Breeding method for postpartum blue crabs

Abstract

The breeding method for the postpartum blue crabs is characterized by comprising a microcirculation preparation for the postpartum blue crabs, and the microcirculation preparation for the postpartum blue crabs at least contains one of the following components in content: rosmarinic acid with the content of 300-400 mg / L, carnosic acid with the content of 250-350 mg / L, oleuropein with the content of 250-350 mg / L and terpene alcohol with the content of 200-300 mg / L. The microcirculation preparation for the postpartum blue crabs can remarkably improve the growth level, the nutrition transportation capacity, the waste metabolism capacity, the tissue repair capacity and the fat and energy storage capacity of the postpartum female crabs, and promote the postpartum cream increase and rejuvenation of the blue crabs.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture, and particularly to a breeding method for mud crabs after parturition. Background Art

[0002] As a high - economic - value aquaculture variety, mud crabs play an important role in the global aquaculture industry. After female mud crabs lay eggs, due to the decline of microcirculation function, problems such as blocked nutrient transportation, accumulation of metabolic wastes, insufficient fat and energy reserves, and limited tissue repair ability often occur. This microcirculation disorder not only damages the hepatopancreas and gonad functions of female crabs, but also causes problems such as insufficient fat reserves, weak constitution, and low efficiency of paste accumulation, thus seriously affecting their overall health, reproductive ability, and market competitiveness. In addition, due to the slow postpartum recovery, the survival rate and breeding efficiency of female crabs are also significantly reduced.

[0003] Currently, the main means for postpartum recovery of female crabs in aquaculture mainly include increasing the feeding amount of high - nutrient feed, improving the aquaculture water quality conditions, and using chemical synthetic additives. However, these measures have limited effects on improving microcirculation function, and the means of increasing the feeding amount of high - nutrient feed and using chemical synthetic additives may cause environmental pollution, increase aquaculture costs, and pose food safety hazards. Especially when dealing with problems such as poor blood circulation and blocked oxygen and nutrient transmission caused by microcirculation disorders, the existing technical means still cannot effectively increase the paste and rejuvenate postpartum female crabs, restricting the comprehensive recovery and improvement of the health level of female crabs. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a breeding method for mud crabs after parturition that can improve the microcirculation function of postpartum mud crabs, promote paste increase, and rejuvenation, in view of the above - mentioned current technical situation.

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

[0006] Furthermore, the microcirculation preparation for mud crabs after parturition further includes the following components: rosmarinic acid, carnosic acid, oleuropein, terpenoid alcohol, wherein the content of rosmarinic acid is 300 - 400 mg / L, the content of carnosic acid is 250 - 350 mg / L, the content of oleuropein is 250 - 350 mg / L, and the content of terpenoid alcohol is 200 - 300 mg / L.

[0007] Furthermore, the postpartum microcirculation preparation for mud crabs further comprises the following components: catechin, quercetin, ursolic acid, and procyanidin, 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 procyanidin is 50-100 mg / L.

[0008] Furthermore, the content of each component in the postpartum microcirculation preparation for mud crabs is as follows: the content of rosmarinic acid is 350 mg / L, the content of carnosic acid is 300 mg / L, the content of oleuropein is 300 mg / L, the content of terpenol is 250 mg / L, the content of catechin is 150 mg / L, the content of quercetin is 150 mg / L, the content of ursolic acid is 75 mg / L, and the content of procyanidin is 75 mg / L.

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

[0010] 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 onions, 5-10 parts of blueberry leaves, and 5-10 parts of grape seeds, and mix them to obtain a raw material mixture; wherein, 1 part = 1 g;

[0011] 2) Ultrasonic extraction: After pulverizing 60-80 parts of the raw material mixture obtained in the above step 1), use 70% ethanol solution as an extraction agent to perform ultrasonic extraction on the pulverized powder to obtain an extraction solution; wherein, 1 part = 1 g;

[0012] 3) Filtration and centrifugation: Perform preliminary filtration on the extraction solution obtained in the above step 2) to obtain a preliminary filtrate solution. After centrifuging the preliminary filtrate solution, discard the lower-layer impurities to obtain a centrifuged solution;

[0013] 4) Concentration: Evaporate and concentrate the centrifuged solution obtained in the above step 3) at 45°C to 1 / 2 of its original volume to obtain a concentrated solution;

[0014] 5) Dilution and storage: Add water to the concentrated solution obtained in the above step 4) for dilution to obtain the postpartum microcirculation preparation for mud crabs. When diluting, the volume ratio of the concentrated solution to water is 1:20. After filling the postpartum microcirculation preparation for mud crabs into a sealed container, store it in an environment at 4°C. The 70% ethanol solution as an extraction agent can dissolve the active ingredients of rosmarinic acid, carnosic acid, oleuropein, terpenol, catechin, quercetin, ursolic acid, and procyanidin; evaporating and concentrating at 45°C can remove the volatile impurities in the extraction solution and can also preserve the above-mentioned active ingredients.

[0015] Furthermore, the post-partum microcirculation preparation of the mud crab is used in combination with the auxiliary liquid. The components and their contents in every 4 - 6 mL of the auxiliary liquid are as follows: Clostridium butyricum 6 - 8 g, L-arginine 4 - 6 g, vitamin C 2 - 4 g, 1×phosphate buffer solution 4 - 6 mL. The post-partum microcirculation preparation of the mud crab and the auxiliary liquid are mixed in a volume ratio of 5:1 to obtain a mixed preparation. L-arginine and vitamin C promote the effective absorption of the post-partum microcirculation preparation of the mud crab by the mud crab, and the Clostridium butyricum component can maintain the stability of the water quality.

[0016] Furthermore, the preparation method of the auxiliary liquid includes the following steps:

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

[0018] 7) Filtration and sterilization: Sterilize the raw material mixed solution obtained in step 6) through a 0.22 μm needle filter to obtain the auxiliary liquid;

[0019] 8) Storage: Put the auxiliary liquid obtained in step 8) into a sealed container and store it in the dark at 4°C.

[0020] Furthermore, it includes the step of applying in stages. Specifically, it is applied in three stages:

[0021] During 3 - 7 days after the mud crab gives birth: Sprinkle once at 18:00 every day, and the amount of each sprinkle is 10 - 15 mL of the mixed preparation per cubic meter of water body;

[0022] During 8 - 14 days after the mud crab gives birth: Sprinkle once at 5:00 and once at 20:00 every day, and the amount of each sprinkle is 15 - 20 mL of the mixed preparation per cubic meter of water body;

[0023] During 15 - 20 days after the mud crab gives birth: Sprinkle once at 5:00 and once at 20:00 every day, and the amount of each sprinkle is 20 - 30 mL of the mixed preparation per cubic meter of water body.

[0024] Furthermore, the application methods in the three stages in the step of applying in stages are as follows:

[0025] During 3 to 7 days after the parturition of the mud crab: Sprinkle once at 18:00 every day, and the amount of each sprinkle is 12.5 ml of the mixed preparation per cubic meter of water body;

[0026] During 8 to 14 days after the parturition of the mud crab: Sprinkle once at 5:00 and once at 20:00 every day, and the amount of each sprinkle is 17.5 ml of the mixed preparation per cubic meter of water body;

[0027] During 15 to 20 days after the parturition of the mud crab: Sprinkle once at 5:00 and once at 20:00 every day, and the amount of each sprinkle is 25 ml of the mixed preparation per cubic meter of water body.

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

[0029] 1. The components in the microcirculation preparation after the parturition of the mud crab can improve the microcirculation function of the mud crab after parturition, promote the increase of fat and rejuvenation. Specifically,

[0030] Rosmarinic acid and procyanidins are mainly used to solve the problem of blocked nutrient transportation after the parturition of the mud crab: Rosmarinic acid can reduce the synthesis of triglycerides, thereby reducing the triglyceride level in the blood, and by inhibiting the activity of HMG-CoA reductase (the rate-limiting enzyme for cholesterol synthesis), reducing cholesterol synthesis, reducing the cholesterol level in the blood, and also increasing the synthesis of nitric oxide (NO), dilating blood vessels, improving blood fluidity, and enhancing the nutrient supply to organs such as the hepatopancreas and gonad; Procyanidins can improve blood circulation and gonad nutrient supply, increase the hepatopancreas index, gonad index, and reduce the triglyceride and total cholesterol levels in hemolymph, thereby optimizing the hepatopancreas function and blood lipid metabolism, enhancing gonad function, and thus solving the problem of blocked nutrient transportation caused by blood lipid accumulation and metabolic disorders;

[0031] Oleuropein and quercetin are mainly used to solve the problem of accumulation of metabolic wastes: Oleuropein can enhance the function of renal tubular cells, promote uric acid excretion, and by stimulating the immune system to increase the levels of antimicrobial peptides and lysozyme, reduce the inflammatory response, and then promote the excretion of urea nitrogen, ammonia, and uric acid in hemolymph, and further increase the levels of antimicrobial peptides and lysozyme, reduce the accumulation of metabolic wastes in the blood, relieve the toxic burden caused by the accumulation of metabolic wastes, and improve the liver detoxification and immune protection functions; Quercetin can activate urea cycle enzymes, enhance the ability of the liver to convert ammonia into urea, accelerate the excretion of urea by the kidneys, and reduce the accumulation of metabolic wastes in the blood;

[0032] Terpenoids and catechins are mainly used to solve the problem of insufficient fat and energy reserves after the parturition of mud crabs: Terpenoids activate fatty acid synthase to maintain the dynamic balance of fat reserves. At the same time, they increase the level of high-density lipoproteins, remove low-density lipoproteins, optimize lipid metabolism and energy supply, thereby improving the efficiency of fat metabolism, enhancing energy reserves, and providing stable energy support for the recovery period of mud crabs; Catechins enhance the reserves of lipids and energy. Their combined effects can effectively solve the problem of insufficient fat and energy reserves after the parturition of mud crabs;

[0033] Carnosic acid and ursolic acid are mainly used to solve the problem of limited tissue repair ability: Carnosic acid accelerates cell proliferation and damage repair by increasing the secretion levels of repair factors (EGF, PDGF, TGF-β), improves tissue regeneration ability, and then accelerates the tissue repair process, enhances cell proliferation and wound healing ability, and improves the tissue repair efficiency of female mud crabs after parturition; Ursolic acid activates fibroblasts, promotes the synthesis of collagen and hydroxyproline, and provides organizational structure support;

[0034] 2. Applying the above-mentioned microcirculation preparation for mud crabs after parturition can significantly improve the growth level, nutrient transport ability, metabolic waste capacity, tissue repair ability, and fat and energy reserve ability of female mud crabs after parturition. The survival rate is increased to 67.34 - 78.13%, and the weight gain rate is increased to 28.30 - 36.03%, promoting the fattening and rejuvenation of mud crabs after parturition. Specific Embodiments

[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 breeding method for mud crabs after parturition in this embodiment is mainly used for the fattening and rejuvenation of mud crabs after parturition; In the natural state, female mud crabs often suffer from problems such as blocked nutrient transport, accumulation of metabolic wastes, insufficient fat and energy reserves, and limited tissue repair ability due to the decline of microcirculation function after spawning. The existing technology mainly improves the growth state of mud crabs by increasing the feeding amount of high-nutrient feed, improving the aquaculture water quality conditions, and using chemically synthesized additives. These methods do not specifically solve the problem of the decline of microcirculation function in mud crabs after parturition, and the effect is poor. Therefore, it will be beneficial to provide a breeding method for mud crabs after parturition that can improve the microcirculation function of mud crabs after parturition, promote fattening and rejuvenation. The following will elaborate on the breeding method for mud crabs after parturition in detail:

[0037] Example 1 Experiment on Rosmarinic Acid Content

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

[0039] I. Ultrasonic Extraction of Rosmarinic Acid

[0040] Step 1: Preparation of Plant Materials

[0041] (1) Sampling: Weigh 22.5, 25, 27.5, 30, and 32.5 grams of rosemary petals respectively. (The plant materials are obtained by purchasing in the market)

[0042] (2) Crushing: Use a crusher (model: LD-1300A high-speed universal crusher, LinDa Machinery Co., Ltd., Wenling, Zhejiang) to coarsely crush the above 5 portions of plant materials respectively, and control the particle size to be 0.5 - 1 mm. The crushed materials are filled into a dry 100 ml airtight glass beaker (100 ml round-bottom glass beaker, Shanghai Heqi Glass Instruments Co., Ltd.) for standby.

