] The ratio of the microcirculation preparation to the auxiliary solution for spawning mud crabs is 5:1. That is, for every 5 ml of microcirculation preparation, 1 ml of auxiliary solution needs to be added and mixed to obtain 6 ml of mixed preparation. The rosmarinic acid content in the 5 mixed preparations after mixing is 300 mg / L, 350 mg / L, 400 mg / L, 200 mg / L and 500 mg / L respectively.

[0067] IV. Experimental Design

[0068] Rosmarinic acid at concentrations of 300 mg / L, 350 mg / L, 400 mg / L, 200 mg / L, and 500 mg / L was extracted from rosemary petals using the above-mentioned ultrasonic extraction combined with 70% ethanol technique. The auxiliary solution was designated as the blank control group, and the concentrations were denoted as follows:

[0069] Component 1: 300 mg / L rosmarinic acid,

[0070] Component 2: 350 mg / L rosmarinic acid,

[0071] Three components: 400 mg / L rosmarinic acid,

[0072] Component 4: 200 mg / L rosmarinic acid,

[0073] Component 5: 500 mg / L rosmarinic acid,

[0074] Control group: auxiliary solution (i.e., 0 mg / L rosmarinic acid).

[0075] V. Aquaculture Experiment

[0076] From September to October 2023, the experiment was conducted in indoor culture ponds at Dongying Kainuo Aquaculture Co., Ltd. Ninety female mud crabs, 1-3 days postpartum, were collected again from the southern coastal region, transported by air to the experimental site, and then centrally disinfected before being placed in two 30m³ tanks. 2 The crabs were temporarily kept indoors at the cement plant for 1-2 days. After the temporary holding, 5 crabs were placed in 18 20m³ containers. 2 Experiments were conducted in laboratory pools. Five postpartum female crabs were placed in each pool, along with five pieces of tile. Pools 1-3 were for component group one, pools 4-6 for component group two, pools 7-9 for component group three, pools 10-12 for component group four, pools 13-15 for component group five, and pools 16-18 for control group one. The purpose of this embodiment was to explore key compounds in a postpartum microcirculation preparation for promoting the recovery of mud crabs after spawning. The experimental materials and sources in this embodiment were the same as in Example 1.

[0077] The dosage and application method of the postpartum microcirculation preparation and auxiliary solution for mud crabs need to be adjusted according to the mud crab's farming stage. The specific adjustment plan is as follows:

[0078] Phase 1 (3 to 7 days postpartum)

[0079] Application time: 6:00 PM daily;

[0080] Application frequency: once daily;

[0081] Dosage: Add 10-15 ml of the mixed preparation per cubic meter of water; preferably 12.5 ml.

[0082] Phase Two (8 to 14 days postpartum)

[0083] Application time: 8:00 PM (8:00 PM) and 5:00 AM (05:00 AM) daily;

[0084] Application frequency: twice daily;

[0085] Dosage: Add 15-20 ml of the mixed preparation per cubic meter of water each time; preferably 17.5 ml.

[0086] Phase 3 (15 to 20 days postpartum)

[0087] Application time: 8:00 PM (8:00 PM) and 5:00 AM (05:00 AM) daily;

[0088] Application frequency: twice daily;

[0089] Dosage: Add 20-30 ml of the mixed preparation per cubic meter of water each time, preferably 25 ml.

[0090] VI. Comparison of Experimental Results

[0091] 1. Analysis of growth index results

[0092] Table 1. Effects of different rosmarinic acid contents in the microcirculation preparation for mud crabs on growth indicators.

[0093] Survival rate (%) Weight gain rate (%) <![CDATA[Condition factor (g / cm 3 )]]> Recovery time (days) One set of components <![CDATA[70.88±2.18 b ]]> <![CDATA[30.48±1.36 b ]]> <![CDATA[47.02±1.90 b ]]> <![CDATA[26.27±1.21 c ]]> Group 2 <![CDATA[78.13±2.10 a ]]> <![CDATA[36.03±1.26 a ]]> <![CDATA[55.63±1.76 a ]]> <![CDATA[23.26±0.86 d ]]> Three groups of components <![CDATA[71.24±2.18 b ]]> <![CDATA[29.78±1.36 b ]]> <![CDATA[45.22±1.71 b ]]> <![CDATA[27.30±1.13 c ]]> Four groups <![CDATA[59.48±2.18 c ]]> <![CDATA[22.98±1.21 c ]]> <![CDATA[32.35±1.82 c ]]> <![CDATA[32.43±1.32 b ]]> Five groups <![CDATA[61.38±2.18 c ]]> <![CDATA[24.98±1.33 c ]]> <![CDATA[34.62±1.36 c ]]> <![CDATA[31.00±1.14 b ]]> Control group <![CDATA[53.63±2.21 d ]]> <![CDATA[17.7±2.14 d ]]> <![CDATA[23.1±2.33 d ]]> <![CDATA[37.63±2.21 a ]]>

[0094] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0095] Table 1 shows that component group two had the following differences in survival rate (78.13±2.10%), weight gain rate (36.03±1.26%), and condition factor (55.63±1.76 g / cm²). 3 The growth rate of the control group was significantly higher than that of component group 1 and component group 3, and the recovery time was the shortest (23.26±0.86 days). There were no significant differences in these indicators between component group 1 and component group 3, but they were better than component group 4 and component group 5. The control group had the worst growth indicators.

[0096] Component Two Advantages: Rosmarinic acid at a concentration of 350 mg / L yields the best results. At this concentration, it promotes the expression of antioxidant enzymes by activating the Nrf2 pathway, reducing oxidative damage to cells by free radicals. Simultaneously, it inhibits the NF-κB pathway, reducing the release of inflammatory factors, alleviating oxidative stress and inflammatory responses, providing a favorable internal environment for cell growth, promoting the growth of mud crabs, increasing survival rate, weight gain, and conditionability, and shortening recovery time. For example, moderate antioxidant and anti-inflammatory effects can maintain normal cell metabolism and proliferation, preventing growth inhibition caused by oxidative damage and inflammation.

[0097] Differences between Component 1 and Components 3 and 2: The contents of Component 1 (300 mg / L) and Component 3 (400 mg / L) deviated from the optimal values. At lower contents (300 mg / L), they could not fully exert their growth-promoting effect; at excessively high contents (400 mg / L), they may cause imbalances in intracellular signaling pathways or produce toxic effects, interfering with normal physiological metabolism and affecting growth performance. Therefore, the growth indicators of Component 1 and Component 3 were inferior to those of Component 2, but since the difference was not significant, there was no statistically significant difference.

[0098] Compared with groups four, five, and control group one: the content of group four (200 mg / L) and group five (500 mg / L) deviated further from the optimal range. Too low a concentration failed to effectively initiate and promote cell division, while too high a concentration exacerbated cellular metabolic disorders, resulting in significantly worse growth indicators compared to groups one and three. The control group, lacking the addition of rosmarinic acid, lacked its growth-promoting and regulatory mechanisms, leaving the cells in a state of natural damage and slow growth, with the worst growth indicators.

[0099] 2. Analysis of Nutritional Transport Indicators

[0100] Table 2. Effects of different rosmarinic acid contents in microcirculation preparations on postpartum nutrient transport indicators in mud crabs.

[0101]

[0102] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0103] As shown in Table 2, Group 2 had the highest levels of hepatopancreatic and gonadal indices (7.8±0.31% and 6.8±0.30%, respectively). Group 2 had the lowest levels of triglycerides and total cholesterol (0.91±0.03mmol / L and 1.64±0.06mmol / L, respectively), and these levels were significantly different from those of other groups. Groups 1 and 3 were similar to and better than Groups 4 and 5. The control group had the worst nutrient transport-related indicators.

[0104] Component 2 Advantages: 350mg / L of rosmarinic acid can better regulate lipid metabolism and promote nutrient absorption and organ function recovery. It enables nutrients to be transported more efficiently to organs such as the liver, pancreas and gonads, promoting their function improvement, as evidenced by increased liver and pancreas indices and gonadal indices, and decreased triglyceride and total cholesterol levels.

[0105] Reasons for differences in other groups: Groups 1 and 3, due to suboptimal content, did not exhibit the same regulatory effects on lipid metabolism and nutrient transport-related enzymes and signaling pathways as Group 2. Both excessively low and high content affected their ability to promote nutrient absorption and enhance organ function, resulting in nutrient transport indicators at an intermediate level. Groups 4 and 5 showed significant deviations in content, greatly interfering with metabolic and transport processes and failing to effectively improve nutrient transport, resulting in poor indicators. The control group, lacking rosmarinic acid, lacked metabolic and regulatory mechanisms related to nutrient transport, resulting in the worst indicators.

[0106] 3. Analysis of indicators related to metabolic waste accumulation

[0107] Table 3. Effects of different rosmarinic acid contents in the microcirculation preparation for mud crabs on indicators related to post-feeding metabolic waste accumulation in mud crabs.

[0108]

[0109] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0110] As shown in Table 3, the contents of urea nitrogen, ammonia, and uric acid were lowest in group two (3.6±0.4 mg / dL, 0.7±0.03 mg / L, and 4.3±0.1 mg / dL, respectively); the contents of antimicrobial peptides and lysozyme were highest in group two (182±12 μg / mL and 177±12 U / mL, respectively); groups one and three were at an intermediate level and were better than groups four and five; and the control group had the most severe accumulation of metabolic waste.

[0111] Component 2 Advantages: The appropriate rosmarinic acid content (350mg / L) helps enhance the metabolic function and immunity of mud crabs by promoting the conversion and excretion of metabolic wastes such as urea nitrogen, ammonia and uric acid, reducing their accumulation in the body; at the same time, it stimulates the immune system to produce more antimicrobial peptides and lysozymes, enhancing the body's ability to clear bacteria and toxins and maintaining the stability of the internal environment.

[0112] Mechanism of difference between groups: The effects of components one and three were relatively overlapping with those of component two, with limited regulatory effects on metabolic enzymes and the immune system. They could not efficiently clear metabolic waste and enhance immunity, remaining at an intermediate level of metabolic waste accumulation. Components four and five showed abnormal levels, severely affecting the balance of metabolism and immune regulation, resulting in excessive waste accumulation and weakened immune function. The control group lacked rosmarinic acid to assist metabolism and immune regulation, leading to a large accumulation of metabolic waste and low immune defense.

[0113] 4. Analysis of related indicators of fat and energy storage

[0114] Table 4: Effects of different rosmarinic acid contents in microcirculation preparations for mud crabs on postpartum fat and energy reserves in mud crabs

[0115]

[0116]

[0117] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0118] As shown in Table 4, the highest high-density lipoprotein level was observed in group 2 (64±3 mg / dL); the lowest low-density lipoprotein level was observed in group 2 (108±4 mg / dL); and the highest fatty acid synthase and carnitine palmitoyltransferase activities were observed in group 2 (1.8±0.2 U / mg and 2.9±0.2 U / mg, respectively); followed by groups 1 and 3; while control group 1 was the worst.

[0119] Component 2 Advantages: 350 mg / L of rosmarinic acid can effectively regulate lipid metabolism and energy-related enzyme activity. At this concentration, it maintains a good lipid metabolism state, providing sufficient energy reserves for mud crabs, and exhibits the best performance in related indicators.

[0120] Components 1 and 3, due to their lower content, had limited ability to maintain lipid metabolism balance and energy reserves, and their indicators were in the middle range. The control group lacked the regulation of lipid and energy metabolism by rosmarinic acid, resulting in disordered lipid metabolism and insufficient energy reserves.

[0121] 5. Analysis of tissue repair-related indicators

[0122] Table 5. Effects of different rosmarinic acid contents in the microcirculation preparation for mud crabs on relevant indicators of postpartum tissue repair in mud crabs.

[0123]

[0124] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0125] As shown in Table 5, the levels of hydroxyproline, epidermal growth factor, platelet-derived growth factor, and transforming growth factor-β in Component 2 were significantly higher than those in other groups; Component 1 and Component 3 were in the middle range; and Control 1 had the lowest levels.

[0126] Component 2 Advantages: This concentration of rosmarinic acid can effectively promote collagen synthesis and growth factor secretion. It accelerates the repair and regeneration of damaged tissues, increases hydroxyproline content, and enhances tissue repair capabilities by stimulating fibroblast proliferation and the release of related growth factors (such as epidermal growth factor, platelet-derived growth factor, and transforming growth factor-β).

[0127] Reasons for differences between groups: Components 1 and 3 had suboptimal content, resulting in weaker promoting effects on cell proliferation and growth factor secretion, and moderate tissue repair effects. Components 4 and 5 had inappropriate content, failing to effectively activate the cellular and molecular mechanisms related to tissue repair, leading to poor repair capacity. The control group, lacking rosmarinic acid, lacked effective stimulation for tissue repair, resulting in the lowest repair-related indicators.

[0128] Therefore, in the mixed formulation, the rosmarinic acid content of 300-400 mg / L has an improving effect on growth indicators, nutrient transport indicators, metabolic waste accumulation indicators, fat and energy storage indicators, and tissue repair-related indicators.

[0129] Example 2: Sage acid content test

[0130] I. Preparation of Mixed Reagents

[0131] Weigh out 17.5, 20, 22.5, 25, and 27.5 grams of sage leaves respectively, and follow steps one to three in Example 1 to obtain mixed formulations with sage acid contents of 250 mg / L, 300 mg / L, 350 mg / L, 200 mg / L, and 400 mg / L respectively. The auxiliary solution was designated as the blank control group, and the results were denoted as follows:

[0132] Component 6: 250 mg / L oxalic acid,

[0133] Component 7: 300 mg / L oxalic acid,

[0134] Component 8: 350 mg / L oxalic acid,

[0135] Component 9: 200 mg / L oxalic acid,

[0136] Component 10: 400 mg / L oxalic acid,

[0137] Control group 2: auxiliary solution (blank group).

[0138] II. Aquaculture Experiment

[0139] From September to October 2023, the experiment was conducted in indoor culture ponds at Dongying Kainuo Aquaculture Co., Ltd. Ninety female mud crabs, 1-3 days postpartum, were collected again from the southern coastal region, transported by air to the experimental site, and then centrally disinfected before being placed in two 30m³ tanks. 2 The crabs were temporarily kept indoors at the cement plant for 1-2 days. After the temporary holding, 5 crabs were placed in 18 20m³ containers. 2 Experiments were conducted in laboratory pools. Five postpartum female crabs were placed in each pool, along with five pieces of tile. Pools 1-3 contained component group six, pools 4-6 contained component group seven, pools 7-9 contained component group eight, pools 10-12 contained component group nine, pools 13-15 contained component group ten, and pools 16-18 served as control group two. The purpose of this embodiment was to explore key compounds in a postpartum microcirculation preparation for promoting the recovery of mud crabs after spawning. The experimental materials and sources in this embodiment were the same as in Example 1.

[0140] III. Comparison of Experimental Results

[0141] 1. Analysis of growth index results

[0142] Table 6. Effects of different sarcopenic acid contents in the microcirculation preparation for mud crabs on growth indicators.

[0143] Survival rate (%) Weight gain rate (%) <![CDATA[Condition factor (g / cm 3 )]]> Recovery time (days) Six groups <![CDATA[70.38±2.18 b ]]> <![CDATA[29.98±1.36 b ]]> <![CDATA[46.52±1.90 b ]]> <![CDATA[26.77±1.21 c ]]> Seven groups <![CDATA[77.63±2.10 a ]]> <![CDATA[35.53±1.26 a ]]> <![CDATA[55.13±1.76 a ]]> <![CDATA[23.76±0.86 d ]]> Group 8 <![CDATA[70.74±2.18 b ]]> <![CDATA[29.28±1.36 b ]]> <![CDATA[44.72±1.71 b ]]> <![CDATA[27.00±1.13 c ]]> Nine groups of components <![CDATA[59.28±2.18 c ]]> <![CDATA[22.48±1.21 c ]]> <![CDATA[31.85±1.82 c ]]> <![CDATA[32.23±1.32 b ]]> 10 groups <![CDATA[60.88±2.18 c ]]> <![CDATA[24.48±1.33 c ]]> <![CDATA[34.12±1.36 c ]]> <![CDATA[31.20±1.14 b ]]> Control Group 2 <![CDATA[53.13±2.21 d ]]> <![CDATA[17.2±2.14 d ]]> <![CDATA[22.6±2.33 d ]]> <![CDATA[38.13±2.21 a ]]>

[0144] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0145] As shown in Table 6, Group 7 performed best in terms of survival rate, weight gain rate, and plumpness, with values ​​significantly higher than other groups (except in specific cases where there was no significant difference); in terms of recovery time, Group 7 had the shortest recovery time.

[0146] Principle Analysis:

[0147] Component seven was superior to components six and eight: the 300 mg / L oxalic acid content was likely closest to the optimal concentration required for postpartum growth in mud crabs. This concentration most effectively promoted the metabolism of mud crabs, enhancing the absorption and utilization efficiency of nutrients; better nutrient supply and metabolic status may have enhanced the immunity of mud crabs, improving their resistance to environmental stress and disease, thereby increasing survival rate. As for recovery time, the more efficient operation of their physiological functions resulted in faster postpartum recovery.

[0148] Components 6 and 8 were superior to components 9 and 10: Although the growth-promoting effects of 250 mg / L and 350 mg / L oxalic acid on growth indicators were not as good as those of 300 mg / L, they could still promote the physiological balance and growth process of mud crabs to a certain extent.

[0149] Components nine and ten were superior to control group two: Compared with control group two, components eight and four contained a certain amount of oxalic acid. This oxalic acid can initiate and regulate growth-related physiological responses in the mud crab to a certain extent. It can promote the mud crab's uptake and utilization of nutrients. At the same time, oxalic acid may participate in the regulation of energy metabolism in the mud crab, making energy production and utilization more efficient, thereby promoting growth, increasing survival rate, weight gain rate and plumpness, and shortening recovery time.

[0150] 2. Analysis of Nutritional Transport Indicators

[0151] Table 7. Effects of different sarcopenic acid contents in microcirculation preparations on postpartum nutrient transport indicators in mud crabs.

[0152]

[0153]

[0154] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0155] As shown in Table 7, the hepatopancreatic and gonadal indices were highest in group 7, decreasing from group 7 to control group 2. Conversely, the triglyceride and total cholesterol levels were lowest in group 7, increasing from group 7 to control group 2. Letters indicate statistical significance; no significant differences were found between groups with the same letter.

[0156] Principle Analysis:

[0157] Component 7 was superior to components 6 and 8: 300 mg / L of sarsaparilla acid had the best regulatory effect on the nutrient transport system of mud crabs. Regarding the hepatopancreas and gonads, it promoted their development, thereby increasing the hepatopancreas and gonadal indices. For example, it promoted the synthesis and storage of proteins related to nutrients within hepatopancreas cells, as well as the development and maturation of gonadal cells. Regarding triglycerides and total cholesterol, 300 mg / L of sarsaparilla acid may have regulated the activity of enzymes related to lipid metabolism, such as promoting the activity of lipases and inhibiting the activity of cholesterol synthases, resulting in lower levels of triglycerides and total cholesterol in the blood. This means more efficient transport and utilization of nutrients in the body, reducing excessive fat accumulation.

[0158] Components 6 and 8 were superior to components 9 and 10: The 250 mg / L and 350 mg / L levels of oxalic acid still promoted the operation of the nutrient transport system to some extent; in contrast, the 200 mg / L level had insufficient promoting effect on the development of the hepatopancreas and gonads, resulting in lower hepatopancreas and gonadal indices; the 400 mg / L level had an excessive or abnormal effect on the regulation of lipid metabolism, interfering with normal nutrient transport and metabolic processes, and similarly leading to unsatisfactory development of the hepatopancreas and gonads and blood lipid levels.

[0159] Components nine and ten were superior to control group two: In the hepatopancreas and gonads, succinic acid increased the absorption and utilization of nutrients by hepatopancreas and gonadal cells; in terms of lipid regulation, succinic acid reduced the synthesis of triglycerides and total cholesterol or promoted their catabolism, thus making components nine and ten, which contained succinic acid, superior to control group two, which did not contain succinic acid, in nutrient transport-related indicators.

[0160] 3. Analysis of indicators related to metabolic waste accumulation

[0161] Table 8. Effects of different sarcopenic acid contents in the microcirculation preparation for mud crabs on indicators related to post-feeding metabolic waste accumulation in mud crabs.

[0162]

[0163] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0164] Table 8 shows that the contents of urea nitrogen, ammonia, and uric acid, from lowest to highest, are: Group 7 < Group 6, Group 8 < Group 9, Group 10 < Control Group 2; the contents of antimicrobial peptides and lysozyme, from highest to lowest, are: Group 7 > Group 6, Group 8 > Group 9, Group 10 > Control Group 2.

[0165] Principle Analysis:

[0166] Component 7 was superior to components 6 and 8: 300 mg / L of oxalic acid most effectively promoted the excretion of metabolic waste in mud crabs. Regarding nitrogen metabolism, it may have enhanced the activity of urea synthase in the liver, allowing ammonia to be converted into urea more rapidly and excreted, thereby reducing the levels of urea nitrogen and ammonia in the blood, while also lowering uric acid levels. Simultaneously, 300 mg / L of oxalic acid significantly enhanced the immune defense capabilities of mud crabs, stimulating immune cells to secrete more antimicrobial peptides and lysozyme.

[0167] Groups 6 and 8 were superior to groups 9 and 10: 250 mg / L and 350 mg / L of oxalic acid effectively maintained the metabolic balance and immune function of mud crabs. In contrast, the 200 mg / L concentration had a weaker promoting effect on metabolic waste excretion and immune regulation. Regarding nitrogen and uric acid metabolism, insufficient activation of related enzymes or inadequate renal excretion led to greater accumulation of urea nitrogen, ammonia, and uric acid. In terms of immunity, immune cell activity was lower, and the secretion of antimicrobial peptides and lysozyme was reduced. The 400 mg / L concentration may have interfered with metabolism and immune regulation, similarly hindering the clearance of metabolic waste and the enhancement of immune function.

[0168] Components 8 and 4 were superior to control group 2: Succinic acid can activate metabolic and immune-related mechanisms in mud crabs. In terms of metabolism, it may promote the expression and activity of enzymes involved in the treatment of metabolic waste in the liver and kidneys, and accelerate the metabolism and excretion of urea nitrogen, ammonia and uric acid. In terms of immunity, succinic acid can stimulate the activity of immune cells and promote the synthesis and secretion of antimicrobial peptides and lysozyme. As a result, components 8 and 4 containing succinic acid were superior to control group 2 without succinic acid in terms of indicators related to metabolic waste accumulation.

[0169] 4. Analysis of related indicators of fat and energy storage

[0170] Table 9. Effects of different sarcopenic acid contents in microcirculation preparations for mud crabs on postpartum fat and energy reserves in mud crabs.

[0171]

[0172] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0173] Table 9 shows that the high-density lipoprotein (HDL) content, ranked from highest to lowest, is: Group 7 > Group 6, Group 8 > Group 9, Group 10 > Control Group 2; the low-density lipoprotein (LDL) content, ranked from lowest to highest, is: Group 7 < Group 6, Group 8 < Group 9 ≈ Group 10 < Control Group 2; and the fatty acid synthase and carnitine palmitoyltransferase (CPLT) activities, ranked from highest to lowest, are: Group 7 > Group 6 ≈ Group 8 > Group 9 ≈ Group 10 > Control Group 2.

[0174] Principle Analysis:

[0175] Component 7 was superior to components 6 and 8: 300 mg / L oxalic acid optimally regulated fat and energy metabolism in mud crabs. For high-density lipoprotein (HDL), it may have promoted HDL synthesis or inhibited its catabolism, leading to increased HDL levels. HDL helps transport cholesterol from peripheral tissues back to the liver for metabolism, thereby reducing blood cholesterol levels and fat deposition on blood vessel walls, contributing to a healthy physiological state. For low-density lipoprotein (LDL), 300 mg / L oxalic acid may have inhibited LDL synthesis or promoted its clearance, resulting in lower LDL levels. Regarding fatty acid synthase (FAS) and carnitine palmitoyltransferase (CPT), 300 mg / L oxalic acid may have increased the activity of FAS and CPT by regulating the expression of related genes. FAS is responsible for fatty acid synthesis, and CPT participates in the transport of fatty acids into mitochondria for β-oxidation for energy. Increased activity of these two enzymes is beneficial for fat synthesis, energy conversion, and storage, providing sufficient energy for the growth and physiological activities of mud crabs.

[0176] Groups 6 and 8 were superior to groups 9 and 10: 250 mg / L and 350 mg / L of oxalic acid were able to maintain the balance of fat and energy metabolism in mud crabs to a certain extent. In contrast, the 200 mg / L concentration had a weaker regulatory effect on enzymes related to fat and energy metabolism, resulting in lower activities of FAS and CPT, and low efficiency in fat synthesis and energy conversion. Simultaneously, it insufficiently regulated HDL and LDL metabolism, leading to lower HDL and higher LDL levels, which is detrimental to rational fat metabolism and energy storage. The 400 mg / L concentration may have had an excessive or abnormal effect on fat and energy metabolism, which is detrimental to its stability.

[0177] Groups 9 and 10, containing oxalic acid, were superior to control group 2. Oxalic acid can activate enzymes and pathways related to fat and energy metabolism in mud crabs. It may initiate a series of signal transduction processes by binding to intracellular receptors, regulating the expression of genes related to fat and energy metabolism. For example, it promotes the transcription of FAS and CPT genes, increasing the synthesis of these two enzymes and thus enhancing their activity, promoting fat synthesis and energy conversion. Regarding HDL and LDL metabolism, oxalic acid may regulate the expression and activity of proteins involved in HDL and LDL synthesis and metabolism in the liver and other tissues, leading to increased HDL levels and decreased LDL levels, thus optimizing fat metabolism. Therefore, groups 8 and 4, containing oxalic acid, were superior to control group 2 (without oxalic acid) in these indicators.

[0178] 5. Analysis of tissue repair-related indicators

[0179] Table 10. Effects of different sarcopenic acid contents in the microcirculation preparation for mud crabs on relevant indicators of postpartum tissue repair in mud crabs.

[0180]

[0181] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0182] Table 10 shows that, for the four indicators closely related to tissue repair—hydroxyproline, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-β (TGF-β)—the data in group 7 were significantly higher than those in other groups (except for specific cases where no significant difference was observed). From group 7 to control group 2, the values ​​of each indicator showed a decreasing trend. The differences between groups were distinguished by letters, with the same letter indicating no significant difference.

[0183] Principle Analysis:

[0184] Component 7 was superior to components 6 and 8: the 300 mg / L caryopsisic acid concentration showed the most significant effect on promoting postpartum tissue repair in mud crabs. Hydroxyproline is an important component of collagen, and 300 mg / L caryopsisic acid may have significantly increased hydroxyproline content by stimulating the activity of fibroblasts and other related cells, thereby promoting collagen synthesis. Regarding epidermal growth factor (EGF), which promotes the proliferation, differentiation, and migration of epidermal cells, 300 mg / L caryopsisic acid may have activated related intracellular signaling pathways, leading to increased EGF secretion by epidermal cells and accelerating epidermal tissue repair and regeneration. Platelet-derived growth factor (PDGF) attracts inflammatory cells and fibroblasts in tissue repair, promoting cell proliferation and extracellular matrix synthesis. 300 mg / L caryopsisic acid may have promoted PDGF secretion by regulating the expression of related genes. Transforming growth factor-β (TGF-β) has multiple functions, including promoting extracellular matrix synthesis and regulating cell proliferation and differentiation. 300 mg / L of oxalic acid may bind to cell surface receptors, initiate a series of intracellular reactions, enhance the expression and secretion of TGF-β, and comprehensively promote the tissue repair process.

[0185] Components six and three were superior to components nine and ten: 250 mg / L and 350 mg / L of oxalic acid could promote the activity of tissue repair-related cells and the secretion of factors to a certain extent; however, the content of 200 mg / L was relatively low and could not fully activate the tissue repair-related mechanisms, resulting in limited secretion of EGF, PDGF, TGF-β, etc., thus affecting the speed and effect of tissue repair; the content of 400 mg / L may have interfered with the normal physiological function of cells, leading to cellular metabolic disorders, which in turn inhibited the normal secretion and function of tissue repair-related factors.

[0186] Components 8 and 4 were superior to control group 2: The presence of succinic acid can activate the tissue repair mechanism of mud crabs. It may act on various cells in the mud crab, such as fibroblasts, epidermal cells, and platelets, promoting these cells to secrete key factors required for tissue repair, thereby increasing the content of hydroxyproline. At the same time, it stimulates epidermal cells to secrete more EGF, promoting the repair of epidermal tissue; it prompts platelets to release PDGF, attracting more cells to participate in the tissue repair process; it induces related cells to secrete TGF-β, regulating cell proliferation and differentiation, and promoting the synthesis of extracellular matrix. Therefore, components 8 and 4 containing succinic acid were significantly superior to control group 2 without succinic acid in tissue repair-related indicators.