[0043] Step 2: Ultrasonic-Assisted Ethanol Extraction

[0044] (3) Solvent Preparation: Use a 70% ethanol solution (purchased in the market) as the extraction solvent. Pour 100 ml of the 70% ethanol solution into 4 500 ml round-bottom glass flasks (500 ml round-bottom glass flasks, Shanghai Heqi Glass Instruments Co., Ltd.).

[0045] (4) Plant Material Blending: Take the 5 portions of crushed rosemary petals respectively and add them into 5 500 ml round-bottom flasks each containing 100 ml of 70% ethanol solution.

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

[0047] Step 3: Filtration and Centrifugation

[0048] (6) Coarse Filtration: Use a filter (model: MS-0.45μm polyethersulfone (PES) membrane filter, Shanghai Mosu Scientific Equipment Co., Ltd.) to preliminarily filter the extract to remove larger particles.

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

[0050] Step 4: Rotary Evaporation and Concentration

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

[0052] Step 5: Preparation of liquid preparation

[0053] (9) Dilution: Dilute 50 ml of the concentrated solution with pure water to 1000 ml, stir well to make the post-partum microcirculation preparation of mud crab.

[0054] (10) Storage: Divide the prepared post-partum microcirculation preparation of mud crab into 2L transparent glass containers and seal them for storage. The containers need to be light-proof and placed in a 4 °C refrigerator to prevent the degradation of active ingredients.

[0055] II. Preparation of auxiliary liquid

[0056] The post-partum microcirculation preparation of mud crab needs to be used together with an auxiliary liquid. The auxiliary liquid 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), aiming to promote the effective absorption of the post-partum microcirculation preparation of mud crab by mud crab and maintain the stability of water quality.

[0057] Experimental materials: Clostridium butyricum: 6 - 8 g, L-arginine: 4 - 6 g, vitamin C: 2 - 4 g, phosphate buffer (PBS): 4 - 6 ml; 500 mL glass beaker (model: GG-17, Shanghai Guanghua Glass Instrument Co., Ltd.), put the glass beaker into a high-pressure sterilizer (DXL-100S automatic high-pressure steam sterilizer, Shandong Dexiang Instrument Co., Ltd.) for high-temperature and high-pressure sterilization treatment, and wait for use.

[0058] Step 5: Preparation of auxiliary liquid

[0059] (11) Dissolve Clostridium butyricum: In a sterile laminar flow hood (model: BSC-1100IIA2-X, Guangdong Keyipu Laboratory Equipment Development Co., Ltd.), take 7 g of Clostridium butyricum powder and add 16.5 ml of sterile pure water; use an electric stirrer (model: IKA RW20 Digital Dissolver, Beijing Labtech Instruments Co., Ltd.) to stir at medium speed for 4 minutes to ensure complete dissolution of Clostridium butyricum and form a uniform suspension.

[0060] (12) Addition of L-arginine and vitamin C: Dissolve 5 g of L-arginine in 12.5 ml of sterile water and stir for 4 minutes until completely dissolved. Then dissolve 2 - 4 g of vitamin C in 10 ml of sterile water and stir for 3 minutes to ensure a clear solution without precipitation. Finally, add the above two solutions to the Clostridium butyricum suspension respectively, and stir slowly while adding to mix evenly.

[0061] (13) Addition of PBS buffer: Take 5 ml of PBS buffer and slowly add it to the above mixture, and continue to stir 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 treatment

[0063] (14) Filtration: Sterile filter the mixed solution through a 0.22 μm needle filter (Model: Syringe Filters 0.22 μm, Zhejiang Sepure Tech Co., Ltd.) to remove bacteria, fungal spores and other microbial contaminants.

[0064] (15) Sub-packaging and storage: Sub-package the filtered auxiliary solution into 500 mL sterile transparent glass cups (Model: GG-17, Shanghai Guanghua Glass Instrument Co., Ltd.), seal and store in a 4°C refrigerator, protected from light to prevent degradation of the active ingredients.

[0065] III. Mixed preparation

[0066] The ratio of the postpartum microcirculation preparation of Scylla serrata to the auxiliary solution is 5:1. That is, for every 5 ml of the microcirculation preparation, 1 ml of the auxiliary solution needs to be added for mixing to obtain 6 ml of the mixed preparation. The rosmarinic acid contents in 5 portions of the mixed preparation are 300 mg / L, 350 mg / L, 400 mg / L, 200 mg / L and 500 mg / L respectively.

[0067] IV. Experimental design

[0068] Extract rosmarinic acid at 300 mg / L, 350 mg / L, 400 mg / L, 200 mg / L and 500 mg / L respectively from rosemary petals by the above ultrasonic combined with 70% ethanol extraction technology. Denote the auxiliary solution as blank control group one, and denote them respectively as:

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

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

[0071] Component group three: 400 mg / L rosmarinic acid,

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

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

[0074] Control Group One: Auxiliary solution (i.e., 0 mg / L rosmarinic acid).

[0075] V. Breeding Experiment

[0076] From September to October 2023, it was carried out in the indoor breeding ponds of Dongying Kenuo Aquaculture Co., Ltd. 90 female mud crabs within 1 - 3 days after parturition were collected again from the southern coastal areas and transported to the experimental site by air. After centralized disinfection treatment, they were put into two indoor cement ponds of 30 m 2 for temporary cultivation for 1 - 2 days. After temporary cultivation, every 5 mud crabs were respectively put into 18 laboratory ponds of 20 m 2 for the experiment. 5 female crabs after parturition were put into one pond, and at the same time, 5 tiles were put into each pond. Among them, ponds No. 1 - 3 were for Component Group One, ponds No. 4 - 6 were for Component Group Two, ponds No. 7 - 9 were for Component Group Three, ponds No. 10 - 12 were for Component Group Four, ponds No. 13 - 15 were for Component Group Five, and ponds No. 16 - 18 were for Control Group One. The purpose of this embodiment is to explore the key compounds in the postpartum microcirculation preparation for mud crabs that are beneficial to the postpartum rejuvenation of mud crabs. The experimental materials and their sources in this embodiment are the same as those in Example 1.

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

[0078] The First Stage (3 - 7 days after parturition)

[0079] Application Time: 6 p.m. (18:00) every day;

[0080] Application Frequency: Once a day;

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

[0082] The Second Stage (8 - 14 days after parturition)

[0083] Application Time: 8 p.m. (20:00) and 5 a.m. (05:00) every day;

[0084] Application Frequency: Twice a day;

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

[0086] The Third Stage (15 - 20 days after parturition)

[0087] Application Time: 8 p.m. (20:00) and 5 a.m. (05:00) every day;

[0088] Application frequency: twice a day;

[0089] Dosage: Add 20 - 30 ml of the mixed preparation to each cubic meter of water body 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 of mud crabs on the growth indexes of mud crabs

[0093] Survival rate (%) Weight gain rate (%) <![CDATA[Condition factor (g / cm 3 )]]> Recovery time (days) Component group 1 <![CDATA[70.88±2.18 b > <![CDATA[30.48±1.36 b > <![CDATA[47.02±1.90 b > <![CDATA[26.27±1.21 c > Component 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 > Component group 3 <![CDATA[71.24±2.18 b > <![CDATA[29.78±1.36 b > <![CDATA[45.22±1.71 b > <![CDATA[27.30±1.13 c > Component group 4 <![CDATA[59.48±2.18 c > <![CDATA[22.98±1.21 c > <![CDATA[32.35±1.82 c > <![CDATA[32.43±1.32 b > Component group 5 <![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 1 <![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] It can be seen from Table 1 that the second component group is significantly higher than the first component group and the third component group in terms of survival rate (78.13 ± 2.10%), weight gain rate (36.03 ± 1.26%), and condition factor (55.63 ± 1.76 g / cm 3 )), and the recovery time (23.26 ± 0.86 days) is the shortest; there is no significant difference between the first component group and the third component group in these indexes, but both are better than the fourth component group and the fifth component group; the growth indexes of the first control group are the worst.

[0096] Advantages of the second component group: The effect is the best when the rosmarinic acid content is 350 mg / L. At this concentration, it promotes the expression of antioxidant enzymes by activating the Nrf2 pathway, reduces the oxidative damage of free radicals to cells, and at the same time inhibits the NF-κB pathway to reduce the release of inflammatory factors, alleviates oxidative stress and inflammatory responses, provides a good internal environment for cell growth, promotes the growth of mud crabs, improves the survival rate, weight gain rate and condition factor, and shortens the recovery time. For example, moderate antioxidant and anti-inflammatory effects can maintain normal cell metabolism and proliferation, and avoid growth inhibition caused by oxidative damage and inflammation.

[0097] Differences between the first component group and the third and second component groups: The contents of the first component group (300 mg / L) and the third component group (400 mg / L) deviate from the optimal value. When the content is relatively low (300 mg / L), the role of promoting growth cannot be fully exerted; when the content is too high (400 mg / L), it may cause imbalance of intracellular signaling pathways or produce toxic effects, interfere with normal physiological metabolism, and affect the growth effect. Therefore, the growth indexes of the two are inferior to those of the second component group, but because the difference is not significant, there is no significant difference.

[0098] Compared with the four-component group, five-component group, and control group 1: The contents of the four-component group (200 mg / L) and five-component group (500 mg / L) deviated more from the appropriate range. Too low a content could not effectively initiate cell division promotion, while too high a content exacerbated cell metabolic disorders, resulting in significantly worse growth indicators than those of the one-component group and three-component group. In control group 1, there was no rosmarinic acid added, lacking its growth promotion and regulation mechanism, and the cells were in a state of natural damage and slow growth, with the worst growth indicators in all aspects.

[0099] 2. Results analysis of nutrient transport indicators

[0100] Table 2 Effects of different rosmarinic acid contents in the microcirculation preparation of mud crabs on the nutrient transport-related indicators after parturition of mud crabs

[0101]

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

[0103] As can be seen from Table 2, in terms of the hepatopancreas index and gonad index, the two-component group (7.8 ± 0.31%, 6.8 ± 0.30%) was the highest; in terms of the triglyceride and total cholesterol levels, the two-component group (0.91 ± 0.03 mmol / L, 1.64 ± 0.06 mmol / L) was the lowest, and there were significant differences from other groups; the one-component group and three-component group were similar and better than the four-component group and five-component group; the nutrient transport-related indicators of control group 1 were the worst.

[0104] Advantages of the two-component group: 350 mg / L of rosmarinic acid could better regulate lipid metabolism, promote the absorption of nutrients and the recovery of organ function. It enabled nutrients to be transported to organs such as the hepatopancreas and gonad more efficiently, promoting the improvement of their functions, manifested as an increase in the hepatopancreas index and gonad index, and a decrease in the triglyceride and total cholesterol levels.

[0105] Reasons for differences in other groups: Since the contents of the one-component group and three-component group were not optimal, their regulation effects on lipid metabolism, nutrient transport-related enzymes, and signaling pathways were not as good as those of the two-component group. Too low or too high a content affected their ability to promote nutrient absorption and organ function enhancement, resulting in nutrient transport indicators at an intermediate level. The contents of the four-component group and five-component group deviated severely, interfering greatly with the metabolic and transport processes and unable to effectively improve nutrient transport, with poor indicators; in control group 1, due to the lack of rosmarinic acid participation, the metabolic and regulatory mechanisms related to nutrient transport were lacking, and the indicators were the worst.

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

[0107] Table 3 Effects of different rosmarinic acid contents in the microcirculation preparation of mud crabs on the indicators related to metabolic waste accumulation after parturition of mud crabs

[0108]

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

[0110] As can be seen from Table 3, in terms of the contents of urea nitrogen, ammonia, and uric acid, the second component group (3.6±0.4mg / dL, 0.7±0.03mg / L, 4.3±0.1mg / dL) is the lowest; in terms of the contents of antibacterial peptides and lysozyme, the second component group (182±12μg / mL, 177±12U / mL) is the highest; the first component group and the third component group are at an intermediate level and are superior to the fourth component group and the fifth component group; the accumulation of metabolic wastes in the first control group is the most serious.

[0111] Advantages of the second component group: An appropriate rosmarinic acid content (350mg / L) helps to enhance the metabolic function and immune capacity of mud crabs, reduces the accumulation of metabolic wastes such as urea nitrogen, ammonia, and uric acid in the body by promoting the transformation and excretion of metabolic wastes, and at the same time stimulates the immune system to produce more antibacterial peptides and lysozyme, enhances the body's ability to clear bacteria and toxins, and maintains the stability of the internal environment.

[0112] Differential mechanisms among different groups: The contents of the first component group and the third component group cross with those of the second component group, and their regulatory effects on metabolic enzymes and the immune system are limited, unable to efficiently clear metabolic wastes and enhance immunity, and are at an intermediate level of metabolic waste accumulation. The contents of the fourth component group and the fifth component group are abnormal, seriously affecting the balance of metabolic and immune regulation, with a large accumulation of wastes and weak immune function. The first control group has no rosmarinic acid to assist in metabolism and immune regulation, with a large accumulation of metabolic wastes and low immune defense ability.

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

[0114] Table 4: Effects of different rosmarinic acid contents in the microcirculation preparation of mud crabs on indicators related to fat and energy reserve after parturition of mud crabs

[0115]

[0116]

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

[0118] As can be seen from Table 4, in terms of the high-density lipoprotein level, the second component group (64±3mg / dL) is the highest; in terms of the low-density lipoprotein level, the second component group (108±4mg / dL) is the lowest; in terms of the activities of fatty acid synthase and carnitine palmitoyl transferase, the second component group (1.8±0.2U / mg, 2.9±0.2U / mg) is the highest; the first component group and the third component group are the second; the first control group is the worst.

[0119] Advantages of Component Two: Rosmarinic acid at 350 mg / L can effectively regulate lipid metabolism and the activities of energy-related enzymes, maintain a good fat metabolism state at this concentration, provide sufficient energy reserves for mud crabs, and show the best relevant indicators.

[0120] Due to the small content, Component One and Component Three have limited ability to maintain fat metabolism balance and energy reserves, and the indicators are in the middle; Control One lacks the regulation of rosmarinic acid on fat and energy metabolism, resulting in disordered fat metabolism and insufficient energy reserves.

[0121] 5. Analysis of Indicators Related to Tissue Repair

[0122] Table 5 Effects of Different Rosmarinic Acid Contents in the Microcirculation Preparation of Mud Crabs on the Indicators Related to Postpartum Tissue Repair of Mud Crabs

[0123]

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

[0125] As can be seen from Table 5, in terms of the contents of hydroxyproline, epidermal growth factor, platelet-derived growth factor, and transforming growth factor-β, Component Two is significantly higher than other groups; Component One and Component Three are in the middle; Control One is the lowest.

[0126] Advantages of Component Two: Rosmarinic acid at this concentration can effectively promote collagen synthesis and growth factor secretion. It accelerates the repair and regeneration of damaged tissues, increases the content of hydroxyproline, and enhances tissue repair ability by stimulating the proliferation of fibroblasts 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 among different groups: The contents of Component One and Component Three are not optimal, and their promoting effects on cell proliferation and growth factor secretion are weak, resulting in medium tissue repair effects. The contents of Component Four and Component Five are inappropriate, and they cannot effectively initiate the cellular and molecular mechanisms related to tissue repair, resulting in poor repair ability. Control One lacks the effective stimulation of rosmarinic acid in tissue repair, and the related repair indicators are the lowest.

[0128] Therefore, within the range of 300 - 400 mg / L of rosmarinic acid content in the mixed preparation, it has promoting effects on growth indicators, nutrient transport indicators, metabolic waste accumulation indicators, fat and energy reserve indicators, and indicators related to tissue repair.

[0129] Example 2 Content Test of Carnosic Acid

[0130] I. Preparation of Mixed Reagents

[0131] Weigh 17.5, 20, 22.5, 25, and 27.5 grams of sage leaves respectively, and obtain mixed preparations with carnosic acid contents of 250 mg / L, 300 mg / L, 350 mg / L, 200 mg / L, and 400 mg / L respectively according to the operations of Steps 1 to 3 in Example 1. Denote the auxiliary solution as the blank control group, and label them respectively as:

[0132] Group Six: 250 mg / L carnosic acid,

[0133] Group Seven: 300 mg / L carnosic acid,

[0134] Group Eight: 350 mg / L carnosic acid,

[0135] Group Nine: 200 mg / L carnosic acid,

[0136] Group Ten: 400 mg / L carnosic acid,

[0137] Control Group Two: Auxiliary solution (blank group).

[0138] II. Breeding experiment

[0139] From September to October 2023, it was carried out in the indoor aquaculture pond of Dongying Kenuo Aquaculture Co., Ltd. Another 90 female mud crabs within 1 to 3 days after giving birth were collected from the southern coastal area, transported to the experimental site by air, and then centrally disinfected. Then they were put into two indoor cement ponds of 30 m 2 for temporary cultivation for 1 to 2 days. After temporary cultivation, every 5 mud crabs were put into 18 laboratory ponds of 20 m 2 for the experiment. 5 female crabs after giving birth were put into one pond, and at the same time, 5 tiles were put into each pond. Among them, ponds 1 to 3 were Group Six, ponds 4 to 6 were Group Seven, ponds 7 to 9 were Group Eight, ponds 10 to 12 were Group Nine, ponds 13 to 15 were Group Ten, and ponds 16 to 18 were Control Group Two. The purpose of this example is to explore the key compounds in the postpartum microcirculation preparation of mud crabs that are beneficial to the postpartum rejuvenation of mud crabs. The experimental materials and sources in this example are the same as those in Example 1.

[0140] III. Comparison of experimental results

[0141] 1. Analysis of growth index results

[0142] Table 6 Effects of different carnosic acid contents in the mud crab microcirculation preparation on the growth index of mud crabs

[0143] Survival rate (%) Weight gain rate (%) <![CDATA[Condition factor (g / cm 3 )]]> Recovery time (days) Component group 6 <![CDATA[70.38±2.18 b > <![CDATA[29.98±1.36 b > <![CDATA[46.52±1.90 b > <![CDATA[26.77±1.21 c > Component group 7 <![CDATA[77.63±2.10 a > <![CDATA[35.53±1.26 a > <![CDATA[55.13±1.76 a > <![CDATA[23.76±0.86 d > Component 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 > Component group 9 <![CDATA[59.28±2.18 c > <![CDATA[22.48±1.21 c > <![CDATA[31.85±1.82 c > <![CDATA[32.23±1.32 b > Component group 10 <![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] It can be seen from Table 6 that in terms of the three indicators of survival rate, weight gain rate and condition factor, Group Seven of the components showed the best performance, and the values were significantly higher than those of other groups (except for specific cases with no significant differences); in terms of recovery time, Group Seven of the components was the shortest.

[0146] Principle analysis:

[0147] Group Seven of the components is superior to Group Six and Group Eight of the components: The content of carnosic acid at 300 mg / L may be closest to the optimal concentration required for the postnatal growth of mud crabs. This concentration can most effectively promote the metabolism of mud crabs, enhance the absorption and utilization efficiency of nutrients; better nutrient supply and metabolic status may enhance the immunity of mud crabs, improve their resistance to environmental stress and diseases, and thus improve the survival rate. For the recovery time, due to the more efficient operation of its physiological functions, the postpartum body recovers faster.

[0148] Group Six and Group Eight of the components are superior to Group Nine and Group Ten of the components: Although the promoting effects of carnosic acid at 250 mg / L and 350 mg / L on growth indicators are not as good as those at 300 mg / L, they can still promote the physiological balance and growth process of mud crabs to a certain extent.

[0149] Group Nine and Group Ten of the components are superior to Control Group Two: Compared with Control Group Two, Group Eight and Group Four of the components contain a certain amount of carnosic acid, which can initiate and regulate the growth-related physiological reactions in mud crabs to a certain extent. It can promote the uptake and utilization of nutrients by mud crabs. At the same time, carnosic acid may participate in the regulation of energy metabolism in mud crabs, making the generation and utilization of energy more efficient, thereby promoting growth, improving the survival rate, weight gain rate and condition factor, and shortening the recovery time.

[0150] 2. Analysis of the results of nutritional transport indicators

[0151] Table 7 Effects of different carnosic acid contents in the microcirculation preparation of mud crabs on the nutritional transport-related indicators of mud crabs after parturition

[0152]

[0153]

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

[0155] It can be seen from Table 7 that in terms of hepatopancreas index and gonad index, Group Seven of the components was the highest, showing a decreasing trend from Group Seven of the components to Control Group Two. The contents of triglyceride and total cholesterol were opposite, with Group Seven of the components being the lowest, showing an increasing trend from Group Seven of the components to Control Group Two. Letters indicate the significance of data differences, and there are no significant differences between groups with the same letter.

[0156] Principle analysis:

[0157] Group Seven is superior to Group Six and Group Eight: The content of 300 mg / L of carnosic acid has the best regulatory effect on the nutrient transport system of mud crabs. In terms of the hepatopancreas and gonads, it promotes the development of the hepatopancreas and gonads, thereby increasing the hepatopancreas index and gonad index. For example, it promotes the synthesis of nutrients and the production of storage-related proteins in hepatopancreas cells, as well as the development and maturation of gonad cells. For triglycerides and total cholesterol, 300 mg / L of carnosic acid may regulate the activities of enzymes related to fat metabolism, such as promoting the activity of lipolytic enzymes and inhibiting the activity of cholesterol synthase, resulting in a decrease in the content of triglycerides and total cholesterol in the blood, which means that the transport and utilization of nutrients in the body are more efficient and the excessive accumulation of fat is reduced.