[0187] Example 3: Olive bitter glycoside content test

[0188] Oleuropein is a phenolic compound extracted from olive leaves, and its main function is to improve the physiological phenomenon of metabolic waste accumulation in female mud crabs. As a bioactive compound, oleuropein significantly affects the postpartum metabolic health of female mud crabs by regulating multiple metabolic pathways. First, regarding the metabolism of urea nitrogen and ammonia in the hemolymph, oleuropein effectively promotes the conversion of ammonia to urea by activating hepatic urea cycle enzymes, and then excretes urea through the kidneys. This process not only promotes urea synthesis but also significantly accelerates the conversion and excretion of ammonia, effectively reducing the accumulation of ammonia in the blood, thereby alleviating the problem of metabolic waste accumulation caused by ammonia poisoning after spawning in female mud crabs and improving their metabolic function. Second, oleuropein further promotes the clearance of metabolic waste by regulating the metabolism of uric acid in the hemolymph. Specifically, by strengthening the function of gill cells in mud crabs, oleuropein promotes the filtration and excretion of uric acid, greatly reducing the accumulation of uric acid in the hemolymph. This process accelerates the clearance of uric acid and reduces its concentration in the hemolymph, thus resolving the problem of metabolic waste accumulation caused by uric acid buildup after spawning in female mud crabs. Furthermore, oleuropein enhances the secretion of antimicrobial peptides and lysozyme by stimulating the immune system. These immune substances play a crucial role in clearing bacteria and toxins from the blood. This effect of oleuropein not only promotes the synthesis of antimicrobial peptides and lysozyme but also accelerates the clearance of bacteria and toxins, thereby improving the immune function of female mud crabs, reducing the accumulation of metabolic waste due to an inefficient immune system, and further improving their metabolic health. In conclusion, oleuropein significantly promotes the clearance of metabolic waste and the recovery of metabolic function after spawning in female mud crabs by regulating multiple metabolic pathways.

[0189] I. Preparation of Mixed Reagents

[0190] Olive leaves weighing 17.5, 20, 22.5, 25, and 27.5 grams respectively were used to obtain mixed preparations with oleuropein contents of 250 mg / L, 300 mg / L, 350 mg / L, 200 mg / L, and 400 mg / L, respectively, following steps one through three of Example 1. The auxiliary solution was designated as the blank control group, and the results were denoted as follows:

[0191] Component group eleven: 250 mg / L oleuropein,

[0192] Component group twelve: 300 mg / L oleuropein,

[0193] Component group 13: 350 mg / L oleuropein,

[0194] Component group fourteen: 200 mg / L oleuropein,

[0195] Component group 15: 400 mg / L oleuropein,

[0196] Control group 3: auxiliary solution (blank group).

[0197] II. Aquaculture Experiment

[0198] From September to October 2023, the experiment was conducted in indoor culture ponds at Dongying Kainuo Aquaculture Co., Ltd. Ninety female mud crabs, 1-3 days postpartum, were collected again from the southern coastal region, transported by air to the experimental site, and then centrally disinfected before being placed in two 30m³ tanks. 2 The crabs were temporarily kept indoors at the cement plant for 1-2 days. After the temporary holding, 5 crabs were placed in 18 20m³ containers. 2 Experiments were conducted in laboratory pools. Five postpartum female crabs were placed in each pool, along with five pieces of tile. Pools 1-3 contained component group eleven, pools 4-6 contained component group twelve, pools 7-9 contained component group thirteen, pools 10-12 contained component group fourteen, pools 13-15 contained component group fifteen, and pools 16-18 served as control groups three. The purpose of this embodiment was to explore key compounds in a postpartum microcirculation preparation for promoting the recovery of mud crabs after spawning. The experimental materials and sources in this embodiment were the same as in Example 1.

[0199] III. Comparison of Experimental Results

[0200] 1. Analysis of growth index results

[0201] Table 11 Effects of different oleuropein contents in the microcirculation preparation for mud crabs on growth indicators of mud crabs

[0202]

[0203]

[0204] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0205] Table 11 shows that component group 12 performed best in terms of survival rate, weight gain rate, and conditionability, with values ​​significantly higher than other groups (except for specific cases where there was no significant difference). Component group 12 had the shortest recovery time. From component group 12 to control group 3, each growth indicator showed a decreasing trend. Differences between groups are distinguished by letters, with the same letter indicating no significant difference.

[0206] Principle Analysis:

[0207] Component 12 is superior to components 11 and 13: The 300 mg / L oleuropein content may best meet the physiological needs of mud crabs during postpartum growth. From a nutritional utilization perspective, it may optimize the absorption mechanism of nutrients in the mud crab's intestines, perhaps by regulating the morphology and function of intestinal villi, increasing the absorption area and efficiency of nutrients, enabling mud crabs to absorb more energy and nutrients for growth, thus resulting in a higher weight gain rate and fullness. In terms of survival, oleuropein may improve the survival rate by regulating the mud crab's immune system, enhancing the activity of immune cells, and increasing resistance to pathogens. During the recovery process, it promotes the balance of hormones in the body, accelerates the recovery of bodily functions, and therefore has the shortest recovery time.

[0208] Components 11 and 13 were superior to those 14 and 15: Oleuropein levels of 250 mg / L and 350 mg / L could still maintain the growth and recovery mechanisms of mud crabs to a certain extent. A level of 200 mg / L might be insufficient to fully activate growth and immune-related signaling pathways in mud crabs. For example, in nutrient absorption signal transduction, the expression of certain key transport proteins might not be effectively initiated, leading to limited nutrient uptake and consequently affecting growth indicators. A level of 400 mg / L might have produced some negative effects, perhaps by overactivating certain metabolic feedback mechanisms, inhibiting normal growth and immune regulation, such as interfering with normal hormone secretion levels, thus hindering the growth and recovery process.

[0209] Components 13 and 14 were superior to control group 3: Compared with control group 3, oleuropein in components 13 and 14 was able to initiate the basic regulatory mechanisms of growth and recovery in mud crabs. Oleuropein may act directly or indirectly at the cellular level of mud crabs, increasing muscle growth and thus improving weight gain. Simultaneously, it activates the activity of immune cells, enhances immune defense, and improves survival rate. In terms of energy metabolism, it may regulate mitochondrial function, improve energy production efficiency, and provide power for growth and recovery, resulting in growth indicators superior to control group 3 without oleuropein.

[0210] 2. Analysis of Nutritional Transport Indicators

[0211] Table 12. Effects of different oleuropein contents in the microcirculation preparation for mud crabs on postpartum nutrient transport indicators of mud crabs.

[0212]

[0213] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0214] Table 12 shows that the hepatopancreatic and gonadal indices were highest in group 12, decreasing from group 12 to control group 3. Conversely, triglyceride and total cholesterol levels were lowest in group 12, increasing from group 12 to control group 3. No significant differences were found between groups with the same letter designation.

[0215] Principle Analysis:

[0216] Component 12 was superior to components 11 and 3: 300 mg / L oleuropein may have the best regulatory effect on the nutrient transport and distribution mechanism of mud crabs. In the hepatopancreas, it may promote the expression of genes related to glycogen synthesis and storage, increase the uptake and conversion capacity of hepatopancreatic cells of nutrients, thereby improving the hepatopancreatic index and optimizing the transport and distribution of nutrients in the body.

[0217] Components 11 and 3 were superior to components 4 and 5: Oleuropein levels of 250 mg / L and 350 mg / L could still maintain normal regulation of nutrient transport and metabolism to a certain extent. A level of 200 mg / L might not adequately activate signaling molecules and enzymes related to nutrient transport and metabolism. For example, in the hepatopancreas, it could not effectively induce the expression of hepatic glycogen synthase, leading to insufficient nutrient storage capacity and a low hepatopancreatic index. In the gonads, it could not adequately stimulate gonadal development-related signaling pathways, resulting in a low gonadal index. Simultaneously, its regulatory effect on lipid metabolism enzymes was weak, leading to higher levels of triglycerides and total cholesterol. A level of 400 mg / L interfered with normal cellular metabolism. For example, it might inhibit the normal activity regulation of lipid metabolism enzymes, leading to lipid metabolism disorders, which also resulted in unsatisfactory nutrient transport and metabolic indicators.

[0218] Components 13 and 4 were superior to control group 3: Oleuropein can activate the relevant pathways of nutrient transport and metabolism in mud crabs. In the hepatopancreas and gonads, it may promote the expression of nutrient transport proteins on the cell membrane and increase the uptake of nutrients by cells. In the gonads, it promotes the absorption of nutrients by gonadal cells, promotes gonadal development, and increases the gonadal index. In terms of blood lipid regulation, it reduces the synthesis of triglycerides and total cholesterol, so components 13 and 14 containing oleuropein are superior to control group 3 without oleuropein in nutrient transport-related indicators.

[0219] 3. Analysis of indicators related to metabolic waste accumulation

[0220] Table 13. Effects of different oleuropein contents in the microcirculation preparation for mud crabs on indicators related to post-feeding metabolic waste accumulation in mud crabs.

[0221]

[0222] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0223] Table 13 shows that the contents of urea nitrogen, ammonia, and uric acid, from low to high, are: Group 12 < Group 11 ≈ Group 13 < Group 14 ≈ Group 15 < Control Group 3; the contents of antimicrobial peptides and lysozyme, from high to low, are: Group 12 > Group 11 ≈ Group 13 > Group 14 ≈ Group 15 > Control Group 3.

[0224] Principle Analysis:

[0225] Component 12 was superior to components 11 and 13: 300 mg / L oleuropein most effectively promoted the excretion of metabolic waste and enhanced immune function in mud crabs. Regarding nitrogen metabolism, it accelerated the conversion of ammonia to urea, thereby reducing the levels of urea nitrogen and ammonia in the blood. For uric acid, it may have promoted renal excretion, possibly by regulating the renal tubular reabsorption and secretion mechanisms of uric acid, thus reducing its accumulation in the body. In terms of immune regulation, 300 mg / L oleuropein may have activated signaling pathways such as NF-κB within immune cells, promoting the transcription and translation of antimicrobial peptide and lysozyme genes, resulting in the secretion of more antimicrobial peptides and lysozyme by immune cells, thereby enhancing the immune defense capabilities of mud crabs. Components eleven and thirteen were superior to those of fourteen and fifteen: 250 mg / L and 350 mg / L oleuropein effectively maintained the metabolic and immune balance in mud crabs; a concentration of 200 mg / L might be insufficient to fully activate key enzymes and signaling pathways related to nitrogen metabolism and immune regulation; for example, in the liver, it could not effectively increase the activity of urea cycle enzymes, leading to the inability of ammonia to be timely converted into urea for excretion, resulting in elevated levels of urea nitrogen and ammonia in the blood. In immune cells, it could not fully activate immune signaling pathways, resulting in lower secretion of antimicrobial peptides and lysozyme. A concentration of 400 mg / L might have excessive or abnormal effects on metabolism and immune regulation; for example, it might interfere with the normal renal regulation of uric acid excretion or overactivate immune feedback inhibition mechanisms, leading to decreased immune cell activity and reduced secretion of antimicrobial peptides and lysozyme, which is also detrimental to the clearance of metabolic waste and the maintenance of immune function.

[0226] Components 13 and 14 were superior to control group 3: Oleuropein can initiate metabolic waste excretion and immune regulation mechanisms in mud crabs. Metabolically, it may activate intracellular metabolic regulatory signaling pathways by binding to receptors on the surface of hepatocytes and kidney cells, promoting the processing and excretion of metabolic waste, such as promoting the metabolic conversion of ammonia by hepatocytes and the excretion of urea nitrogen and uric acid by kidney cells. Immunely, oleuropein may activate immune cells, such as macrophages and lymphocytes, promoting their secretion of antimicrobial peptides and lysozymes. For example, by stimulating pattern recognition receptors on the surface of macrophages, it initiates intracellular immune responses, promoting the synthesis and release of antimicrobial peptides and lysozymes. This resulted in components 13 and 4, containing oleuropein, being superior to control group 3 (without oleuropein) in metabolic waste accumulation-related indicators.

[0227] 4. Analysis of related indicators of fat and energy storage

[0228] Table 14. Effects of different oleuropein contents in the microcirculation preparation for mud crabs on postpartum fat and energy reserves in mud crabs.

[0229]

[0230] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0231] Table 14 shows that the high-density lipoprotein (HDL) content, ranked from highest to lowest, is: Group 12 > Group 11 ≈ Group 13 > Group 14 ≈ Group 15 > Control Group 3; the low-density lipoprotein (LDL) content, ranked from lowest to highest, is: Group 12 < Group 11 ≈ Group 13 < Group 14 ≈ Group 15 < Control Group 3; and the fatty acid synthase (FAS) and carnitine palmitoyltransferase (CTLT) activities, ranked from highest to lowest, are: Group 12 > Group 11 ≈ Group 13 > Group 14 ≈ Group 15 > Control Group 3.

[0232] Mechanism Analysis: Component 12 was superior to components 11 and 3: 300 mg / L oleuropein was most effective in regulating fat and energy metabolism in mud crabs. For high-density lipoprotein (HDL), it may increase HDL levels by regulating the synthesis and secretion of related proteins such as apolipoprotein AI in the liver, promoting HDL assembly and secretion. HDL can transport cholesterol from peripheral tissues back to the liver for metabolism, reducing blood cholesterol levels and fat deposition on blood vessel walls, thus helping to maintain normal physiological function. For low-density lipoprotein (LDL), it may increase LDL clearance and reduce LDL levels by inhibiting the degradation of LDL receptor-related proteins in the liver. Regarding fatty acid synthase (FAS) and carnitine palmitoyltransferase (CPT), 300 mg / L oleuropein may increase the activity of these two enzymes by regulating intracellular signaling pathways, such as activating AMPK-mTOR signaling pathways, promoting the expression of FAS and CPT genes. FAS is responsible for fatty acid synthesis, while CPT participates in the transport of fatty acids into the mitochondria for β-oxidation to provide energy. Increased activity of these two enzymes is beneficial for fat synthesis, energy conversion and storage, providing sufficient energy for the growth and physiological activities of mud crabs.

[0233] Components elixir elixir (250 mg / L and 350 mg / L) were superior to those elixir elixir monnieri (200 mg / L and 350 mg / L, respectively) in maintaining the balance of fat and energy metabolism in mud crabs to some extent. However, a concentration of 200 mg / L might not adequately activate signaling pathways and gene expression related to fat and energy metabolism. For example, in regulating HDL and LDL metabolism, it might not effectively regulate the synthesis and degradation of related proteins, resulting in lower HDL and higher LDL levels. Similarly, in regulating FAS and CPT activity, it might not adequately activate related signaling pathways, leading to lower FAS and CPT activity and lower efficiency in fat synthesis and energy conversion. A concentration of 400 mg / L might have an excessive or abnormal effect on fat and energy metabolism. For instance, it might overactivate lipolysis pathways, leading to reduced fat synthesis, or cause an imbalance in the metabolic regulation of HDL and LDL, which is also detrimental to the stability of fat and energy metabolism.

[0234] Components 13 and 14 were superior to control group 3: Oleuropein can increase the synthesis of two enzymes, FAS and CPT, thereby improving enzyme activity and promoting fat synthesis and energy conversion. In terms of HDL and LDL metabolism, oleuropein may regulate the expression and activity of proteins involved in HDL and LDL synthesis and metabolism in the liver and other tissues, resulting in increased HDL content and decreased LDL content, thus optimizing fat metabolism. Therefore, components 13 and 4 containing oleuropein were superior to control group 3 without oleuropein in these indicators.

[0235] 5. Analysis of tissue repair-related indicators

[0236] Table 15. Effects of different oleuropein contents in the microcirculation preparation for mud crabs on relevant indicators of postpartum tissue repair in mud crabs.

[0237]

[0238] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0239] As shown in Table 15, component group 12 exhibited the highest values ​​for the four key tissue repair indicators: hydroxyproline, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-β (TGF-β), demonstrating the best performance. From component group 12 to control group 3, the values ​​of each indicator generally showed a decreasing trend.

[0240] Principle Analysis:

[0241] Component 12 was superior to components 11 and 13: the concentration of 300 mg / L oleuropein had the strongest promoting effect on postpartum tissue repair in mud crabs. Regarding hydroxyproline, a key amino acid in collagen, 300 mg / L oleuropein may significantly enhance fibroblast activity, strongly promote collagen synthesis, and thus significantly increase hydroxyproline content. As for epidermal growth factor (EGF), this concentration of oleuropein can stimulate epidermal cells to secrete large amounts of EGF, effectively accelerating the repair and regeneration of epidermal tissue. For platelet-derived growth factor (PDGF), it causes platelets and other related cells to secrete large amounts of PDGF, attracting more inflammatory cells and fibroblasts to migrate to damaged tissue sites, strongly promoting cell proliferation and extracellular matrix synthesis. Transforming growth factor-β (TGF-β) has many important functions, including regulating cell proliferation and differentiation and promoting extracellular matrix synthesis. 300 mg / L oleuropein may, by binding to specific receptors on the cell surface, greatly enhance the expression and secretion levels of TGF-β, comprehensively and deeply promoting the tissue repair process.

[0242] Components 11 and 13 were superior to those 14 and 15: Oleuropein levels of 250 mg / L and 350 mg / L could still effectively promote the activity of tissue repair-related cells and the secretion of key factors to a certain extent. However, the 200 mg / L level, being relatively low, could not fully activate a series of key mechanisms involved in tissue repair. For example, in fibroblasts, it might not provide sufficient energy and raw materials for collagen synthesis, or it might not effectively activate collagen synthesis-related enzymes, resulting in low hydroxyproline levels. In epidermal cells, platelets, and other cells, the stimulation of related growth factor secretion was insufficient, resulting in limited secretion of growth factors such as EGF, PDGF, and TGF-β, severely affecting the speed and effectiveness of tissue repair. A level of 400 mg / L might interfere with normal cellular physiological functions to some extent. For example, it might overactivate certain signaling pathways, disrupting intracellular signal balance, leading to cellular metabolic disorders, which in turn inhibits the normal secretion and function of tissue repair-related factors, resulting in unsatisfactory tissue repair effects.

[0243] Components 13 and 4 were superior to control group 3: Oleuropein effectively initiated the tissue repair mechanism of mud crabs. It may act on various key cells in the mud crab, such as fibroblasts, epidermal cells, and platelets, significantly promoting the secretion of key factors necessary for tissue repair by these cells. Specifically, oleuropein may regulate the intracellular gene expression regulatory network, causing fibroblasts to significantly increase collagen synthesis, thereby greatly increasing the content of hydroxyproline. At the same time, it strongly stimulates epidermal cells to secrete more EGF, effectively promoting the rapid repair of epidermal tissue; it causes platelets to release large amounts of PDGF, actively attracting more cells to participate in the tissue repair process; and it induces related cells to secrete large amounts of TGF-β, finely regulating the cell proliferation and differentiation process, and significantly promoting the synthesis of extracellular matrix. Therefore, components 13 and 4 containing oleuropein were significantly superior to control group 3 without oleuropein in tissue repair-related indicators.

[0244] Example 4: Terpene alcohol content experiment

[0245] Terpenols are terpenoid compounds extracted from perilla leaves, and their main function is to improve the physiological phenomenon of insufficient fat and energy reserves in female mud crabs. As a bioactive compound, terpenols significantly affect lipid metabolism, fat synthesis and breakdown, and energy supply in female mud crabs by regulating multiple metabolic pathways. First, in the metabolism of high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in the hemolymph, terpenols effectively promote the reverse transport of cholesterol by increasing HDL levels, while clearing LDL from the blood and preventing lipid deposition. This process not only promotes HDL synthesis but also accelerates LDL clearance, significantly reducing its accumulation in the blood, thereby optimizing lipid metabolism and alleviating the problem of nutrient transport obstruction caused by insufficient fat and energy reserves. Second, terpenols also have an important impact on fat synthesis by regulating the activity of fatty acid synthase (FAS) in the hepatopancreas. Specifically, terpenols can reduce fat synthesis and maintain the balance of fat metabolism. While this process promotes the synthesis and storage of fatty acids, it also reduces excessive fat storage, preventing imbalances in fat metabolism and thus addressing the problem of insufficient fat reserves in female mud crabs after spawning. Furthermore, terpenoids activate carnitine palmitoyltransferase (CPT) in the hepatopancreas, promoting the entry of long-chain fatty acids into mitochondria for β-oxidation, accelerating fat breakdown and releasing more energy. This process not only promotes the synthesis of fatty acid oxidases but also significantly accelerates the β-oxidation of long-chain fatty acids, providing a continuous energy supply for female mud crabs after spawning and resolving the problem of slow physiological recovery due to insufficient energy reserves. In conclusion, terpenoids, by regulating multiple metabolic pathways, have a positive impact on lipid metabolism, fat synthesis and breakdown, and energy supply in female mud crabs, promoting their physiological health and recovery.

[0246] I. Preparation of Mixed Reagents

[0247] Weigh out 12.5, 15, 17.5, 20, and 22.5 grams of perilla leaves respectively, and follow steps one to three in Example 1 to obtain mixed preparations with terpene alcohol contents of 200 mg / L, 250 mg / L, 300 mg / L, 150 mg / L, and 350 mg / L respectively. The auxiliary solution was designated as the blank control group, and the results were recorded as follows:

[0248] Component group sixteen: 200 mg / L terpene alcohol,

[0249] Component group seventeen: 250 mg / L terpene alcohol,

[0250] Component group 18: 300 mg / L terpene alcohol,

[0251] Component 19: 150 mg / L terpene alcohol,

[0252] Component group 20: 350 mg / L terpene alcohol,

[0253] Control group 4: auxiliary solution (blank group).

[0254] II. Aquaculture Experiment

[0255] From September to October 2023, the experiment was conducted in indoor culture ponds at Dongying Kainuo Aquaculture Co., Ltd. Ninety female mud crabs, 1-3 days postpartum, were collected again from the southern coastal region, transported by air to the experimental site, and then centrally disinfected before being placed in two 30m³ tanks. 2 The crabs were temporarily kept indoors at the cement plant for 1-2 days. After the temporary holding, 5 crabs were placed in 18 20m³ containers. 2 Experiments were conducted in laboratory pools. Five postpartum female crabs were placed in each pool, along with five pieces of tile. Pools 1-3 contained component group 16, pools 4-6 contained component group 17, pools 7-9 contained component group 18, pools 10-12 contained component group 19, pools 13-15 contained component group 20, and pools 16-18 served as the control group (group 4). The purpose of this embodiment was to explore key compounds in a postpartum microcirculation preparation for promoting the recovery of mud crabs after spawning. The experimental materials and sources in this embodiment were the same as in Example 1.

[0256] III. Comparison of Experimental Data

[0257] 1. Analysis of growth index results

[0258] Table 16 Effects of different terpene alcohol contents in the microcirculation preparation for mud crabs on growth indicators of mud crabs

[0259] Survival rate (%) Weight gain rate (%) <![CDATA[Condition factor (g / cm 3 )]]> Recovery time (t, days) Sixteen groups of components <![CDATA[67.34±2.07 b ]]> <![CDATA[28.96±1.29 b ]]> <![CDATA[44.67±1.81 b ]]> <![CDATA[27.41±1.15 c ]]> Components 17 groups <![CDATA[74.22±1.99 a ]]> <![CDATA[34.23±1.20 a ]]> <![CDATA[52.85±1.67 a ]]> <![CDATA[22.10±0.82 d ]]> 18 groups <![CDATA[67.68±2.07 b ]]> <![CDATA[28.30±1.29 b ]]> <![CDATA[42.96±1.62 b ]]> <![CDATA[25.94±1.07 c ]]> 19 groups <![CDATA[56.51±2.07 c ]]> <![CDATA[21.83±1.15 c ]]> <![CDATA[30.73±1.73 c ]]> <![CDATA[30.81±1.26 b ]]> 20 groups <![CDATA[58.31±2.07 c ]]> <![CDATA[23.73±1.27 c ]]> <![CDATA[32.90±1.29 c ]]> <![CDATA[29.45±1.08 b ]]> Four control groups <![CDATA[50.95±2.10 d ]]> <![CDATA[16.82±2.03 d ]]> <![CDATA[21.95±2.22 d ]]> <![CDATA[35.75±2.10 a ]]>

[0260] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0261] Table 16 shows that, in terms of survival rate, weight gain rate, and conditionability, the values ​​of component group 17 were significantly higher than those of other groups (except where there was no significant difference). Component group 17 also had the shortest recovery time. Overall, the growth indicators showed a trend of gradually worsening from component group 17 to control group 4. Differences between groups were distinguished by letters, with the same letter indicating no significant difference.

[0262] Principle Analysis:

[0263] Component 17 was superior to components 16 and 18: the terpene alcohol content of 250 mg / L was likely the most suitable for the growth needs of mud crabs after spawning, accelerating their metabolism and improving the efficiency of food digestion and absorption, thus providing more energy and nutrients for growth and recovery, resulting in a higher weight gain rate and plumpness; at the same time, this suitable terpene alcohol content may also have enhanced the immune function of mud crabs, improving their resistance to disease and environmental stress, resulting in a higher survival rate; in terms of recovery time, due to the promoting effect of terpene alcohol on various bodily functions, the recovery speed of mud crabs after spawning was accelerated and the recovery time was shortened.

[0264] Components 16 and 18 were superior to components 19 and 20: Although the terpene alcohol content of 200 mg / L and 300 mg / L was not as effective as that of 250 mg / L, it could still maintain the growth and recovery mechanisms of the mud crab to a certain extent; the terpene alcohol content of 150 mg / L may be too low and cannot fully activate the physiological processes related to growth and immunity in the mud crab; the terpene alcohol content of 350 mg / L may be too high and have a certain negative impact on the physiological functions of the mud crab, such as overstimulating the endocrine system, which leads to the inhibition of growth and recovery processes;

[0265] Components 19 and 20 were superior to control group 4: Compared with control group 4, components 18 and 4 contained a certain amount of terpene alcohols, which can activate the growth and immune regulation mechanisms in the mud crab. Terpene alcohols promoted the absorption of nutrients by the mud crab and improved the utilization rate of nutrients. At the same time, they may have activated the activity of immune cells and enhanced the immune defense ability of the mud crab, thereby improving the survival rate. In terms of energy metabolism, terpene alcohols may have regulated the intracellular energy metabolism pathways, making energy production and utilization more efficient, providing power for the growth and recovery of the mud crab. Therefore, they were superior to control group 4 without terpene alcohols in terms of growth indicators.

[0266] 2. Analysis of Nutritional Transport Indicators

[0267] Table 17. Effects of different terpene alcohol contents in the microcirculation preparation for mud crabs on postpartum nutrient transport indicators of mud crabs.

[0268] Hepatopancreatic index (%) Gonadal index (%) Triglycerides (mmol / L) Total cholesterol (mmol / L) Sixteen groups of components <![CDATA[6.0±0.28 b ]]> <![CDATA[4.94±0.24 b ]]> <![CDATA[1.37±0.05 c ]]> <![CDATA[1.99±0.07 c ]]> Components 17 groups <![CDATA[7.4±0.29 a ]]> <![CDATA[6.46±0.29 a ]]> <![CDATA[0.87±0.03 d ]]> <![CDATA[1.56±0.06 d <!-- 22 -->]]> 18 groups <![CDATA[6.1±0.26 b ]]> <![CDATA[5.13±0.19 b ]]> <![CDATA[1.38±0.06 c ]]> <![CDATA[2.07±0.07 c ]]> 19 groups <![CDATA[5.2±0.24 c ]]> <![CDATA[4.37±0.29 c ]]> <![CDATA[1.54±0.04 b ]]> <![CDATA[2.20±0.05 b ]]> 20 groups <![CDATA[5.0±0.71 c ]]> <![CDATA[4.47±0.23 c ]]> <![CDATA[1.56±0.02 b ]]> <![CDATA[2.25±0.04 b ]]> Four control groups <![CDATA[3.2±0.23 d ]]> <![CDATA[2.66±0.25 d ]]> <![CDATA[1.74±0.04 a ]]> <![CDATA[2.46±0.05 a ]]>

[0269] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0270] Table 17 shows that the hepatopancreatic index and gonadal index were highest in group 17, decreasing from group 17 to control group 4. Conversely, the triglyceride and total cholesterol levels were lowest in group 17, increasing from group 17 to control group 4. The significance of differences between groups was indicated by letters; the same letter indicates no significant difference.

[0271] Principle Analysis: Component 17 is superior to components 16 and 18: 250 mg / L of terpene alcohol may have played an optimal regulatory role in the nutrient transport and distribution system of mud crabs; in the hepatopancreas, it may have promoted the expression of genes related to glycogen synthesis and storage, increased the uptake and conversion capacity of hepatopancreatic cells of nutrients, thereby improving the hepatopancreatic index. For the gonads, it may have stimulated signaling pathways related to gonadal cell proliferation and differentiation, promoting gonadal development and increasing the gonadal index. In terms of lipid regulation, it may have lowered the levels of triglycerides and total cholesterol in the blood by regulating the activity of key enzymes in lipid metabolism, such as upregulating the activity of lipases to promote triglyceride breakdown, while downregulating the activity of cholesterol synthases to reduce total cholesterol synthesis, thus optimizing the transport and distribution of nutrients in the body.

[0272] Components 16 and 18 were superior to those 19 and 20: Terpenoid content of 200 mg / L and 300 mg / L still promoted normal regulation of nutrient transport and metabolism to some extent; 150 mg / L was insufficient to induce the expression of hepatic glycogen synthase in the hepatopancreas, leading to insufficient storage capacity of nutrients in the hepatopancreas and a lower hepatopancreatic index. In the gonads, it failed to adequately stimulate gonadal development, resulting in a low gonadal index; simultaneously, its regulatory effect on lipid metabolism enzymes was weak, leading to higher triglyceride and total cholesterol levels; 350 mg / L may have overactivated certain feedback inhibition mechanisms or interfered with normal cellular metabolism, for example, it may have inhibited the normal activity regulation of lipid metabolism enzymes, leading to lipid metabolism disorders, which also resulted in unsatisfactory nutrient transport and metabolic indicators.

[0273] Components 19 and 20 were superior to the control group 4: Terpenoids can promote nutrient transport and metabolism in mud crabs; in the hepatopancreas and gonads, they may have promoted the expression of nutrient transport proteins on cell membranes, increasing cellular uptake of nutrients, such as promoting the uptake of glucose, amino acids, and other nutrients by hepatopancreas cells, thus increasing the hepatopancreas index. In the gonads, they promoted the absorption of nutrients by gonadal cells, promoted gonadal development, and increased the gonadal index; in terms of lipid regulation, they reduced the synthesis of triglycerides and total cholesterol or promoted their catabolism, making components 19 and 20, which contain terpenoids, superior to the control group 4 without terpenoids in nutrient transport-related indicators.