[0158] Group Six and Group Eight are superior to Group Nine and Group Ten: The content of 250 mg / L and 350 mg / L of carnosic acid can still promote the operation of the nutrient transport system to a certain extent; in contrast, the content of 200 mg / L has insufficient promotion effect on the development of the hepatopancreas and gonads, resulting in lower hepatopancreas index and gonad index; the content of 400 mg / L has an excessive or abnormal effect on fat metabolism regulation, interfering with the normal nutrient transport and metabolism processes, also resulting in unsatisfactory development of the hepatopancreas and gonads and blood lipid levels.

[0159] Group Nine and Group Ten are superior to Control Group Two: In the hepatopancreas and gonads, carnosic acid increases the absorption and utilization of nutrients by hepatopancreas and gonad cells; in terms of blood lipid regulation, carnosic acid reduces the synthesis of triglycerides and total cholesterol or promotes their catabolism, so that Group Nine and Group Ten containing carnosic acid are superior to Control Group Two without carnosic acid in the nutrient transport-related indicators.

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

[0161] Table 8 Effects of different carnosic acid contents in the mud crab microcirculation preparation on the indicators related to the accumulation of postpartum metabolic wastes in mud crabs

[0162]

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

[0164] It can be seen from Table 8 that the contents of urea nitrogen, ammonia and uric acid are sorted from low to high as Group Seven < Group Six, Group Eight < Group Nine, Group Ten < Control Group Two; the contents of antibacterial peptides and lysozymes are sorted from high to low as Group Seven > Group Six, Group Eight > Group Nine, Group Ten > Control Group Two.

[0165] Principle analysis:

[0166] Group VII is superior to Groups VI and VIII: 300 mg / L of carnosic acid can most effectively promote the excretion process of metabolic wastes in mud crabs. In terms of nitrogen metabolism, it may enhance the activity of urea synthase in the liver, enabling ammonia to be more rapidly converted into urea and excreted from the body, thereby reducing the contents of urea nitrogen and ammonia in the blood, and at the same time reducing the content of uric acid. Meanwhile, 300 mg / L of carnosic acid significantly enhances the immune defense ability of mud crabs, stimulating immune cells to secrete more antibacterial peptides and lysozymes.

[0167] Groups VI and VIII are superior to Groups IX and X: 250 mg / L and 350 mg / L of carnosic acid can better maintain the metabolic balance and immune function of mud crabs. In contrast, a content of 200 mg / L has a weaker promoting effect on the excretion of metabolic wastes and immune regulation. In terms of nitrogen metabolism and uric acid metabolism, due to insufficient activation of related enzymes or inadequate improvement of renal excretion function, there is more accumulation of urea nitrogen, ammonia, and uric acid; in terms of immunity, the activity of immune cells is lower, and the secretion amounts of antibacterial peptides and lysozymes are less. A content of 400 mg / L may have caused certain interference to metabolism and immune regulation, which is also not conducive to the clearance of metabolic wastes and the improvement of immune function.

[0168] Groups VIII and IV are superior to Control Group II: Carnosic acid can activate the metabolic and immune-related mechanisms of mud crabs. In terms of metabolism, it may promote the expression and activity of enzymes involved in the treatment of metabolic wastes in the liver and kidneys, accelerating the metabolism and excretion of urea nitrogen, ammonia, and uric acid; in terms of immunity, carnosic acid can stimulate the activity of immune cells, promoting the synthesis and secretion of antibacterial peptides and lysozymes, so that Groups VIII and IV containing carnosic acid are superior to Control Group II without carnosic acid in terms of indicators related to the accumulation of metabolic wastes.

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

[0170] Table 9 Effects of different carnosic acid contents in the microcirculation preparation of mud crabs on indicators related to 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] It can be seen from Table 9 that the high-density lipoprotein content is ranked from high to low as Group VII > Groups VI and VIII > Groups IX and X > Control Group II; the low-density lipoprotein content is ranked from low to high as Group VII < Groups VI and VIII < Groups IX and X ≈ Control Group II; the activities of fatty acid synthase and carnitine palmitoyl transferase are ranked from high to low as Group VII > Groups VI ≈ VIII > Groups IX ≈ X > Control Group II.

[0174] Principle analysis:

[0175] Component group seven is superior to component groups six and eight: 300 mg / L of carnosic acid can optimally regulate the fat and energy metabolism of mud crabs. For high-density lipoprotein (HDL), it may promote the synthesis of HDL or inhibit its catabolism, resulting in an increase in the HDL content. HDL helps transport cholesterol in peripheral tissues back to the liver for metabolism, thereby reducing the cholesterol content in the blood, decreasing the deposition of fat on the blood vessel walls, and being beneficial to maintaining a healthy physiological state. For low-density lipoprotein (LDL), 300 mg / L of carnosic acid may inhibit the synthesis of LDL or promote its clearance, resulting in a decrease in the LDL content. In terms of fatty acid synthase (FAS) and carnitine palmitoyl transferase (CPT), 300 mg / L of carnosic acid may increase the activities of FAS and CPT by regulating the expression of related genes; FAS is responsible for the synthesis of fatty acids, and CPT is involved in the transport of fatty acids into mitochondria for β-oxidation to provide energy. The increase in the activities of these two enzymes is beneficial to the synthesis of fat, energy conversion, and reserve, providing sufficient energy for the growth and physiological activities of mud crabs.

[0176] Component groups six and eight are superior to groups nine and ten: 250 mg / L and 350 mg / L of carnosic acid can maintain the balance of fat and energy metabolism in mud crabs to a certain extent. In contrast, a content of 200 mg / L has a weak regulatory effect on the enzymes related to fat and energy metabolism, resulting in lower activities of FAS and CPT, lower efficiency of fat synthesis and energy conversion. At the same time, the regulation of HDL and LDL metabolism is insufficient, resulting in a lower HDL content and a higher LDL content, which is not conducive to the reasonable metabolism of fat and energy reserve. A content of 400 mg / L may have an excessive or abnormal impact on fat and energy metabolism, which is not conducive to the stability of fat and energy metabolism.

[0177] Component groups nine and ten are superior to control group two: Carnosic acid can activate the enzymes and pathways related to fat and energy metabolism in mud crabs. It may bind to receptors in cells, initiate a series of signal transduction processes, and regulate the expression of genes related to fat and energy metabolism. For example, it promotes the transcription of FAS and CPT genes, increases the synthesis amount of these two enzymes, thereby increasing the enzyme activities, and promoting fat synthesis and energy conversion. In terms of HDL and LDL metabolism, carnosic acid may regulate the expression and activities of related proteins involved in the synthesis and metabolism of HDL and LDL in the liver and other tissues, resulting in an increase in the HDL content and a decrease in the LDL content, optimizing fat metabolism. Therefore, component groups eight and four containing carnosic acid are superior to control group two without carnosic acid in these indicators.

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

[0179] Table 10 Effects of Different Carnosic Acid Contents in the Microcirculation Preparation of Scylla serrata on the Indexes Related to Postpartum Tissue Repair of Scylla serrata

[0180]

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

[0182] As can be seen from Table 10, in the four indexes closely related to tissue repair, namely hydroxyproline, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-β (TGF-β), the data of Group Seven were significantly higher than those of other groups (except for specific cases without significant differences). From Group Seven to Control Group Two, the values of each index showed a decreasing trend, and the differences between groups were distinguished by letters. The same letter indicates no significant difference.

[0183] Principle analysis:

[0184] Group Seven is superior to Group Six and Group Eight: The content of 300 mg / L of carnosic acid has the most significant promoting effect on the postpartum tissue repair of Scylla serrata. Hydroxyproline is an important component of collagen. The carnosic acid with a content of 300 mg / L may promote the synthesis of collagen by stimulating the activities of related cells such as fibroblasts, thereby significantly increasing the content of hydroxyproline. For epidermal growth factor (EGF), it can promote the proliferation, differentiation, and migration of epidermal cells. The carnosic acid with a content of 300 mg / L may activate the signal transduction pathway in related cells, enabling epidermal cells to secrete more EGF and accelerating the repair and regeneration of epidermal tissue. Platelet-derived growth factor (PDGF) can attract inflammatory cells, fibroblasts, etc. in tissue repair, promoting cell proliferation and the synthesis of extracellular matrix. The carnosic acid with a content of 300 mg / L may promote the secretion of PDGF by regulating the expression of related genes. Transforming growth factor-β (TGF-β) has multiple functions such as promoting the synthesis of extracellular matrix, regulating cell proliferation and differentiation, etc. The carnosic acid with a content of 300 mg / L 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] Group Six and Group Three are superior to Group Nine and Group Ten: The contents of 250 mg / L and 350 mg / L of carnosic acid can promote the activities of tissue repair-related cells and the secretion of factors to a certain extent; however, the content of 200 mg / L is relatively low and cannot fully activate the related mechanisms of tissue repair, 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 a certain interference with the normal physiological functions of cells, leading to cell metabolic disorders and instead inhibiting the normal secretion and function of tissue repair-related factors.

[0186] Group VIII and Group IV are superior to Control Group II: The presence of carnosic acid can initiate the tissue repair mechanism of mud crabs. It may act on various cells in the mud crabs, such as fibroblasts, epidermal cells, platelets, etc., 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; prompts platelets to release PDGF, attracting more cells to participate in the tissue repair process; induces related cells to secrete TGF-β, regulating cell proliferation and differentiation, and promoting the synthesis of extracellular matrix. Therefore, Group VIII and Group IV containing carnosic acid are significantly superior to Control Group II without carnosic acid in terms of tissue repair-related indicators.

[0187] Example 3 Oleuropein Content Test

[0188] Oleuropein is a phenolic compound extracted from olive leaves, and its main effect is to improve the physiological phenomenon of metabolic waste accumulation in female mud crabs. As a biologically active compound, oleuropein has a significant impact on the metabolic health of female mud crabs after parturition by regulating multiple metabolic pathways in their bodies. First, in terms of the metabolism of urea nitrogen and ammonia in hemolymph, oleuropein effectively promotes the conversion of ammonia to urea by activating liver urea cycle enzymes and excretes urea out of the body through the excretion function of the kidneys. This process not only promotes the synthesis of urea 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 in female mud crabs after parturition and improving their metabolic function. Second, oleuropein also promotes the clearance of metabolic waste by regulating the metabolism of uric acid in hemolymph. Specifically, oleuropein promotes the filtration and excretion of uric acid by enhancing the function of gill cells in mud crabs, greatly reducing the accumulation of uric acid in hemolymph. This process speeds up the clearance rate of uric acid and reduces its concentration in hemolymph, thus resolving the problem of metabolic waste accumulation caused by uric acid accumulation in female mud crabs after parturition. In addition, oleuropein also enhances the secretion of antimicrobial peptides and lysozymes by stimulating the immune system. These immune substances play an important role in clearing bacteria and toxins in the blood. This effect of oleuropein not only promotes the synthesis of antimicrobial peptides and lysozymes but also accelerates the clearance of bacteria and toxins, thereby improving the immune function of female mud crabs and reducing the problem of metabolic waste accumulation caused by inefficient immune system, further improving their metabolic health. In summary, oleuropein significantly promotes the clearance of metabolic waste and the recovery of metabolic function in female mud crabs after parturition by regulating multiple metabolic pathways.

[0189] I. Preparation of Mixed Reagents

[0190] Weigh 17.5, 20, 22.5, 25, and 27.5 grams of olive leaves respectively, and obtain mixed preparations with oleuropein contents of 250 mg / L, 300 mg / L, 350 mg / L, 200 mg / L, and 400 mg / L respectively according to the operations in Steps 1 to 3 of Example 1. Denote the auxiliary liquid as the blank control group, and denote them respectively as:

[0191] Group 11: 250 mg / L oleuropein,

[0192] Group 12: 300 mg / L oleuropein,

[0193] Group 13: 350 mg / L oleuropein,

[0194] Group 14: 200 mg / L oleuropein,

[0195] Group 15: 400 mg / L oleuropein,

[0196] Control Group 3: Auxiliary liquid (blank group).

[0197] II. Breeding experiment

[0198] It was carried out in the indoor breeding pond of Dongying Kenuo Aquaculture Co., Ltd. from September to October 2023. Collect 90 female mud crabs within 1 - 3 days after giving birth from the southern coastal area again, disinfect them centrally after air transportation to the experimental site, and then put them into 2 indoor cement ponds of 30 m 2 for temporary cultivation for 1 - 2 days. After temporary cultivation, put 5 female mud crabs into each of the 18 laboratory ponds of 20 m 2 for the experiment. Put 5 female crabs after giving birth into one pond, and put 5 tiles into each pond at the same time. Among them, Ponds 1 - 3 are Group 11, Ponds 4 - 6 are Group 12, Ponds 7 - 9 are Group 13, Ponds 10 - 12 are Group 14, Ponds 13 - 15 are Group 15, and Ponds 16 - 18 are Control Group 3. The purpose of this example is to explore the key compounds in the microcirculation preparation for mud crabs after giving birth that are beneficial to the postnatal rejuvenation of mud crabs. The experimental materials and their sources in this example are the same as those 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 of mud crabs on the growth indexes of mud crabs

[0202]

[0203]

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

[0205] As shown in Table 11, in terms of survival rate, weight gain rate and fatness, group 12 performed best, with values ​​significantly higher than other groups (except for specific cases where there was no significant difference). In terms of recovery time, group 12 had the shortest time. From group 12 to group 3, each growth index showed a decreasing trend, with letters used to distinguish differences between groups, and the same letters indicated no significant difference.

[0206] Principle analysis:

[0207] Component group 12 is superior to component groups 11 and 13: the oleuropein content of 300 mg / L may best meet the physiological needs of blue crabs for postpartum growth. From the perspective of nutrient utilization, it may optimize the blue crab intestine's absorption mechanism of nutrients, perhaps by regulating the morphology and function of intestinal villi, increasing the absorption area and efficiency of nutrients, allowing blue crabs to absorb more energy and nutrients for growth, which is reflected in a higher weight gain rate and fatness; in terms of survival guarantee, oleuropein may improve the survival rate by regulating the blue crab's immune system, enhancing the activity of immune cells, and improving resistance to pathogens; and in the recovery process, it promotes the balanced regulation of hormones in the body and accelerates the recovery of body functions, so the recovery time is the shortest.

[0208] Component groups 11 and 13 are better than groups 14 and 15: oleuropein contents of 250mg / L and 350mg / L can still maintain the growth and recovery mechanisms of blue crabs to a certain extent. The content of 200mg / L may not be able to fully activate the growth and immune-related signaling pathways of blue crabs due to insufficient dosage. For example, in the nutrient absorption signal transduction, the expression of certain key transport proteins cannot be effectively activated, resulting in limited nutrient intake, which in turn affects growth indicators. The content of 400mg / L may have certain negative effects, perhaps over-activating certain metabolic feedback mechanisms, inhibiting normal growth and immune regulation, such as interfering with the normal secretion level of hormones, which hinders the growth and recovery process.

[0209] Component groups 13 and 14 are superior to control groups 3: Compared with control groups 3, oleuropein in component groups 13 and 14 can activate the basic regulatory mechanism for growth and recovery of blue crabs. Oleuropein may act directly or indirectly at the cellular level of blue crabs to increase muscle growth, thereby increasing the rate of weight gain. At the same time, it activates the activity of immune cells, enhances immune defense, and improves survival rate. In terms of energy metabolism, it may regulate the function of mitochondria, improve energy production efficiency, provide power for growth and recovery, and make the growth indicators better than the three control groups without oleuropein.

[0210] 2. Analysis of nutrient transport index results

[0211] Table 12 Effects of Different Oleuropein Contents in the Microcirculation Preparation of Scylla serrata on the Indicators Related to Postpartum Nutrient Transport in Scylla serrata

[0212]

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

[0214] As can be seen from Table 12, in terms of hepatopancreas index and gonad index, the content of Component Twelve was the highest, showing a decreasing trend from Component Twelve to Control Three. The contents of triglyceride and total cholesterol were opposite, with the content of Component Twelve being the lowest, showing an increasing trend from Component Twelve to Control Three. By marking the data differences with letters, there were no significant differences between groups with the same letter.

[0215] Principle analysis:

[0216] Component Twelve was superior to Component Eleven and Three: 300 mg / L of oleuropein might have the best regulatory effect on the nutrient transport and distribution mechanism of Scylla serrata. In the hepatopancreas, it might promote the expression of genes related to the synthesis and storage of glycogen, increase the uptake and transformation ability of hepatopancreas cells to nutrients, thus improving the hepatopancreas index and optimizing the transport and distribution of nutrients in the body.

[0217] Component Eleven and Three were superior to Four and Five: The contents of 250 mg / L and 350 mg / L of oleuropein could still maintain the normal regulation of nutrient transport and metabolism to a certain extent. The content of 200 mg / L might not be able to fully activate the signal molecules and enzymes related to nutrient transport and metabolism. For example, in the hepatopancreas, it could not effectively induce the expression of glycogen synthase, resulting in insufficient storage capacity of the hepatopancreas for nutrients and a lower hepatopancreas index. In the gonad, it could not fully stimulate the signal pathway related to gonad development, resulting in a lower gonad index. At the same time, the regulatory effect on lipid metabolism enzymes was weak, resulting in higher contents of triglyceride and total cholesterol. The content of 400 mg / L interfered with the normal metabolism of cells. For example, it might inhibit the normal activity regulation of lipid metabolism enzymes, leading to lipid metabolism disorders and also making the nutrient transport and metabolism indicators not ideal.

[0218] Component Thirteen and Four were superior to Control Three: Oleuropein could activate the related pathways of nutrient transport and metabolism in Scylla serrata. In the hepatopancreas and gonad, it might promote the expression of nutrient transporter proteins on the cell membrane, increasing the uptake of nutrients by cells; in the gonad, it promoted the absorption of nutrients by gonad cells, promoted gonad development, and increased the gonad index; in terms of blood lipid regulation, it reduced the synthesis of triglyceride and total cholesterol, making Component Thirteen and Fourteen containing oleuropein superior to Control Three without oleuropein in the indicators related to nutrient transport.

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

[0220] Table 13 Effects of different oleuropein contents in the microcirculation preparation of mud crabs on indicators related to the accumulation of metabolic waste after parturition in mud crabs

[0221]

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

[0223] It can be seen from Table 13 that the contents of urea nitrogen, ammonia and uric acid are sorted from low to high as Group Twelve < Group Eleven ≈ Group Thirteen < Group Fourteen ≈ Group Fifteen < Control Group Three; the contents of antibacterial peptides and lysozyme are sorted from high to low as Group Twelve > Group Eleven ≈ Group Thirteen > Group Fourteen ≈ Group Fifteen > Control Group Three.

[0224] Principle analysis:

[0225] Group Twelve is superior to Group Eleven and Group Thirteen: Oleuropein at 300 mg / L can most effectively promote the excretion of metabolic waste and the improvement of immune function in mud crabs. In terms of nitrogen metabolism, it accelerates the process of ammonia conversion to urea, thus reducing the contents of urea nitrogen and ammonia in the blood; for uric acid, it may promote the excretion function of the kidneys for uric acid, perhaps by regulating the reabsorption and secretion mechanisms of the renal tubules for uric acid, reducing the accumulation of uric acid in the body; in terms of immune regulation, oleuropein at 300 mg / L may activate signaling pathways such as NF-κB in immune cells, promoting the transcription and translation of genes for antibacterial peptides and lysozyme, enabling immune cells to secrete more antibacterial peptides and lysozyme, and enhancing the immune defense ability of mud crabs. Group Eleven and Group Thirteen are superior to Group Fourteen and Group Fifteen: Oleuropein at 250 mg / L and 350 mg / L can better maintain the metabolic and immune balance of mud crabs; the content of 200 mg / L may be insufficient in dosage to fully activate the key enzymes and signaling pathways related to nitrogen metabolism and immune regulation; for example, in the liver, it cannot effectively increase the activity of urea cycle enzymes, resulting in the inability of ammonia to be promptly converted to urea and excreted, and the increase in the contents of urea nitrogen and ammonia in the blood. In immune cells, it cannot fully activate the immune signaling pathway, resulting in less secretion of antibacterial peptides and lysozyme. The content of 400 mg / L may have an excessive or abnormal impact on metabolism and immune regulation. For example, it may interfere with the normal excretion regulation mechanism of the kidneys for uric acid, or overactivate the immune feedback inhibition mechanism, leading to a decrease in the activity of immune cells and a reduction in the secretion of antibacterial peptides and lysozyme, which is also not conducive to the clearance of metabolic waste and the maintenance of immune function.

[0226] Component groups thirteen and fourteen are superior to control group three: Oleuropein can initiate the metabolic waste excretion and immune regulation mechanisms of mud crabs. In terms of metabolism, it may bind to receptors on the surfaces of hepatocytes and renal cells, activate the intracellular metabolic regulation signaling pathway, and promote the processing and excretion of metabolic wastes. For example, it promotes the metabolic conversion of ammonia by hepatocytes and the excretion of urea nitrogen and uric acid by renal cells. In terms of immunity, oleuropein may activate immune cells such as macrophages and lymphocytes, and promote them to secrete antimicrobial peptides and lysozymes. For example, by stimulating the pattern recognition receptors on the surface of macrophages, it initiates the intracellular immune response and promotes the synthesis and release of antimicrobial peptides and lysozymes, making component groups thirteen and fourteen containing oleuropein superior to control group three in terms of indicators related to the accumulation of metabolic wastes.

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

[0228] Table 14 Effects of different oleuropein contents in the microcirculation preparation of mud crabs on indicators related to postpartum fat and energy reserve of mud crabs

[0229]

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

[0231] As can be seen from Table 14, the high-density lipoprotein content is ranked from high to low as component group twelve > component group eleven ≈ component group thirteen > component group fourteen ≈ component group fifteen > control group three; the low-density lipoprotein content is ranked from low to high as component group twelve < component group eleven ≈ component group thirteen < component group fourteen ≈ component group fifteen < control group three; the activities of fatty acid synthase and carnitine palmitoyltransferase are ranked from high to low as component group twelve > component group eleven ≈ component group thirteen > component group fourteen ≈ component group fifteen > control group three.