[0274] 3. Analysis of indicators related to metabolic waste accumulation

[0275] Table 18. Effects of different terpene alcohol contents in the microcirculation preparation for mud crabs on indicators related to post-feeding metabolic waste accumulation in mud crabs.

[0276] Blood urea nitrogen (mg / dL) Ammonia (mg / L) Uric acid (mg / dL) Antimicrobial peptides (μg / mL) Lysozyme (U / mL) Sixteen groups of components <![CDATA[4.47±0.09 c ]]> <![CDATA[0.86±0.04 c ]]> <![CDATA[4.56±0.29 c ]]> <![CDATA[165.3±9.5 b ]]> <![CDATA[157.7±9.5 b ]]> Components 17 groups <![CDATA[3.42±0.38 d ]]> <![CDATA[0.67±0.03 d ]]> <![CDATA[4.09±0.09 d ]]> <![CDATA[172.9±11.4 a ]]> <![CDATA[168.2±11.4 a ]]> 18 groups <![CDATA[4.28±0.29 c ]]> <![CDATA[0.86±0.06 c ]]> <![CDATA[4.66±0.09 c ]]> <![CDATA[166.3±12.4 b ]]> <![CDATA[155.6±10.5 b ]]> 19 groups <![CDATA[5.23±0.29 b ]]> <![CDATA[1.33±0.04 b ]]> <![CDATA[5.23±0.09 b ]]> <![CDATA[154.9±9.5 c ]]> <![CDATA[144.4±6.6 c ]]> 20 groups <![CDATA[5.04±0.09 b ]]> <![CDATA[1.22±0.03 b ]]> <![CDATA[5.32±0.19 b ]]> <![CDATA[156.8±13.3 c ]]> <![CDATA[141.6±8.6 c ]]> Four control groups <![CDATA[6.27±0.09 a ]]> <![CDATA[1.62±0.03 a ]]> <![CDATA[5.99±0.09 a ]]> <![CDATA[139.7±7.6 d ]]> <![CDATA[116.9±6.6 d ]]>

[0277] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0278] Table 18 shows that the contents of urea nitrogen, ammonia, and uric acid, from low to high, are: Group 17 < Group 16 ≈ Group 18 < Group 19, Group 20 < Control Group 4; the contents of antimicrobial peptides and lysozyme, from high to low, are: Group 17 > Group 16, Group 18 > Group 19, Group 20 > Control Group 4.

[0279] Principle Analysis:

[0280] Component 17 was superior to components 16 and 18: 250 mg / L of terpene alcohol most effectively promoted the excretion of metabolic waste and enhanced immune function in mud crabs. Regarding nitrogen metabolism, it may accelerate the conversion of ammonia to urea by regulating the activity of urea cycle-related enzymes in the liver, such as arginase and ornithine carbamoyltransferase, thereby reducing the levels of urea nitrogen and ammonia in the blood. For uric acid, it may promote renal excretion, possibly by regulating the reabsorption and secretion mechanisms of uric acid in the renal tubules, thus reducing uric acid accumulation in the body. In terms of immune regulation, 250 mg / L of terpene alcohol may activate signaling pathways such as NF-κB in immune cells, promoting the transcription and translation of antimicrobial peptide and lysozyme genes, resulting in the secretion of more antimicrobial peptides and lysozyme by immune cells, thereby enhancing the immune defense capabilities of mud crabs.

[0281] Components 16 and 18 were superior to those 19 and 20: terpenoids at 200 mg / L and 300 mg / L could better maintain the metabolic and immune balance of mud crabs; a content of 150 mg / L might be insufficient, failing to effectively increase the activity of urea cycle enzymes in the liver, resulting in ammonia not being converted into urea for excretion in a timely manner, leading to increased urea nitrogen and ammonia levels in the blood; in immune cells, it could not fully activate immune signaling pathways, resulting in lower secretion of antimicrobial peptides and lysozyme; a content of 350 mg / L might have excessive or abnormal effects on metabolism and immune regulation, for example, it might interfere with the normal renal excretion regulation mechanism of uric acid, or overactivate the immune feedback inhibition mechanism, leading to decreased immune cell activity and reduced secretion of antimicrobial peptides and lysozyme, which is also detrimental to the clearance of metabolic waste and the maintenance of immune function.

[0282] Components 19 and 20 were superior to control group 4: Terpenoids can initiate the excretion of metabolic waste and immune regulation mechanisms in mud crabs. In terms of metabolism, they may activate intracellular metabolic regulatory signaling pathways by binding to receptors on the surface of hepatocytes and kidney cells, promoting the processing and excretion of metabolic waste, such as promoting the metabolic conversion of ammonia by hepatocytes and the excretion of urea nitrogen and uric acid by kidney cells. In terms of immunity, terpenoids may activate immune cells, such as macrophages and lymphocytes, and promote their secretion of antimicrobial peptides and lysozyme, so that components 19 and 20 containing terpenoids are superior to control group 4 without terpenoids in terms of indicators related to metabolic waste accumulation.

[0283] 4. Analysis of related indicators of fat and energy storage

[0284] Table 19. Effects of different terpene alcohol contents in the microcirculation preparation for mud crabs on postpartum fat and energy reserves in mud crabs.

[0285]

[0286] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0287] As shown in Table 19, among the indicators related to fat and energy reserves, component group 17 had the highest HDL content and the lowest LDL content, with the strongest FAS and CPT activities. From component group 17 to control group 4, HDL content gradually decreased, LDL content gradually increased, and FAS and CPT activities gradually weakened.

[0288] Principle Analysis:

[0289] Component 17 was superior to components 16 and 18: 250 mg / L of terpene alcohol may have the best effect on the fat and energy reserves of mud crabs through multiple mechanisms, thereby increasing the expression of HDL, LDL, FAS and CPT, increasing fatty acid synthesis, and storing energy for the body.

[0290] Components 16 and 18 were superior to those 19 and 20: 200 mg / L and 300 mg / L of terpenoids could maintain the homeostasis of fat and energy metabolism to a certain extent; the 150 mg / L content may be insufficient, resulting in lower FAS and CPT activity and low efficiency in fat synthesis and energy conversion. 350 mg / L of terpenoids inhibited fat synthesis and also caused an imbalance in the regulation of HDL and LDL metabolism, which was detrimental to fat and energy storage and normal metabolism.

[0291] Components 19 and 20 were superior to control group 4: Terpenoids can initiate the basic regulatory mechanisms of fat and energy metabolism in mud crabs. For example, they can directly or indirectly act on PPAR family transcription factors. PPARα can regulate the expression of genes such as fatty acid transporters, fatty acid binding proteins, and CPT-1, promoting fatty acid uptake and oxidation. PPARγ can regulate genes such as FAS to participate in fatty acid synthesis. In terms of HDL and LDL metabolism, terpenoids may optimize fat metabolism by regulating the activity of related metabolic enzymes and transporters in the liver and peripheral tissues, increasing HDL content and decreasing LDL levels. Therefore, components 19 and 20 containing terpenoids were superior to control group 4 without terpenoids in these indicators.

[0292] 5. Analysis of tissue repair-related indicators

[0293] Table 20. Effects of different terpene alcohol contents in the microcirculation preparation for mud crabs on relevant indicators of postpartum tissue repair in mud crabs.

[0294]

[0295] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0296] As shown in Table 20, among the tissue repair-related indicators, the components in group 17 had the highest levels of hydroxyproline, EGF, PDGF, and TGF-β, indicating their strongest tissue repair ability. From group 17 to the control group 4, the values ​​of each indicator gradually decreased.

[0297] Principle Analysis:

[0298] Component 17 was superior to components 16 and 3: 250 mg / L terpene alcohol significantly promoted postpartum tissue repair in mud crabs. Regarding hydroxyproline synthesis, it may promote the activity of proline hydroxylase in fibroblasts. This enzyme hydroxylates proline to hydroxyproline, a key step in collagen synthesis, thereby increasing hydroxyproline content and promoting collagen fiber formation and tissue repair. For EGF, terpene alcohol may activate the EGFR-Ras-Raf-MEK-ERK signaling cascade in epidermal cells, promoting EGF gene transcription and translation, leading to increased EGF secretion from epidermal cells, accelerating epidermal cell proliferation, migration, and differentiation, and promoting wound healing. Regarding PDGF secretion, terpene alcohol may regulate the PI3K-Akt signaling pathway in related cells such as platelets and macrophages, prompting these cells to secrete more PDGF, recruiting fibroblasts and smooth muscle cells to the injury site, and promoting cell proliferation and extracellular matrix synthesis. For TGF-β, terpenoids may activate the Smad signaling pathway by binding to TGF-β receptors on the cell surface, thereby enhancing the expression and secretion of TGF-β, regulating cell proliferation, differentiation, and the synthesis and remodeling of the extracellular matrix, and comprehensively promoting tissue repair.

[0299] Components 16 and 18 were superior to those 19 and 20: terpenoids at 200 mg / L and 300 mg / L promoted tissue repair; 150 mg / L may be insufficient, leading to limited synthesis of hydroxyproline and limited secretion of EGF, PDGF, and TGF-β, thus affecting the tissue repair process; 350 mg / L of terpenoids may interfere with intracellular signal transduction, such as overactivating certain signaling pathways and triggering feedback inhibition, resulting in reduced secretion of related growth factors, or having toxic effects on normal cell metabolism, thus inhibiting tissue repair.

[0300] Components 19 and 20 were superior to the control group 4: Terpenoids can promote tissue repair in mud crabs, promote the expression of collagen genes in fibroblasts, and increase hydroxyproline content. Simultaneously, they stimulate epidermal cells and platelets to secrete growth factors such as EGF, PDGF, and TGF-β, initiating a cellular cascade reaction for tissue repair, promoting cell proliferation, migration, and extracellular matrix synthesis. Therefore, components 19 and 20, containing terpenoids, outperformed the control group 4 (which did not contain terpenoids) in tissue repair-related indicators.

[0301] Example 5: Concentration Experiment of Four Components: Rosmarinic Acid, Sargenin, Oleurotin, and Terpenol

[0302] I. Preparation of Mixed Reagents

[0303] First, weigh out the following five groups of plant materials:

[0304] 22.5g rosemary petals, 17.5g sage leaves, 17.5g olive leaves, 12.5g perilla leaves;

[0305] 25g rosemary petals, 20g sage leaves, 20g olive leaves, 15g perilla leaves;

[0306] 27.5g rosemary petals, 22.5g sage leaves, 22.5g olive leaves, 17.5g perilla leaves;

[0307] 30g rosemary petals, 25g sage leaves, 25g olive leaves, 20g perilla leaves;

[0308] 32.5 grams of rosemary petals, 27.5 grams of sage leaves, 27.5 grams of olive leaves, and 22.5 grams of perilla leaves were used to prepare five groups of mixed reagents with different concentrations, following steps one through three of Example 1. The auxiliary solution was designated as the blank control group, and the concentrations were denoted as follows:

[0309] Component 21: 300 mg / L rosmarinic acid + 250 mg / L caryopsisic acid + 250 mg / L oleuropein + 200 mg / L terpene alcohol,

[0310] Component group 22: 350 mg / L rosmarinic acid + 300 mg / L caryopsisic acid + 300 mg / L oleuropein + 250 mg / L terpene alcohol,

[0311] Component group 23: 400 mg / L rosmarinic acid + 350 mg / L caryopsisic acid + 350 mg / L oleuropein + 300 mg / L terpene alcohol,

[0312] Composition 24: 200 mg / L rosmarinic acid + 200 mg / L sauropic acid + 200 mg / L oleuropein + 150 mg / L terpene alcohol,

[0313] Component group 25: 500 mg / L rosmarinic acid + 400 mg / L caryopsisic acid + 400 mg / L oleuropein + 350 mg / L terpene alcohol,

[0314] Control group 5: auxiliary solution (blank group).

[0315] II. Aquaculture Experiment

[0316] From March to April 2024, the experiment was conducted in indoor culture ponds at Dongying Kainuo Aquaculture Co., Ltd. Ninety female mud crabs, 1-3 days postpartum, were collected again from the southern coastal region. After being transported by air to the experimental site and centrally disinfected, they were temporarily held in two 30m² indoor cement plants for 1-2 days. After temporary holding, five mud crabs were placed in 18 20m² laboratory ponds for the experiment. Five postpartum female crabs were placed in each pond, along with five pieces of tile. Ponds 1-3 contained component group 21, ponds 4-6 contained component group 22, ponds 7-9 contained component group 23, ponds 10-12 contained component group 24, ponds 13-15 contained component group 25, and ponds 16-18 served as the control group (group 5). The purpose of this embodiment was to explore key compounds in a postpartum microcirculation preparation for promoting the recovery of mud crabs after spawning. The experimental materials and sources of this embodiment were the same as in Example 1.

[0317] III. Comparison of Experimental Results

[0318] 1. Analysis of growth index results

[0319] Table 21 Effects of different sarcophic acid contents in microcirculation preparations on growth indicators of mud crabs

[0320]

[0321]

[0322] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0323] Table 21 shows that the survival rate (86.95±1.96%), weight gain rate (46.03±1.28%), and condition factor (62.21±1.63 g / cm³) of component 22 were... 3 The highest performance was observed in group 22, with the shortest recovery time (17.44±0.55 days). Groups 21 and 23 showed the next best performance, with no significant difference between them. Groups 24 and 25 performed poorly, while control group 5 performed the worst. The indicators of each group showed a decreasing trend from group 22 to control group 5.

[0324] Principle Analysis: The advantage of group 22 likely stems from the suitable component ratio (350 mg / L rosmarinic acid + 300 mg / L sarsaparilla acid + 300 mg / L oleuropein + 250 mg / L terpene alcohol), which may maximize the promotion of growth and metabolism in mud crabs. Rosmarinic acid and sarsaparilla acid enhance cell metabolism and proliferation; oleuropein may improve nutrient utilization by enhancing intestinal absorption and metabolism; and terpene alcohol further enhances growth efficiency. In contrast, excessively high (e.g., group 25) or excessively low (e.g., group 24) component concentrations may lead to increased metabolic stress or insufficient nutrient supply, limiting growth efficiency. The control group 5, lacking effective bioactive components, showed significantly lower growth indicators than the experimental groups.

[0325] Table 22 Effects of different component contents in the microcirculation preparation for mud crabs on postpartum nutrient transport indicators of mud crabs.

[0326]

[0327] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0328] As shown in Table 22, group 22 had the highest hepatopancreatic index (8.24±0.24%) and gonadal index (7.32±0.33%), and the lowest triglyceride (0.71±0.12mmol / L) and total cholesterol (1.45±0.04mmol / L); groups 21 and 23 were next; groups 24 and 25 showed significant decreases in their indices, and control group 5 was the worst.