[0232] Principle analysis: Component 12 is superior to Component 11 and Component 3: Oleuropein at 300 mg / L is the most effective in regulating the fat and energy metabolism of mud crabs. For high-density lipoprotein (HDL), it may promote the assembly and secretion of HDL by regulating the synthesis and secretion of related proteins such as apolipoprotein A-I in the liver, thereby increasing the content of HDL. HDL can transport cholesterol in peripheral tissues back to the liver for metabolism, reduce the cholesterol content in the blood, and reduce the deposition of fat on the blood vessel wall, which is beneficial to maintaining normal physiological functions. For low-density lipoprotein (LDL), it may increase the clearance of LDL and reduce the LDL content by inhibiting the degradation of LDL receptor-related proteins in the liver. In terms of fatty acid synthase (FAS) and carnitine palmitoyltransferase (CPT), oleuropein at 300 mg / L may regulate the intracellular signal transduction pathway, such as activating signal pathways such as AMPK-mTOR, promoting the expression of FAS and CPT genes, and increasing the activities of these two enzymes. FAS is responsible for fatty acid synthesis, and CPT is involved in the transport of fatty acids into mitochondria for β-oxidation to provide energy. The increase in the activities of these two enzymes is beneficial to fat synthesis, energy conversion, and storage, providing sufficient energy for the growth and physiological activities of mud crabs.

[0233] Component 11 and Component 13 are superior to Component 14 and Component 15: Oleuropein at 250 mg / L and 350 mg / L can maintain the balance of fat and energy metabolism of mud crabs to a certain extent. The content of 200 mg / L may not be able to fully activate the signal pathways and gene expressions related to fat and energy metabolism. For example, in regulating the metabolism of HDL and LDL, it cannot effectively regulate the synthesis and degradation of related proteins, resulting in a lower HDL content and a higher LDL content. In regulating the activities of FAS and CPT, the related signal pathways cannot be fully activated, resulting in lower activities of FAS and CPT and low efficiency of fat synthesis and energy conversion. The content of 400 mg / L may have an excessive or abnormal impact on fat and energy metabolism. For example, it may over-activate the fat decomposition pathway, resulting in reduced fat synthesis, or the metabolic regulation of HDL and LDL is out of balance, which is also not conducive to the stability of fat and energy metabolism.

[0234] Component 13 and Component 14 are superior to Control Group 3: Oleuropein can increase the synthesis amount of these two enzymes, FAS and CPT, thereby increasing the enzyme activities and promoting fat synthesis and energy conversion; in the metabolism of HDL and LDL, oleuropein may regulate the expression and activities of related proteins involved in the synthesis and metabolism of HDL and LDL in the liver and other tissues, resulting in an increase in HDL content and a decrease in LDL content, optimizing fat metabolism. Therefore, Component 13 and Group 4 containing oleuropein are superior to Control Group 3 without oleuropein in these indicators.

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

[0236] Table 15 Effects of Different Oleuropein Contents in the Microcirculation Preparation of Scylla serrata on the Indexes Related to Postpartum Tissue Repair of Scylla serrata

[0237]

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

[0239] As can be seen from Table 15, among the four key indexes of tissue repair, namely hydroxyproline, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-β (TGF-β), the twelfth component group showed the highest values, demonstrating the most excellent performance. From the twelfth component group to the third control group, the values of each index showed a decreasing trend overall.

[0240] Principle analysis:

[0241] The twelfth component group is superior to the eleventh and thirteenth component groups: The concentration of 300mg / L of oleuropein has the strongest promoting effect on the postpartum tissue repair of Scylla serrata. In terms of hydroxyproline, which is a key amino acid in collagen, 300mg / L of oleuropein may greatly enhance the activity of fibroblasts, effectively promote the synthesis of collagen, and thus significantly increase the content of hydroxyproline; in terms of epidermal growth factor (EGF), oleuropein at this concentration can promote a large amount of EGF secretion by epidermal cells, effectively accelerating the repair and regeneration process of epidermal tissue; for platelet-derived growth factor (PDGF), it enables platelets and other related cells to secrete a large amount of PDGF, attracting more inflammatory cells, fibroblasts, etc. to migrate to the damaged tissue site, effectively promoting cell proliferation and the synthesis of extracellular matrix; and transforming growth factor-β (TGF-β) has many important functions such as regulating cell proliferation, differentiation, and promoting the synthesis of extracellular matrix. 300mg / L of oleuropein may bind to specific receptors on the cell surface, greatly enhancing the expression and secretion levels of TGF-β, and comprehensively and deeply promoting the tissue repair process.

[0242] Components 11 and 13 are better than 14 and 15: 250mg / L and 350mg / L oleuropein content can still effectively promote the activity of tissue repair-related cells and the secretion of key factors to a certain extent; however, the content of 200mg / L is relatively low and cannot fully activate a series of key mechanisms involved in tissue repair. For example, in fibroblasts, it may not provide sufficient energy and raw materials for collagen synthesis, or it may not effectively activate enzymes related to collagen synthesis, resulting in low hydroxyproline content; in epidermal cells, platelets and other cells, the secretion stimulation of related growth factors is insufficient, resulting in limited secretion of growth factors such as EGF, PDGF, TGF-β, etc., which seriously affects the speed and effect of tissue repair; 400mg / L content may interfere with the normal physiological function of cells to a certain extent. For example, it may over-activate certain signal pathways, break the signal balance in cells, lead to cell metabolism disorders, and inhibit the normal secretion and function of tissue repair-related factors, making the tissue repair effect unsatisfactory.

[0243] Component groups 13 and 4 are better than control groups 3: Oleuropein can effectively activate the tissue repair mechanism of blue crabs. It may play a role in a variety of key cells in the blue crab, such as fibroblasts, epidermal cells, platelets, etc., and significantly promote these cells to secrete key factors necessary for tissue repair. Specifically, oleuropein may regulate the gene expression regulatory network in the cells, so that fibroblasts significantly increase the synthesis of collagen, 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 prompts platelets to release a large amount of PDGF, actively attracting more cells to participate in the process of tissue repair; it induces related cells to secrete a large amount of TGF-β, finely regulates the proliferation and differentiation process of cells, and significantly promotes the synthesis of extracellular matrix. Therefore, component groups 13 and 4 containing oleuropein are significantly better than the control groups without oleuropein in terms of tissue repair related indicators.

[0244] Example 4 Terpene alcohol content experiment

[0245] Terpenol is a terpene compound extracted from perilla leaves, and its main effect is to improve the physiological phenomenon of insufficient fat and energy reserves in female mud crabs. As a biologically active compound, terpenol has a significant impact on blood lipid metabolism, fat synthesis and decomposition, and energy supply in female mud crabs by regulating multiple metabolic pathways. First, in terms of the metabolism of high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in hemolymph, terpenol effectively promotes the reverse transport of cholesterol by increasing the level of HDL, while clearing LDL in the blood and preventing lipid deposition. This process not only promotes the synthesis of HDL but also accelerates the clearance of LDL, significantly reducing its accumulation in the blood, thus optimizing blood lipid metabolism and alleviating the problem of blocked nutrient transport caused by insufficient fat and energy reserves. Second, terpenol also has an important impact on fat synthesis by regulating the activity of fatty acid synthase (FAS) in the hepatopancreas. Specifically, terpenol can reduce fat synthesis and maintain the balance of fat metabolism. Although this process promotes the synthesis and storage of fatty acids, it also reduces the excessive storage of fat, avoiding fat metabolism imbalance, thus solving the problem of insufficient fat reserves in female mud crabs after parturition. In addition, terpenol promotes the entry of long-chain fatty acids into mitochondria for β-oxidation by activating carnitine palmitoyltransferase (CPT) in the hepatopancreas, accelerating fat decomposition and releasing more energy. This process not only promotes the synthesis of fatty acid oxidase but also significantly accelerates the β-oxidation of long-chain fatty acids, providing continuous energy supply for female mud crabs after parturition and solving the problem of slow physiological recovery caused by insufficient energy reserves. In summary, terpenol has a positive impact on the blood lipid metabolism, fat synthesis and decomposition, and energy supply of female mud crabs by regulating multiple metabolic pathways, promoting their physiological health and recovery.

[0246] I. Preparation of mixed reagents

[0247] Weigh 12.5, 15, 17.5, 20, and 22.5 grams of perilla leaves respectively, and obtain mixed preparations with terpenol contents of 200 mg / L, 250 mg / L, 300 mg / L, 150 mg / L, and 350 mg / L respectively according to the operations in Steps 1 to 3 of Example 1. Denote the auxiliary solution as the blank control group, and denote them respectively as:

[0248] Component Group 16: 200 mg / L terpenol,

[0249] Component Group 17: 250 mg / L terpenol,

[0250] Component Group 18: 300 mg / L terpenol,

[0251] Component Group 19: 150 mg / L terpenol,

[0252] Component Group Twenty: 350 mg / L terpineol,

[0253] Control Group Four: Auxiliary solution (blank group).

[0254] II. Breeding Experiment

[0255] From September to October 2023, it was carried out in the indoor breeding pond of Dongying Kenuo Aquaculture Co., Ltd. Another 90 female mud crabs within 1 - 3 days after giving birth were collected from the southern coastal area, transported to the experimental site by air, and then centrally disinfected. Then they were put into two indoor cement ponds of 30 m 2 for temporary cultivation for 1 - 2 days. After temporary cultivation, every 5 paste crabs were respectively put into 18 laboratory ponds of 20 m 2 for the experiment. 5 female crabs after giving birth were put into one pond, and at the same time, 5 tiles were put into each pond. Among them, ponds 1 - 3 were Component Group Sixteen, ponds 4 - 6 were Component Group Seventeen, ponds 7 - 9 were Component Group Eighteen, ponds 10 - 12 were Component Group Nineteen, ponds 13 - 15 were Component Group Twenty, and ponds 16 - 18 were Control Group Four. The purpose of this example is to explore the key compounds in the mud crab postpartum microcirculation preparation that are beneficial to the postpartum rejuvenation of mud crabs. The experimental materials and their sources in this example are the same as those in Example 1.

[0256] III. Comparison of Experimental Data

[0257] 1. Analysis of Growth Index Results

[0258] Table 16 Effects of Different Terpineol Contents in Mud Crab Microcirculation Preparation on Mud Crab Growth Indexes

[0259] Survival rate (%) Weight gain rate (%) <![CDATA[Condition factor (g / cm 3 )]]> Recovery time (t, days) Component group 16 <![CDATA[67.34±2.07 b > <![CDATA[28.96±1.29 b > <![CDATA[44.67±1.81 b > <![CDATA[27.41±1.15 c > Component group 17 <![CDATA[74.22±1.99 a > <![CDATA[34.23±1.20 a > <![CDATA[52.85±1.67 a > <![CDATA[22.10±0.82 d > Component group 18 <![CDATA[67.68±2.07 b > <![CDATA[28.30±1.29 b > <![CDATA[42.96±1.62 b > <![CDATA[25.94±1.07 c > Component group 19 <![CDATA[56.51±2.07 c > <![CDATA[21.83±1.15 c > <![CDATA[30.73±1.73 c > <![CDATA[30.81±1.26 b > Component group 20 <![CDATA[58.31±2.07 c > <![CDATA[23.73±1.27 c > <![CDATA[32.90±1.29 c > <![CDATA[29.45±1.08 b > Control group 4 <![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] It can be seen from Table 16 that in the three growth indexes of survival rate, weight gain rate, and condition factor, the values of Component Group Seventeen are significantly higher than those of other groups (except for cases with no significant differences), and the recovery time of Component Group Seventeen is the shortest. Overall, there is a trend that from Component Group Seventeen to Control Group Four, each growth index gradually deteriorates, and the differences between groups are distinguished by letters. The same letter indicates no significant difference.

[0262] Principle Analysis:

[0263] Group 17 is superior to Group 16 and Group 18: The content of terpineol at 250 mg / L may be most suitable for the growth requirements of mud crabs after parturition, accelerating the metabolism of mud crabs, improving the digestion and absorption efficiency of food, and thus providing more energy and nutrients for the growth and recovery of the body, manifested as higher weight gain rate and condition factor; at the same time, this appropriate content of terpineol may also enhance the immune function of mud crabs, improve their resistance to diseases and environmental stress, resulting in higher survival rate; in terms of recovery time, due to the promoting effect of terpineol on various body functions, the speed of the body's recovery after parturition of mud crabs is accelerated and the recovery time is shortened.

[0264] Group 16 and Group 18 are superior to Group 19 and Group 20: Although the content of terpineol at 200 mg / L and 300 mg / L is not as effective as that at 250 mg / L, it can still maintain the growth and recovery mechanism of mud crabs to a certain extent; the content of terpineol at 150 mg / L may be too low to fully activate the physiological processes related to growth and immunity in mud crabs; the content of terpineol at 350 mg / L may be too high, having a certain negative impact on the physiological functions of mud crabs. For example, it overstimulates the endocrine system, resulting in the inhibition of the growth and recovery process.

[0265] Group 19 and Group 20 are superior to Control Group 4: Compared with Control Group 4, Group 18 and Group 4 contain a certain amount of terpineol, which can initiate the growth and immune regulation mechanisms in mud crabs; terpineol promotes the absorption of nutrients by mud crabs and improves the utilization rate of nutrients. At the same time, it may activate the activity of immune cells and enhance the immune defense ability of mud crabs, thus increasing the survival rate; in terms of energy metabolism, terpineol may regulate the energy metabolism pathway in cells, making the generation and utilization of energy more efficient, providing power for the growth and recovery of mud crabs, so it is superior to Control Group 4 without terpineol in terms of growth indicators.

[0266] 2. Results analysis of nutritional transport indicators

[0267] Table 17 Effects of different terpineol contents in the microcirculation preparation of mud crabs on the nutritional transport-related indicators of mud crabs after parturition

[0268] Hepatosomatic index (%) Gonadosomatic index (%) Triglyceride (mmol / L) Total cholesterol (mmol / L) Component group 16 <![CDATA[6.0±0.28 b > <![CDATA[4.94±0.24 b > <![CDATA[1.37±0.05 c > <![CDATA[1.99±0.07 c > Component group 17 <![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 -->]]> Component group 18 <![CDATA[6.1±0.26 b > <![CDATA[5.13±0.19 b > <![CDATA[1.38±0.06 c > <![CDATA[2.07±0.07 c > Component group 19 <![CDATA[5.2±0.24 c > <![CDATA[4.37±0.29 c > <![CDATA[1.54±0.04 b > <![CDATA[2.20±0.05 b > Component group 20 <![CDATA[5.0±0.71 c > <![CDATA[4.47±0.23 c > <![CDATA[1.56±0.02 b > <![CDATA[2.25±0.04 b > Control group 4 <![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] It can be seen from Table 17 that in terms of hepatopancreas index and gonad index, the values of Group 17 are the highest, showing a decreasing trend from Group 17 to Control Group 4. On the contrary, for the contents of triglyceride and total cholesterol, Group 17 is the lowest, showing an increasing trend from Group 17 to Control Group 4. By using letters to mark the significant differences in data between groups, the same letter indicates no significant difference.

[0271] Principle analysis: Component group 17 is superior to component groups 16 and 18: Terpineol at 250 mg / L may play an optimal regulatory role in the nutrient transport and distribution system of mud crabs; in the hepatopancreas, it may promote the expression of genes related to the synthesis and storage of glycogen, increasing the ability of hepatopancreas cells to uptake and transform nutrients, thereby increasing the hepatopancreas index. For the gonads, it may stimulate the signal pathways related to the proliferation and differentiation of gonadal cells, promoting gonadal development and increasing the gonad index. In terms of blood lipid regulation, it may regulate the activity of key enzymes in lipid metabolism, such as upregulating the activity of lipolytic enzymes to promote the decomposition of triglycerides, while downregulating the activity of cholesterol synthase to reduce the synthesis of total cholesterol, resulting in a decrease in the content of triglycerides and total cholesterol in the blood and optimizing the transport and distribution of nutrients in the body.

[0272] Component groups 16 and 18 are superior to component groups 19 and 20: Terpineol contents of 200 mg / L and 300 mg / L can still promote the normal regulation of nutrient transport and metabolism to a certain extent; a content of 150 mg / L cannot effectively induce the expression of glycogen synthase in the hepatopancreas, resulting in insufficient storage capacity of the hepatopancreas for nutrients and a lower hepatopancreas index. In the gonads, it cannot sufficiently stimulate gonadal development, resulting in a low gonad index; at the same time, the regulatory effect on lipid-metabolizing enzymes is weak, leading to higher triglyceride and total cholesterol contents; a content of 350 mg / L may overactivate certain feedback inhibition mechanisms or interfere with the normal metabolism of cells. For example, it may inhibit the normal activity regulation of lipid-metabolizing enzymes, leading to lipid metabolism disorders and also making the nutrient transport and metabolism indicators unsatisfactory.

[0273] Component groups 19 and 20 are superior to control group 4: Terpineol can promote the nutrient transport and metabolism of mud crabs; in the hepatopancreas and gonads, it may promote the expression of nutrient transporter proteins on the cell membrane, increasing the uptake of nutrients by cells. For example, it promotes the uptake of nutrients such as glucose and amino acids by hepatopancreas cells, increasing the hepatopancreas index. In the gonads, it promotes the absorption of nutrients by gonadal cells, promoting gonadal development and increasing the gonad index; in terms of blood lipid regulation, it reduces the synthesis of triglycerides and total cholesterol or promotes their catabolism, making component groups 19 and 20 containing terpineol superior to control group 4 without terpineol in terms of nutrient transport-related indicators.

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

[0275] Table 18 Effects of different terpineol contents in the microcirculation preparation of mud crabs on indicators related to the accumulation of postpartum metabolic wastes in mud crabs

[0276] Urea nitrogen (mg / dL) Ammonia (mg / L) Uric acid (mg / dL) Antibacterial peptide (μg / mL) Lysozyme (U / mL) Component group 16 <![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 > Component group 17 <![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 > Component group 18 <![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 > Component group 19 <![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 > Component group 20 <![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 > Control group 4 <![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] As can be seen from Table 18, the contents of urea nitrogen, ammonia, and uric acid are sorted from low to high as Group Seventeen < Group Sixteen ≈ Group Eighteen < Group Nineteen, Group Twenty < Control Group Four; the contents of antibacterial peptides and lysozyme are sorted from high to low as Group Seventeen > Group Sixteen, Group Eighteen > Group Nineteen, Group Twenty > Control Group Four.

[0279] Principle analysis:

[0280] Group Seventeen is superior to Group Sixteen and Group Eighteen: 250 mg / L of terpenol can most effectively promote the excretion of metabolic wastes and the improvement of immune function in mud crabs. In terms of nitrogen metabolism, it may accelerate the conversion of ammonia to urea by regulating the activities of urea cycle-related enzymes in the liver, such as arginase and ornithine carbamoyltransferase, thereby reducing the contents of urea nitrogen and ammonia in the blood; for uric acid, it may promote the excretion function of the kidneys for uric acid, perhaps by regulating the reabsorption and secretion mechanisms of uric acid in the renal tubules, reducing the accumulation of uric acid in the body. In immune regulation, 250 mg / L of terpenol may activate signal pathways such as NF-κB in immune cells, promoting the transcription and translation of antibacterial peptide and lysozyme genes, enabling immune cells to secrete more antibacterial peptides and lysozymes, and enhancing the immune defense ability of mud crabs.

[0281] Group Sixteen and Group Eighteen are superior to Group Nineteen and Group Twenty: 200 mg / L and 300 mg / L of terpenol can better maintain the metabolic and immune balance of mud crabs; the content of 150 mg / L may be insufficient in dose and unable to effectively increase the activity of urea cycle enzymes in the liver, resulting in the failure of ammonia to be promptly converted into urea and excreted, and the increase in the contents of urea nitrogen and ammonia in the blood; in immune cells, the immune signal pathway cannot be fully activated, resulting in less secretion of antibacterial peptides and lysozymes; the content of 350 mg / L may have an excessive or abnormal impact on metabolism and immune regulation. For example, it may interfere with the normal excretion regulation mechanism of uric acid in the kidneys, or overactivate the immune feedback inhibition mechanism, leading to a decrease in the activity of immune cells and a reduction in the secretion of antibacterial peptides and lysozymes, which is also not conducive to the clearance of metabolic wastes and the maintenance of immune function.

[0282] Component groups 19 and 20 are superior to control group 4: Terpenol can initiate the metabolic waste excretion and immune regulation mechanisms of mud crabs. In terms of metabolism, it may bind to receptors on the surfaces of hepatocytes and renal cells, activate the intracellular metabolic regulation signaling pathway, and promote the processing and excretion of metabolic wastes. For example, it promotes the metabolic conversion of ammonia by hepatocytes and the excretion of urea nitrogen and uric acid by renal cells. In terms of immunity, terpenol may activate immune cells such as macrophages and lymphocytes, and promote them to secrete antibacterial peptides and lysozymes, making component groups 19 and 20 containing terpenol superior to control group 4 in terms of indicators related to metabolic waste accumulation.

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

[0284] Table 19 Effects of different terpenol contents in the microcirculation preparation of mud crabs on indicators related to postpartum fat and energy reserve of mud crabs

[0285]

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

[0287] It can be seen from Table 19 that among the indicators related to fat and energy reserve, the component group 17 has the highest HDL content, the lowest LDL content, and the strongest FAS and CPT activities. From component group 17 to control group 4, the HDL content gradually decreases, the LDL content gradually increases, and the FAS and CPT activities also gradually weaken.

[0288] Principle analysis:

[0289] Component group 17 is superior to component groups 16 and 18: Terpenol at 250 mg / L may have the best effect on the fat and energy reserve 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] Component groups 16 and 18 are superior to component groups 19 and 20: Terpenol at 200 mg / L and 300 mg / L can maintain the homeostasis of fat and energy metabolism to a certain extent; the content of 150 mg / L may be insufficient in dosage, resulting in low FAS and CPT activities and low efficiency of fat synthesis and energy conversion. Terpenol at 350 mg / L inhibits fat synthesis and at the same time causes imbalance in the metabolic regulation of HDL and LDL, which is instead unfavorable for the reserve and normal metabolism of fat and energy.