[0329] Principle Analysis: The advantage of group 22 likely stems from the rational combination of its components, synergistically regulating nutrient metabolism pathways to optimize nutrient transport and storage efficiency. Rosmarinic acid and sarsaparilla acid may improve the nutrient absorption and storage capacity of hepatocellular, pancreatic, and gonadal cells by enhancing vascular permeability and blood circulation. Oleuropein may reduce serum triglyceride and total cholesterol levels by inhibiting HMG-CoA reductase activity and decreasing cholesterol synthesis. Terpenoids may enhance the cell membrane's ability to absorb nutrients by regulating lipid metabolism and membrane protein activity. In contrast, inappropriate component concentrations (such as group 24 and group 25) may cause metabolic burden and reduce nutrient transport efficiency. The control group 5, lacking active ingredients, had the worst nutrient transport-related indicators.

[0330] Table 23. Effects of different component contents in the microcirculation preparation for mud crabs on indicators related to post-feeding metabolic waste accumulation in mud crabs.

[0331]

[0332] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0333] As shown in Table 23, group 22 had the lowest levels of urea nitrogen (2.84±0.18 mg / dL), ammonia (0.32±0.02 mg / L), and uric acid (3.24±0.22 mg / dL), while it had the highest levels of antimicrobial peptides (186.9±12 μg / mL) and lysozyme (183.3±10.4 U / mL). Groups 21 and 23 were next in these levels. Groups 24 and 25 showed significant accumulation of metabolic waste, with control group 5 being the most severely affected.

[0334] Principle Analysis: The advantages of group 22 may stem from the dual regulation of metabolic enzymes and the immune system by the components. Rosmarinic acid and sarsaparilla acid may promote the excretion of ammonia and uric acid and reduce the accumulation of metabolic waste by enhancing the activity of ornithine transaminase and urea cycle enzymes in the liver; oleuropein may accelerate the excretion of metabolic waste by enhancing kidney function; terpenoids enhance the body's ability to fight infection and clear toxins by stimulating immune cells to secrete antimicrobial peptides and lysozyme; inappropriate components (such as group 24 and group 25) may lead to increased metabolic stress or impaired immune function, affecting waste clearance; control group 5 lacks metabolic and immune regulatory mechanisms and has the most severe waste accumulation.

[0335] Table 24. Effects of different component contents in the microcirculation preparation for mud crabs on postpartum fat and energy reserves in mud crabs.

[0336]

[0337]

[0338] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0339] As shown in Table 24, group 22 had the highest HDL (68.14±3.11 mg / dL) and CPT activity (3.27±0.11 U / mg), the lowest LDL (101.8±2.9 mg / dL), and the strongest FAS activity (2.13±0.42 U / mg); groups 21 and 23 were next; groups 24 and 25 showed significant decreases in their indicators, and control group 5 was the worst.

[0340] Mechanism analysis: The synergistic effect of component group 22 is manifested in the following ways: rosmarinic acid and sarsaparilla acid jointly inhibit ACC activity and promote fatty acid β-oxidation; terpenoids activate the PPARα pathway and balance fat synthesis and decomposition; oleuropein enhances HDL synthesis; the fat metabolism efficiency of components group 21 and 23 is partially limited due to the deviation of some component concentrations; the concentration imbalance of components group 24 and 25 leads to reduced HDL synthesis and LDL accumulation; the control group 5 lacks regulatory mechanisms and exhibits disordered fat metabolism.

[0341] Table 25. Effects of different component contents in the microcirculation preparation for mud crabs on relevant indicators of postpartum tissue repair in mud crabs.

[0342]

[0343] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0344] As shown in Table 25, the contents of hydroxyproline (7.82±0.42μg / g), EGF (52.21±2.03pg / mL), PDGF (132.7±4.1pg / mL), and TGF-β (222.7±8.8pg / mL) were highest in group 22; followed by groups 21 and 23; the repair capacity of groups 24 and 25 was significantly weakened, and the control group 5 was the worst.

[0345] Mechanism Analysis: The complex components of Component 22 promote tissue repair through multiple synergistic pathways: rosmarinic acid activates the TGF-β / Smad pathway, sagebate promotes EGF secretion, oleuropein enhances PDGF recruitment, and terpenoids regulate cell proliferation and differentiation. Components 21 and 23, due to concentration discrepancies in some components, did not achieve optimal levels of repair factor secretion. Components 24 and 25, due to concentration imbalances, resulted in limited repair factor secretion. The control group (Group 5) lacked active ingredients to initiate the repair mechanism, and all indicators showed a lag.

[0346] Example 6: Postpartum farming method for mud crabs 1

[0347] The method for improving microcirculation function in female mud crabs after spawning, as described in this embodiment, includes the following steps:

[0348] I. Microcirculation Preparations for Mud Crabs After Laying

[0349] Terpenoids, phenols, and flavonoids were extracted from natural plants including 27.5g rosemary petals, 22.5g sage leaves, 22.5g olive leaves, 17.5g perilla leaves, 12.5g green tea leaves, 12.5g red onion, 7.5g blueberry leaves, and 7.5g grape seeds. The terpenoids and phenols included rosmarinic acid (350mg / L), sage acid (300mg / L), oleuropein (300mg / L), terpene alcohol (250mg / L), catechin (150mg / L), quercetin (150mg / L), ursolic acid (75mg / L), and proanthocyanidins (75mg / L). These substances were extracted and purified using an ultrasonic-assisted 70% ethanol extraction method to prepare a postpartum microcirculation preparation for mud crabs.

[0350] The microcirculation preparation for mud crabs contains terpenoids and phenolic compounds extracted from natural plant sources such as rosemary petals, sage leaves, olive leaves, perilla leaves, green tea leaves, red onions, blueberry leaves, and grape seeds. These compounds include active ingredients such as rosmarinic acid, sage acid, oleuropein, terpene alcohols, catechins, quercetin, ursolic acid, and proanthocyanidins. By influencing hepatopancreatic index, gonadal index, and other related indicators, these compounds can effectively promote the transport of nutrients in female mud crabs after spawning and alleviate physiological disorders caused by the accumulation of metabolic waste, lack of fat and energy reserves, and weakened tissue repair function.

[0351] The specific steps for preparing a postpartum microcirculation preparation for mud crabs are as follows:

[0352] Step 1: Preparation of plant materials

[0353] (1) Ingredients: Weigh out 27.5 parts rosemary petals, 22.5 parts sage leaves, 22.5 parts olive leaves, 17.5 parts perilla leaves, 17.5 parts green tea leaves, 12.5 parts red onion, 7.5 parts blueberry leaves, and 7.5 parts grape seeds. (The plant materials can be purchased from the market.)

[0354] (2) Grinding: The above plant materials were coarsely ground using a grinder (model: LD-1300A high-speed universal grinder, Zhejiang Wenling Linda Machinery Co., Ltd.), with the particle size controlled to be 0.5-1 mm. The ground material was then placed into a dry 100ml sealed glass beaker (100ml round-bottom glass beaker, Shanghai Heqi Glass Instrument Co., Ltd.) for later use.

[0355] Step 2: Ultrasonic-assisted ethanol extraction

[0356] (3) Solvent preparation: Use 70% ethanol solution (commercially available) as the extraction solvent. Dispense 100ml of 70% ethanol solution into four 500ml round-bottom glass flasks (500ml round-bottom glass flasks, Shanghai Heqi Glass Instrument Co., Ltd.).

[0357] (4) Plant material preparation: Take crushed rosemary petals, sage leaves, olive leaves, perilla leaves, green tea leaves, red onion, blueberry leaves and grape seeds respectively, and add them to four 500ml round bottom flasks containing 100ml of 70% ethanol solution.

[0358] (5) Ultrasonic extraction: Place the four flasks into an ultrasonic extractor (model: YM-T2000CT multi-purpose constant temperature ultrasonic extractor, Shanghai Yuming Instrument Co., Ltd.), and set the extraction conditions according to the following parameters: ultrasonic power: 300 watts, ultrasonic frequency: 40 kHz, temperature: 50℃, extraction time: 40 minutes. After extraction, cool the extract to room temperature.

[0359] Step 3: Filtration and centrifugation

[0360] (6) Coarse filtration: The extract was initially filtered using a filter (model: MS-0.45μm polyethersulfone (PES) membrane filter, Shanghai Mosu Scientific Instruments Co., Ltd.) to remove larger particles.

[0361] (7) Centrifugation: Transfer the coarsely filtered extract to a centrifuge (model: TG16-WS benchtop high-speed centrifuge, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.), and centrifuge using the following parameters: speed: 10000 rpm, time: 15 minutes. After centrifugation, collect the supernatant and discard any remaining impurities.

[0362] Step 4: Rotary evaporation concentration

[0363] (8) Concentration: Transfer the supernatant after centrifugation to a rotary evaporator (model: RE-52AA rotary evaporator, Shanghai Yarong Biochemical Instrument Factory) for concentration. Set the following parameters: water bath temperature: 45℃, rotation speed: 80rpm. Evaporate and concentrate to approximately 50ml of concentrate to ensure that the active ingredients in the liquid are retained.

[0364] Step 5: Preparation of liquid formulation

[0365] (9) Dilution: Dilute 50ml of concentrate with purified water to 1000ml, stir thoroughly to make a postpartum microcirculation preparation for blue crabs.

[0366] (10) Storage: Dispense the prepared mud crab postpartum microcirculation preparation into 2L transparent glass containers and seal them for storage. The containers should be kept away from light and placed in a refrigerator at 4°C to prevent degradation of the active ingredients.

[0367] II. Auxiliary Fluid

[0368] The postpartum microcirculation preparation for mud crabs needs to be used simultaneously with an auxiliary solution. This auxiliary solution contains 7g of Clostridium butyricum, 5g of L-arginine, 3g of vitamin C, and 5ml of 1×phosphate buffer (1×PBS), which aims to promote the effective absorption of the postpartum microcirculation preparation by mud crabs and maintain water quality stability.

[0369] Experimental materials: Clostridium butyricum: 7g, L-arginine: 5g, vitamin C: 3g, phosphate buffered saline (PBS): 4-6ml; 500mL glass beaker (model: GG-17, Shanghai Guanghua Glass Instrument Co., Ltd.). The glass beaker was placed in an autoclave (DXL-100S automatic high-pressure steam sterilizer, Shandong Dexiang Instrument Co., Ltd.) for high-temperature and high-pressure sterilization, and then set aside for later use.

[0370] Step 2: Preparation of auxiliary solution

[0371] (11) Dissolving Clostridium butyricum: In a sterile laminar flow hood (model: BSC-1100IIA2-X, Guangdong Keyipu Laboratory Equipment Research and Development Co., Ltd.), take 7g of Clostridium butyricum powder and add 16.5ml of sterile purified water; use an electric stir bar (model: IKA RW20 Digital Dissolver, Beijing Labtech Instrument Co., Ltd.) to stir at medium speed for 4 minutes to ensure that Clostridium butyricum is completely dissolved and forms a uniform suspension.

[0372] (12) Add L-arginine and vitamin C: Dissolve 5g of L-arginine in 12.5ml of sterile water and stir for 4 minutes until completely dissolved. Then dissolve 3g of vitamin C in 10ml of sterile water and stir for 3 minutes to ensure that the solution is transparent and free of precipitate. Finally, add the two solutions to the butyric acid clostridium suspension while stirring slowly until well mixed.

[0373] (13) Add PBS buffer: Take 5 ml of PBS buffer and slowly add it to the above mixture. Continue stirring for 4 minutes to ensure that all components are fully dissolved and mixed evenly to form a stable solution.

[0374] Step 3: Filtration and Aseptic Processing

[0375] (14) Filtration: The mixed solution was aseptically filtered through a 0.22μm needle filter (model: Syringe Filters 0.22μm, Zhejiang Saifen Technology Co., Ltd.) to remove bacteria, fungal spores and other microbial contaminants.

[0376] (15) Dispensing and storage: Dispense the filtered auxiliary solution into 500mL sterile transparent glass cups (model: GG-17, Shanghai Guanghua Glass Instrument Co., Ltd.), seal and store in a refrigerator at 4℃, away from light to prevent degradation of active ingredients.

[0377] III. Mixed Formulations

[0378] The ratio of the microcirculation preparation to the auxiliary solution for spawning mud crabs is 5:1, that is, for every 5ml of microcirculation preparation, 1ml of auxiliary solution needs to be added for mixing to obtain 6ml of mixed preparation.

[0379] IV. Steps for staged application of mixed formulations

[0380] The dosage and application method of the postpartum microcirculation preparation and auxiliary solution for mud crabs need to be adjusted according to the mud crab's farming stage. The specific adjustment plan is as follows:

[0381] Phase 1 (3 to 7 days postpartum)

[0382] Application time: 6:00 PM daily;

[0383] Application frequency: once daily;

[0384] Dosage: Add 10-15 ml of the mixed preparation per cubic meter of water; preferably 12.5 ml.

[0385] Phase Two (8 to 14 days postpartum)

[0386] Application time: 8:00 PM (8:00 PM) and 5:00 AM (05:00 AM) daily;

[0387] Application frequency: twice daily;

[0388] Dosage: Add 15-20 ml of the mixed preparation per cubic meter of water each time; preferably 17.5 ml.

[0389] Phase 3 (15 to 20 days postpartum)

[0390] Application time: 8:00 PM (8:00 PM) and 5:00 AM (05:00 AM) daily;

[0391] Application frequency: twice daily;

[0392] Dosage: Add 20-30 ml of the mixed preparation per cubic meter of water each time, preferably 25 ml.

[0393] Example 7: Postpartum farming method for mud crabs 2

[0394] The breeding method is basically the same as that in Example 1, except that:

[0395] I. Microcirculation Preparations for Mud Crabs After Laying

[0396] Terpenoids, phenols, and flavonoids were extracted from natural plants including 25g rosemary petals, 20g sage leaves, 20g olive leaves, 15g perilla leaves, 10g green tea leaves, 10g red onion, 5g blueberry leaves, and 5g grape seeds. Among these, the terpenoids, phenols, and flavonoids contained 300mg / L rosmarinic acid, 250mg / L salvia oleuropein, 200mg / L terpene alcohol, 100mg / L catechin, 100mg / L quercetin, 50mg / L ursolic acid, and 50mg / L proanthocyanidins. These substances were extracted and purified using an ultrasonic-assisted 70% ethanol extraction method to prepare a postpartum microcirculation preparation for mud crabs.

[0397] The specific steps for configuration are as follows:

[0398] Step 1: Preparation of plant materials

[0399] (1) Ingredients: Weigh out 25g rosemary petals, 20g sage leaves, 20g olive leaves, 15g perilla leaves, 10g green tea leaves, 10g red onion, 5g blueberry leaves, and 5g grape seeds. (The plant materials can be purchased from the market.)

[0400] II. Auxiliary Fluid

[0401] The postpartum microcirculation preparation for mud crabs needs to be used simultaneously with an auxiliary solution. This auxiliary solution contains 6g of Clostridium butyricum, 4g of L-arginine, 2g of vitamin C, and 4ml of 1×phosphate buffer (1×PBS), which aims to promote the effective absorption of the postpartum microcirculation preparation by mud crabs and maintain water quality stability.

[0402] Experimental materials: Clostridium butyricum: 6g, L-arginine: 4g, vitamin C: 2g, phosphate-buffered saline (PBS): 4ml;

[0403] Step 2: Preparation of auxiliary solution

[0404] (11) Dissolving Clostridium butyricum: In a sterile laminar flow hood (model: BSC-1100IIA2-X, Guangdong Keyipu Laboratory Equipment Research and Development Co., Ltd.), take 6g of Clostridium butyricum powder and add 8ml of sterile purified water; use an electric stir bar (model: IKARW20 Digital Dissolver, Beijing Labtech Instrument Co., Ltd.) to stir at medium speed for 3 to 5 minutes to ensure that Clostridium butyricum is completely dissolved and forms a uniform suspension.

[0405] (12) Add L-arginine and vitamin C: Dissolve 4g of L-arginine in 10ml of sterile water and stir for 3-5 minutes until completely dissolved. Then dissolve 2g of vitamin C in 8ml of sterile water and stir for 2-4 minutes to ensure that the solution is transparent and free of precipitate. Finally, add the two solutions to the butyric acid clostridium suspension while stirring slowly until well mixed.

[0406] (13) Add PBS buffer: Take 4 ml of PBS buffer and slowly add it to the above mixture. Continue stirring for 3 to 5 minutes to ensure that all components are fully dissolved and mixed evenly to form a stable solution.

[0407] Example 8: Postpartum farming method for mud crabs 3

[0408] The breeding method is basically the same as that in Example 1, except that:

[0409] I. Microcirculation Preparations for Mud Crabs After Laying

[0410] Terpenoids, phenols, and flavonoids were extracted from natural plants including 30g rosemary petals, 25g sage leaves, 25g olive leaves, 20g perilla leaves, 15g green tea leaves, 15g red onion, 10g blueberry leaves, and 10g grape seeds. Among these, the terpenoids, phenols, and flavonoids contained 400mg / L rosmarinic acid, 350mg / L salvia oleuropein, 300mg / L terpene alcohol, 200mg / L catechin, 200mg / L quercetin, 100mg / L ursolic acid, and 100mg / L proanthocyanidins. These substances were extracted and purified using an ultrasonic-assisted 70% ethanol extraction method to prepare a postpartum microcirculation preparation for mud crabs.

[0411] The specific steps for configuration are as follows:

[0412] Step 1: Preparation of plant materials

[0413] (1) Ingredients: Weigh out 30g rosemary petals, 25g sage leaves, 25g olive leaves, 20g perilla leaves, 15g green tea leaves, 15g red onion, 10g blueberry leaves, and 10g grape seeds. (The plant materials can be purchased from the market.)

[0414] II. Auxiliary Fluid

[0415] The postpartum microcirculation preparation for mud crabs needs to be used simultaneously with an auxiliary solution. This auxiliary solution contains 8g of Clostridium butyricum, 6g of L-arginine, 4g of vitamin C, and 6ml of 1×phosphate buffer (1×PBS), which aims to promote the effective absorption of the postpartum microcirculation preparation by mud crabs and maintain water quality stability.

[0416] Experimental materials: Clostridium butyricum: 8g, L-arginine: 6g, vitamin C: 4g, phosphate-buffered saline (PBS): 6ml;

[0417] Step 2: Preparation of auxiliary solution

[0418] (11) Dissolving Clostridium butyricum: In a sterile laminar flow hood (model: BSC-1100IIA2-X, Guangdong Keyipu Laboratory Equipment Research and Development Co., Ltd.), take 8g of Clostridium butyricum powder and add 25ml of sterile purified water; use an electric stir bar (model: IKARW20 Digital Dissolver, Beijing Labtech Instrument Co., Ltd.) to stir at medium speed for 3-5 minutes to ensure that Clostridium butyricum is completely dissolved and forms a uniform suspension.

[0419] (12) Add L-arginine and vitamin C: Dissolve 6g of L-arginine in 10ml of sterile water and stir for 3-5 minutes until completely dissolved. Then dissolve 4g of vitamin C in 8ml of sterile water and stir for 2-4 minutes to ensure that the solution is transparent and free of precipitate. Finally, add the two solutions to the butyric acid clostridium suspension while stirring slowly until well mixed.

[0420] (13) Add PBS buffer: Take 6 ml of PBS buffer and slowly add it to the above mixture. Continue stirring for 3 to 5 minutes to ensure that all components are fully dissolved and mixed evenly to form a stable solution.

[0421] Example 9: Concentration Experiment of the Whole Components of the Postpartum Microcirculation Preparation for Blue Crabs

[0422] The purpose of this embodiment is to explore the component ratio and optimal concentration of a postpartum microcirculation preparation for promoting the recovery of mud crabs after spawning. The experimental materials and sources of this embodiment are the same as those in Embodiment 1.

[0423] I. Preparation of Postpartum Microcirculation Formulas for Mud Crabs with Different Component Ratios

[0424] 1.1 Components: Twenty-six groups: The plant content of the microcirculation preparation for postpartum crabs prepared by the ultrasonic-assisted 70% ethanol extraction method in Example 7 includes 25g of rosemary petals, 20g of sage leaves, 20g of olive leaves, 15g of perilla leaves, 10g of green tea leaves, 10g of red onion, 5g of blueberry leaves, and 5g of grape seeds. The concentrations of the compounds finally extracted according to the method of Example 7 are: rosmarinic acid 300mg / L, sage acid 250mg / L, oleuropein 250mg / L, terpene alcohol 200mg / L, catechin 100mg / L, quercetin 100mg / L, ursolic acid 50mg / L, and proanthocyanidins 50mg / L.

[0425] 1.2 Components: Twenty-seven components were extracted and purified using ultrasound-assisted 70% ethanol extraction in Example 6 to obtain a microcirculation preparation for postpartum crabs. The plant content included 27.5g of rosemary petals, 22.5g of sage leaves, 22.5g of olive leaves, 17.5g of perilla leaves, 12.5g of green tea leaves, 12.5g of red onion, 7.5g of blueberry leaves, and 7.5g of grape seeds. The concentrations of the compounds extracted according to the method in Example 6 were as follows: rosmarinic acid 350mg / L, sage acid 300mg / L, oleuropein 300mg / L, terpene alcohol 250mg / L, catechin 150mg / L, quercetin 150mg / L, ursolic acid 75mg / L, and proanthocyanidins 75mg / L.

[0426] 1.3 Components (28 groups): In Example 8, the plant content of the preparation for the postpartum microcirculation of mud crabs was extracted and purified by ultrasonic-assisted 70% ethanol extraction. The plant content included 30g of rosemary petals, 25g of sage leaves, 25g of olive leaves, 20g of perilla leaves, 15g of green tea leaves, 15g of red onion, 10g of blueberry leaves, and 10g of grape seeds. The concentrations of the compounds extracted according to the method in Example 8 were as follows: 400mg / L of rosmarinic acid, 350mg / L of salvia oleuropein, 350mg / L of oleuropein, 300mg / L of terpene alcohol, 200mg / L of catechin, 200mg / L of quercetin, 100mg / L of ursolic acid, and 100mg / L of proanthocyanidins.

[0427] II. Comparative Experiment of Different Components of Postpartum Microcirculation Preparations for Mud Crabs

[0428] From May to June 2023, the experiment was conducted in indoor culture ponds at Dongying Kainuo Aquaculture Co., Ltd. Seventy-five female mud crabs, 1-3 days postpartum, were collected from the southern coastal region, transported by air to the experimental site, and centrally disinfected. They were then temporarily held in two 30m² indoor cement plants for 1-2 days. After temporary holding, five mud crabs were placed in twelve 20m² laboratory ponds for the experiment. Five postpartum female crabs were placed in each pond, along with five pieces of tile. Ponds 1-3 contained twenty-six components, ponds 4-6 contained twenty-seven components, ponds 7-9 contained twenty-eight components, and ponds 10-12 were the control group (without using the mud crab postpartum microcirculation preparation and auxiliary solution). The specific usage methods of the mud crab postpartum microcirculation preparation and auxiliary solution for each experimental group are as follows: First stage (3-7 days postpartum): Every evening at 6 pm, 12.5 ml of the mixed preparation prepared in a 5:1 ratio was added per cubic meter of water, once a day. Phase Two (8-14 days postpartum): At 8 PM and 5 AM the following morning, add 17.5 ml of the mixed preparation (5:1 ratio) per cubic meter of water twice daily. Phase Three (15-20 days postpartum): At 8 PM and 5 AM the following morning, add 25 ml of the mixed preparation (5:1 ratio) per cubic meter of water twice daily. During the rearing period, feed with clams, razor clams, etc. Clean up all uneaten feed 3 hours after feeding. Change one-third of the water every three days, 4-5 hours after applying the postpartum microcirculation preparation and auxiliary solution. The experiment lasted 40 days. After the experiment, the survival rate, weight gain rate, fatness, and recovery time of the female crabs in each group were measured and calculated, and the groups were compared and analyzed. Recovery time was mainly measured by the following three aspects: activity level, feeding status, and growth rate. First, regarding activity levels, we observe the activity levels of the mud crabs after treatment. Generally, mud crabs that recover faster exhibit higher activity and foraging behavior. Second, regarding feeding, we meticulously record the time required for the mud crabs to resume feeding from the start of treatment. Generally, mud crabs that recover quickly can resume feeding in a shorter period. Finally, growth rate is also an important indicator of recovery time. We assess whether the mud crabs have returned to normal growth by regularly measuring growth parameters such as weight or plumpness.

[0429] Crabs were collected from each pond for analysis. First, they were weighed using an electronic balance (FA1204, Shanghai Precision Scientific Instruments Co., Ltd.) (accurate to 0.01g). Before weighing, the crabs were gently wiped dry with a towel. Then, their growth indicators were measured using vernier calipers (LG-150, Shanghai Precision Measuring & Cutting Tools Co., Ltd.). After the growth indicators were measured, the crabs were anesthetized on ice. A 2.0mL disposable sterile syringe, rinsed with anticoagulant, was used to extract 1.5mL of hemolymph sample from the base of the carapace. The hemolymph was mixed with anticoagulant at a 1:1 (volume ratio) ratio and centrifuged at 8000r / min for 10min at 4℃. The supernatant was used for hemolymph-related indicator determination.

[0430] III. Comparative Experimental Results Analysis

[0431] After the 40-day breeding experiment, it is necessary to test and analyze the growth indicators, nutrient transport-related indicators, metabolic waste accumulation-related indicators, fat and energy storage-related indicators, and tissue repair-related indicators of the female mud crabs after spawning.

[0432] 1. Analysis of growth index test results

[0433] Table 26 Effects of different compositions and dosages of postpartum microcirculation preparations on postpartum growth indicators of mud crabs.

[0434] 26 groups 27 groups 28 groups Six control groups Survival rate (%) <![CDATA[82.33±3.21 b ]]> <![CDATA[91.00±3.08 a ]]> <![CDATA[80.67±3.15 b ]]> <![CDATA[65.33±2.81 c ]]> Weight gain rate (%) <![CDATA[34.1±3.02 b ]]> <![CDATA[46.8±3.12 a ]]> <![CDATA[36.7±3.00 b ]]> <![CDATA[17.7±2.41 c ]]> <![CDATA[Condition factor (g / cm 3 )]]> <![CDATA[60.2±2.65 b ]]> <![CDATA[61.0±2.45 a ]]> <![CDATA[59.8±2.71 b ]]> <![CDATA[33.1±2.33 c ]]> Recovery time (t) <![CDATA[27.33±2.12 b ]]> <![CDATA[18.83±2.05 c ]]> <![CDATA[25.50±2.10 b ]]> <![CDATA[35.33±3.20 a ]]>

[0435] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0436] As shown in Table 26, groups 26, 27, and 28 showed significant differences from the control group 6 in survival rate, weight gain rate, condition factor, and recovery time. This indicates that within the range of effective component content parameters (rosmarinic acid 300–400 mg / L, carrageenan 250–350 mg / L, oleuropein 250–350 mg / L, terpene alcohol 200–300 mg / L, catechin 100–200 mg / L, quercetin 100–200 mg / L, ursolic acid 50–100 mg / L, and proanthocyanidins 50–100 mg / L), these components significantly promoted the post-spawning growth of mud crabs.

[0437] In addition, regarding the survival rate, group 27 (91.00% ± 3.08%) had the highest survival rate and was significantly better than other groups; in terms of weight gain rate, group 27 (46.8% ± 3.12%) also had the highest; and in terms of body condition, group 27 (61.0 ± 2.45 g / cm³) had the highest. 3The component group showed the best performance; in terms of recovery time, the 27th component group had the shortest recovery time (18.83±2.05 days); overall, the 27th component group showed the best performance in growth indicators, with the following specific advantages:

[0438] 1) Appropriate Plant Extract Content: The effective components in the postpartum microcirculation preparation for mud crabs come from plant extracts such as rosmarinic acid, carrageenan, oleuropein, terpene alcohol, catechin, quercetin, ursolic acid, and proanthocyanidins. Component 27 uses 8-10 grams of plant materials, which is more abundant than component 26 (5-7 grams), allowing it to better improve microcirculation and promote growth. While component 28 (11-13 grams) contains more plant materials, it may have issues such as excessively high extract concentrations or imbalances in other component ratios, affecting its effectiveness. For example, catechins and proanthocyanidins in plant extracts can regulate metabolism and promote growth; appropriate levels are beneficial for the growth and development of mud crabs, while too much or too little is detrimental.

[0439] 2) Matching the physiological needs of mud crabs: Mud crabs need to recover their bodily functions after spawning. Appropriate doses of plant extracts can better meet their nutritional needs, promote metabolism, increase weight gain and plumpness, enhance immunity, improve survival rate and shorten recovery time. The amount of plant raw materials in the twenty-seven groups is more in line with the physiological recovery needs of mud crabs after spawning, while other groups cannot achieve such ideal results.

[0440] 2. Analysis of nutrient delivery index test results

[0441] Table 27 Effects of different compositions and dosages of postpartum microcirculation preparations on postpartum nutrient transport indicators in mud crabs.

[0442]

[0443]

[0444] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0445] As shown in Table 27, the hepatopancreatic index, gonadal index, triglycerides, and total cholesterol of components 26, 27, and 28 were significantly different from those of control group 6. This indicates that the effective components of each component have a significant promoting effect on the post-spawning nutrient transport of mud crabs within the range of their effective component content parameters.

[0446] Furthermore, in terms of hepatopancreatic and gonadal indices, group 27 (8.4% ± 0.25% and 7.3% ± 0.30%) were significantly higher than other groups; while in terms of triglyceride and total cholesterol levels, group 27 (0.82 ± 0.04 mmol / L and 1.52 ± 0.05 mmol / L) were the lowest and significantly lower than other groups. This indicates that group 27 performed best in nutrient transport-related indicators, and had a significant effect on promoting hepatopancreatic and gonadal function and optimizing lipid metabolism. Specific advantages are as follows:

[0447] 3) Excellent synergistic effect of active ingredients: Many components in the plant extracts, such as rosmarinic acid and proanthocyanidins, regulate lipid metabolism, promote nutrient absorption, and aid in organ function recovery. The proportions of plant materials in the twenty-seven groups ensure a relatively suitable ratio of these active ingredients, resulting in better synergistic effects. This more effectively promotes the digestion, absorption, and storage of nutrients by the hepatopancreas, increasing the hepatopancreatic index; it also contributes to gonadal development and increases the gonadal index. Other groups may have had less balanced component ratios, affecting the effectiveness of this synergistic effect.

[0448] 4) Reduced metabolic burden: Appropriate plant extract content helps lower triglyceride and total cholesterol levels, reducing the metabolic burden on the crab and enabling more efficient transport and utilization of nutrients, thus promoting the recovery of overall physiological functions. Component 27 performed exceptionally well in this regard; other components may not have achieved the same metabolic regulatory effect due to inappropriate amounts of plant materials.

[0449] 3. Analysis of the test results of metabolic waste accumulation indicators

[0450] Table 28 Effects of different compositions and dosages of postpartum microcirculation preparations on indicators related to postpartum metabolic waste accumulation in mud crabs.

[0451] 26 groups 27 groups 28 groups Six control groups Blood urea nitrogen (mg / dL) <![CDATA[3.3±0.2 b ]]> <![CDATA[2.8±0.1 c ]]> <![CDATA[3.4±0.2 b ]]> <![CDATA[5.9±0.3 a ]]> Ammonia (mg / L) <![CDATA[1.0±0.1 b ]]> <![CDATA[0.6±0.1 c ]]> <![CDATA[1.1±0.1 b ]]> <![CDATA[1.9±0.2 a ]]> Uric acid (mg / dL) <![CDATA[4.6±0.2 b ]]> <![CDATA[4.1±0.1 c ]]> <![CDATA[4.8±0.2 b ]]> <![CDATA[6.8±0.3 a ]]> Antimicrobial peptides (μg / mL) <![CDATA[182±10 b ]]> <![CDATA[195±12 a ]]> <![CDATA[185±11 b ]]> <![CDATA[155±8 c ]]> Lysozyme (U / mL) <![CDATA[168±10 b ]]> <![CDATA[180±12 a ]]> <![CDATA[171±9 b ]]> <![CDATA[125±7 c ]]>

[0452] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0453] As shown in Table 28, the components 26, 27, and 28 showed significant differences from the control group 6 in terms of urea nitrogen, ammonia, uric acid, antimicrobial peptides, and lysozyme. This indicates that within the range of effective component content parameters, the components have a significant promoting effect on the removal of metabolic waste after spawning in mud crabs.

[0454] Furthermore, among the indicators related to metabolic waste accumulation, the levels of urea nitrogen, ammonia, and uric acid in group 27 (2.8±0.1 mg / dL, 0.6±0.1 mg / L, and 4.1±0.1 mg / dL, respectively) were the lowest and significantly lower than other groups; while the levels of antimicrobial peptides and lysozyme in group 27 (195±12 μg / mL and 180±12 U / mL, respectively) were the highest and significantly higher than other groups. This indicates that group 27 can most effectively reduce the accumulation of metabolic waste in the mud crab and enhance its immune function, with the following specific advantages:

[0455] 5) Regulating the balance of metabolism and immune function: Quercetin has antioxidant, anti-inflammatory, and immunomodulatory effects. The appropriate amount of plant materials in the twenty-seven components allows these components to better regulate the metabolic processes in the crab, promoting the liver's clearance of metabolic wastes such as urea nitrogen, ammonia, and uric acid, thereby reducing their accumulation in the body. Simultaneously, it effectively stimulates the crab's immune system to produce more antimicrobial peptides and lysozyme, enhancing immune function; other groups, due to differences in the amount of plant materials, cannot achieve this balance between metabolic regulation and immune enhancement.

[0456] 6) Maintaining internal environmental stability: Lower accumulation of metabolic waste helps maintain the stability of the internal environment of the mud crab, reduces the damage of harmful substances to the body, and further promotes the recovery of its physiological functions; the twenty-seven components have obvious advantages in this regard, providing a good internal environment for the healthy recovery of the mud crab.

[0457] 4. Analysis of test results for fat and energy storage indicators

[0458] Table 29 Effects of different compositions and dosages of postpartum microcirculation preparations on postpartum fat and energy reserves in mud crabs.

[0459]

[0460] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0461] As shown in Table 29, the HDL, LDL, FAS, and CPT in groups 26, 27, and 28 were significantly different from the control group 6. This indicates that the effective components in each group have a significant promoting effect on fat and energy reserves in mud crabs after spawning.

[0462] Furthermore, in terms of high-density lipoprotein (HDL) levels, group 27 (62±3 mg / dL) had the highest level, significantly higher than other groups; in terms of low-density lipoprotein (LDL) levels, group 27 (96±4 mg / dL) had the lowest level, significantly lower than other groups; and in terms of fatty acid synthase and carnitine palmitoyltransferase activities, group 27 (1.7±0.1 U / mg and 2.7±0.1 U / mg, respectively) had the highest levels, significantly higher than other groups. This indicates that group 27 performed best in fat and energy storage-related indicators, demonstrating a significant improvement in lipid metabolism and energy storage. Specific advantages are as follows:

[0463] 7) Optimization of lipid metabolism pathways: Terpenoids, catechins, and other components in plant extracts can regulate lipid metabolism, affecting HDL and LDL levels, as well as the activity of fatty acid synthase and carnitine palmitoyltransferase. The amount of plant raw materials in the twenty-seven groups allows these components to function in an appropriate ratio, promoting HDL synthesis, reducing LDL levels, and optimizing lipid metabolism balance, thereby contributing to fat synthesis and energy storage. Other groups in this embodiment may experience imbalances in lipid metabolism regulation due to differences in the amount of plant raw materials, affecting indicators related to fat and energy storage.

[0464] 8) Meeting energy needs and reserves: After spawning, mud crabs need to restore their energy reserves. The proportion of plant extract components in component group 27 is more conducive to promoting the activity of enzymes related to fat synthesis and energy reserves, enabling mud crabs to accumulate energy more effectively and meet their energy needs for growth and recovery. Other components in this embodiment are not as effective as component group 27 in this regard.

[0465] 5. Analysis of Tissue Repair Indicator Test Results

[0466] Table 30 Effects of different compositions and dosages of postpartum microcirculation preparations on postpartum tissue repair indicators in mud crabs.

[0467]

[0468] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05);

[0469] Table 30 shows that the components 26, 27, and 28 had significant differences from the control group 6 in terms of hydroxyproline, epidermal growth factor, platelet-derived growth factor, and transforming growth factor-β. This indicates that the components have a significant promoting effect on tissue repair after spawning in mud crabs within the range of effective component content parameters.

[0470] Furthermore, in terms of hydroxyproline content, group 27 (8.2±0.2 μg / g) had the highest level and was significantly higher than other groups; the levels of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-β (TGF-β) in group 27 (56±2 pg / mL, 127±4 pg / mL, and 214±10 pg / mL, respectively) were all significantly higher than other groups. This indicates that group 27 performed best in promoting tissue repair-related indicators and can effectively accelerate the repair and regeneration of postpartum tissues in mud crabs. Specific advantages are as follows:

[0471] 9) Promotes cell proliferation and repair: Components such as ursolic acid and caryopsisic acid in plant extracts can promote collagen synthesis and stimulate growth factor secretion, thereby accelerating tissue repair. The appropriate amount of plant materials in component group 27 allows these components to function better, promoting hydroxyproline synthesis, increasing collagen content, and providing a foundation for tissue repair; simultaneously stimulating the secretion of growth factors such as EGF, PDGF, and TGF-β, enhancing cell proliferation and regeneration capacity; other groups may have had less appropriate amounts of plant materials, affecting the effectiveness of these components in promoting tissue repair.

[0472] 10) Improved overall physiological state promotes repair: By improving the overall physiological state of the crab, such as blood circulation and excretion of metabolic waste (as shown in the table above, the good performance of component group 27 in other indicators), favorable conditions are created for tissue repair, enabling the tissue repair process to proceed more smoothly; the advantage of component group 27 in improving overall physiological state indirectly promotes the improvement of tissue repair-related indicators, while the comprehensive effect of other groups in this regard is not as good as that of component group 27.

[0473] 3.6 Summary of Results

[0474] In summary, components 26 to 28 of this embodiment showed significantly better performance than control group 6 in terms of specific detection parameters for growth indicators, nutrient transport indicators, metabolic waste accumulation indicators, fat and energy storage indicators, and tissue repair indicators. In particular, component 27 showed significantly better performance than components 26 and 28 in all these parameters. Therefore, it can be concluded that the effective component content in the postpartum microcirculation preparation for mud crabs is 300–400 mg / L for rosmarinic acid, 250–350 mg / L for sarsaparilla acid, 250–350 mg / L for oleuropein, and 200–300 mg / L for terpene alcohol. When the concentrations of active ingredients in the postpartum microcirculation preparation of mud crabs are within the range of 100-200 mg / L for catechins, 100-200 mg / L for quercetin, 50-100 mg / L for ursolic acid, and 50-100 mg / L for proanthocyanidins, and when applied in conjunction with an auxiliary solution, it is beneficial for the female crabs to increase their roe and recover after spawning. The effect is even better when the effective ingredient concentrations in the preparation are rosmarinic acid 350 mg / L, caryopsisic acid 300 mg / L, oleuropein 300 mg / L, terpene alcohol 250 mg / L, catechin 150 mg / L, quercetin 150 mg / L, ursolic acid 75 mg / L, and proanthocyanidins 75 mg / L.

Claims

1. A method for raising mud crabs after spawning, characterized in that: The product includes a postpartum microcirculation preparation for mud crabs, wherein the postpartum microcirculation preparation for mud crabs contains at least one of the following components and contents: rosmarinic acid at a content of 300-400 mg / L, caryopsisic acid at a content of 250-350 mg / L, oleuropein at a content of 250-350 mg / L, and terpene alcohol at a content of 200-300 mg / L. It also includes the following components: rosmarinic acid, caryopsisic acid, oleuropein, terpene alcohol, catechin, quercetin, ursolic acid, and proanthocyanidins, wherein the content of rosmarinic acid is 300~400mg / L, the content of caryopsisic acid is 250~350mg / L, the content of oleuropein is 250~350mg / L, the content of terpene alcohol is 200~300mg / L, the content of catechin is 100~200mg / L, the content of quercetin is 100~200mg / L, the content of ursolic acid is 50~100mg / L, and the content of proanthocyanidins is 50~100mg / L; The postpartum microcirculation preparation for mud crabs is used in combination with the auxiliary solution. The components and contents of each 4-6 mL of the auxiliary solution are: 6-8 g of Clostridium butyricum, 4-6 g of L-arginine, 2-4 g of vitamin C, and 4-6 mL of 1× phosphate buffer. The postpartum microcirculation preparation for mud crabs and the auxiliary solution are mixed in a volume ratio of 5:1 to obtain the mixed preparation.

2. The method for raising mud crabs after spawning according to claim 1, characterized in that: The content of each component in the postpartum microcirculation preparation of mud crab is as follows: rosmarinic acid 350 mg / L, carrageenan 300 mg / L, oleuropein 300 mg / L, terpene alcohol 250 mg / L, catechin 150 mg / L, quercetin 150 mg / L, ursolic acid 75 mg / L, and proanthocyanidins 75 mg / L.

3. The method for raising mud crabs after spawning according to claim 1, characterized in that: The preparation method of the postpartum microcirculation preparation for mud crabs includes the following steps: 1) Mixing: Weigh 25-30 parts of rosemary petals, 20-25 parts of sage leaves, 20-25 parts of olive leaves, 15-20 parts of perilla leaves, 10-15 parts of green tea leaves, 10-15 parts of red onion, 5-10 parts of blueberry leaves and 5-10 parts of grape seeds, and mix them to obtain the raw material mixture. 2) Ultrasonic extraction: After pulverizing 60-80 parts of the raw material mixture obtained in step 1), ultrasonic extraction is performed on the pulverized powder using 70% ethanol solution as the extraction agent to obtain an extraction solution; 3) Filtration and centrifugation: The extraction solution obtained in step 2) is initially filtered to obtain a pre-filtered solution. After centrifuging the pre-filtered solution, the lower layer impurities are discarded to obtain the centrifuged solution. 4) Concentration: The centrifuged solution obtained in step 3) is evaporated and concentrated to 1 / 2 of its volume to obtain a concentrated solution; 5) Dilution and storage: The concentrate obtained in step 4) is diluted with water to obtain the postpartum microcirculation preparation of mud crab. The volume ratio of concentrate to water during dilution is 1:

20. The postpartum microcirculation preparation of mud crab is placed in a sealed container and stored at 4°C.

4. The method for raising mud crabs after spawning according to claim 1, characterized in that: The method for preparing the auxiliary liquid includes the following steps: 6) Raw material dissolution: In a sterile laminar flow hood, weigh 6-8 parts by weight of Clostridium butyricum powder, add 8-25 parts by weight of sterile water and stir thoroughly to obtain a Clostridium butyricum suspension; weigh 4-6 parts by weight of L-arginine powder and dissolve in 10-15 parts by weight of sterile water and stir thoroughly to obtain an L-arginine solution; weigh 2-4 parts by weight of vitamin C powder and dissolve in 8-12 parts by weight of sterile water and stir thoroughly to obtain a vitamin C solution; mix the obtained Clostridium butyricum suspension, L-arginine solution, vitamin C solution and 1×PBS buffer at a volume ratio of 8-25:10-15:8-12:4-6 to obtain a raw material mixture solution; 7) Filtration and sterilization: The raw material mixture obtained in step 6) is aseptically filtered through a 0.22μm needle filter to obtain the auxiliary solution; 8) Storage: After the auxiliary liquid obtained in step 8) is placed into a sealed container, it is stored in a light-proof environment at 4°C.

5. The method for raising mud crabs after spawning according to claim 4, characterized in that, This includes a phased application process, specifically, three phases of application: During the 3-7 days after the mud crabs spawn: apply once a day at 18:00, each time applying 10-15ml of the mixed preparation per cubic meter of water. During the 8-14 days postpartum period of mud crabs: apply the mixture once at 5:00 and once at 20:00 each day, with each application being 15-20 ml of the mixture per cubic meter of water. During the 15-20 days after the mud crabs spawn: apply the mixture once at 5:00 and once at 20:00 every day, with each application being 20-30 ml of the mixture per cubic meter of water.

6. The method for raising mud crabs after spawning according to claim 5, characterized in that, The application methods for the three stages in the phased application process are as follows: During the 3-7 days after the mud crabs spawn: apply once a day at 18:00, each time applying 12.5ml of the mixed preparation per cubic meter of water; During the 8-14 days postpartum period of mud crabs: apply the mixture once at 5:00 and once at 20:00 each day, with each application being 17.5 ml of the mixture per cubic meter of water. During the 15-20 days after the mud crabs spawn: apply the mixture once at 5:00 and once at 20:00 every day, with each application being 25ml of the mixture per cubic meter of water.

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