[0291] Components Nineteen and Twenty are superior to Control Group Four: Terpenol can initiate the basic regulatory mechanisms of fat and energy metabolism in mud crabs. For example, it acts directly or indirectly on the transcription factors of the PPAR family. PPARα can regulate the expression of genes such as fatty acid transporter, fatty acid binding protein, and CPT-1, promoting fatty acid uptake and oxidation; PPARγ can regulate genes such as FAS involved in fatty acid synthesis; in terms of HDL and LDL metabolism, terpenol may optimize fat metabolism by regulating the activities of related metabolic enzymes and transporters in the liver and peripheral tissues, increasing the content of HDL and reducing the level of LDL. Therefore, Components Nineteen and Twenty containing terpenol are superior to Control Group Four without terpenol in these indicators.

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

[0293] Table 20 Effects of different terpenol contents in the microcirculation preparation of mud crabs on indicators related to postpartum tissue repair of mud crabs

[0294]

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

[0296] As can be seen from Table 20, among the indicators related to tissue repair, the contents of hydroxyproline, EGF, PDGF, and TGF-β in Component Seventeen are the highest, indicating that its tissue repair ability is the strongest. From Component Seventeen to Control Group Four, the values of each indicator gradually decrease.

[0297] Principle analysis:

[0298] Component 17 is superior to Component 16 and Component 3: The promoting effect of 250 mg / L terpenol on the postpartum tissue repair of mud crabs is significant. In terms of hydroxyproline synthesis, it may promote the activity of proline hydroxylase in fibroblasts. This enzyme can hydroxylate proline to hydroxyproline, which is a key step in collagen synthesis, thereby increasing the content of hydroxyproline, facilitating the formation of collagen fibers and tissue repair. For EGF, terpenol may activate the EGFR-Ras-Raf-MEK-ERK signal cascade in epidermal cells, promoting the transcription and translation of the EGF gene, enabling epidermal cells to secrete more EGF, accelerating the proliferation, migration, and differentiation of epidermal cells, and promoting wound healing. In terms of PDGF secretion, terpenol may regulate the PI3K-Akt signaling pathway in related cells such as platelets and macrophages, prompting these cells to secrete more PDGF, recruiting fibroblasts, smooth muscle cells, etc. to the injury site, and promoting cell proliferation and extracellular matrix synthesis. For TGF-β, terpenol may bind to the TGF-β receptor on the cell surface, activate the Smad signaling pathway, enhance the expression and secretion of TGF-β, regulate cell proliferation, differentiation, and the synthesis and remodeling of the extracellular matrix, and comprehensively promote tissue repair.

[0299] Component 16 and Component 18 are superior to Component 19 and Component 20: 200 mg / L and 300 mg / L terpenol can promote tissue repair; a content of 150 mg / L may, due to insufficient dosage, lead to limited synthesis of hydroxyproline and limited secretion of EGF, PDGF, and TGF-β, affecting the tissue repair process; 350 mg / L terpenol may interfere with intracellular signal transduction. For example, after over-activating certain signaling pathways, feedback inhibition is triggered, resulting in reduced secretion of related growth factors, or it may have a toxic effect on normal cell metabolism, inhibiting tissue repair.

[0300] Component 19 and Component 20 are superior to Control Group 4: Terpenol can promote the tissue repair of mud crabs, promote the expression of collagen genes in fibroblasts, and increase the content of hydroxyproline. At the same time, it stimulates epidermal cells, platelets, etc. to secrete growth factors such as EGF, PDGF, and TGF-β, initiating the cell cascade reaction of tissue repair, promoting cell proliferation, migration, and extracellular matrix synthesis, making Component 19 and Component 20 containing terpenol superior to Control Group 4 without terpenol in terms of tissue repair-related indicators.

[0301] Example 5 Experiment on the Concentrations of Four Components: Rosmarinic Acid, Carnosic Acid, Oleuropein, and Terpenol

[0302] I. Preparation of the Mixed Reagent

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

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

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

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

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

[0308] 32.5 g of rosemary petals, 27.5 g of sage leaves, 27.5 g of olive leaves, 22.5 g of perilla leaves. Five groups of mixed reagents with different concentrations were obtained according to the operations of Step 1 to Step 3 in Example 1. The auxiliary solution was recorded as the blank control group and was respectively recorded as:

[0309] Component Group 21: 300 mg / L rosmarinic acid + 250 mg / L caffeic acid + 250 mg / L oleuropein + 200 mg / L terpenol,

[0310] Component Group 22: 350 mg / L rosmarinic acid + 300 mg / L caffeic acid + 300 mg / L oleuropein + 250 mg / L terpenol,

[0311] Component Group 23: 400 mg / L rosmarinic acid + 350 mg / L caffeic acid + 350 mg / L oleuropein + 300 mg / L terpenol,

[0312] Component Group 24: 200 mg / L rosmarinic acid + 200 mg / L caffeic acid + 200 mg / L oleuropein + 150 mg / L terpenol,

[0313] Component Group 25: 500 mg / L rosmarinic acid + 400 mg / L caffeic acid + 400 mg / L oleuropein + 350 mg / L terpenol,

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

[0315] II. Breeding experiment

[0316] It was carried out in the indoor aquaculture ponds of Dongying Kenuo Aquaculture Co., Ltd. from March to April 2024. Again, 90 female mud crabs within 1 - 3 days after giving birth were collected from the southern coastal areas and transported by air to the experimental site, where they were centrally disinfected. Then they were placed in two indoor cement ponds of 30 m² for temporary rearing for 1 - 2 days. After temporary rearing, every 5 fat crabs were respectively placed into 18 laboratory ponds of 20 m² for the experiment. Five postpartum female crabs were placed in one pond, and at the same time, 5 tiles were placed into each pond. Among them, ponds 1 - 3 were group twenty - one, ponds 4 - 6 were group twenty - two, ponds 7 - 9 were group twenty - three, ponds 10 - 12 were group twenty - four, ponds 13 - 15 were group twenty - five, and ponds 16 - 18 were control group five. The purpose of this example is to explore the key compounds in the postpartum microcirculation preparation of mud crabs that are beneficial to the postpartum rejuvenation of mud crabs. The experimental materials and their sources in this example are the same as those in Example 1.

[0317] III. Comparison of experimental results

[0318] 1. Analysis of growth index results

[0319] Table 21 Effects of different carnosic acid contents in the mud crab microcirculation preparation on the growth indexes of mud crabs

[0320]

[0321]

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

[0323] It can be seen from Table 21 that the survival rate (86.95 ± 1.96%), weight gain rate (46.03 ± 1.28%), and condition factor (62.21 ± 1.63 g / cm 3 ) of group twenty - two were the highest, and the recovery time (17.44 ± 0.55 days) was the shortest; group twenty - one and group twenty - three were the second, with no significant difference; group twenty - four and group twenty - five performed worse, and control group five was the worst. The indexes of each group showed a decreasing trend from group twenty - two to control group five.

[0324] Principle analysis: The advantages of Component Group 22 may stem from the appropriate ingredient ratio (350 mg / L rosmarinic acid + 300 mg / L carnosic acid + 300 mg / L oleuropein + 250 mg / L terpenol), which may maximize the growth and metabolism of mud crabs. Rosmarinic acid and carnosic acid enhance cell metabolism and proliferation ability; oleuropein may improve intestinal absorption and metabolism ability, thereby increasing nutrient utilization rate; terpenol further enhances growth efficiency. In contrast, too high (such as Component Group 25) or too low (such as Component Group 24) ingredient concentration may lead to increased metabolic stress or insufficient nutrient supply, restricting growth efficiency. Control Group 5 lags significantly behind the experimental groups due to the lack of effective bioactive ingredients.

[0325] Table 22 Effects of different ingredient contents in the mud crab microcirculation preparation on the indicators related to postpartum nutrient transportation of mud crabs

[0326]

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

[0328] As can be seen from Table 22, Component Group 22 has the highest hepatopancreas index (8.24 ± 0.24%) and gonad index (7.32 ± 0.33%), and the lowest triglyceride (0.71 ± 0.12 mmol / L) and total cholesterol (1.45 ± 0.04 mmol / L); Component Group 21 and Component Group 23 are the next; the indicators of Component Group 24 and Component Group 25 decrease significantly, and Control Group 5 is the worst.

[0329] Principle analysis: The advantages of Component Group 22 may stem from the reasonable combination of its ingredients, which optimizes the nutrient transportation and storage efficiency by synergistically regulating the nutrient metabolism pathway. Rosmarinic acid and carnosic acid may improve the nutrient absorption and storage ability of hepatopancreas and gonad cells by enhancing vascular permeability and blood circulation. Oleuropein may reduce serum triglyceride and total cholesterol levels by inhibiting the activity of HMG-CoA reductase and decreasing cholesterol synthesis. Terpenol may enhance the uptake ability of cell membranes for nutrients by regulating lipid metabolism and membrane protein activity. In contrast, improper ingredient concentration (such as Component 24 and Component 25) may cause metabolic burden and reduce nutrient transportation efficiency. Control Group 5 has the worst indicators related to nutrient transportation due to the lack of active ingredients.

[0330] Table 23 Effects of different ingredient contents in the mud crab microcirculation preparation on the indicators related to postpartum metabolic waste accumulation of mud crabs

[0331]

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

[0333] As can be seen from Table 23, the contents of urea nitrogen (2.84±0.18mg / dL), ammonia (0.32±0.02mg / L) and uric acid (3.24±0.22mg / dL) in Group 22 were the lowest, while the contents of antibacterial peptide (186.9±12μg / mL) and lysozyme (183.3±10.4U / mL) were the highest; Group 21 and Group 23 were the next; the accumulation of metabolic wastes in Group 24 and Group 25 was significant, and that in Control Group 5 was the most serious.

[0334] Principle analysis: The advantages of Group 22 may stem from the dual regulation of metabolic enzymes and the immune system by its components. Rosmarinic acid and carnosic acid may promote the excretion of ammonia and uric acid and reduce the accumulation of metabolic wastes by enhancing the activities of ornithine aminotransferase and urea cycle enzymes in the liver; oleuropein may accelerate the excretion of metabolic wastes by enhancing kidney function; terpenol may improve the body's ability to resist infection and remove toxins by stimulating immune cells to secrete antibacterial 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 regulation mechanisms, and the accumulation of wastes is the most serious.

[0335] Table 24 Effects of different component contents in the microcirculation preparation of mud crab on the indexes related to postpartum fat and energy reserve of mud crab

[0336]

[0337]

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

[0339] As can be seen from Table 24, the HDL (68.14±3.11mg / dL) and CPT activity (3.27±0.11U / mg) in Group 22 were the highest, the LDL (101.8±2.9mg / dL) was the lowest, and the FAS activity (2.13±0.42U / mg) was the strongest; Group 21 and Group 23 were the next; the indexes in Group 24 and Group 25 decreased significantly, and those in Control Group 5 were the worst.

[0340] Principle analysis: The synergistic effect of Component Twenty-two is reflected in that rosmarinic acid and carnosic acid jointly inhibit the activity of ACC and promote fatty acid β-oxidation; terpenol activates the PPARα pathway to balance fat synthesis and decomposition; oleuropein enhances HDL synthesis; the fat metabolism efficiency of Component Twenty-one and Component Twenty-three is partially limited due to the deviation of some component concentrations; the imbalance of concentrations in Component Twenty-four and Component Twenty-five leads to a decrease in HDL synthesis and LDL accumulation; Control Group Five lacks a regulatory mechanism and has disordered fat metabolism.

[0341] Table 25 Effects of different component contents in the microcirculation preparation of mud crabs on the indexes related to postpartum tissue repair of mud crabs

[0342]

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

[0344] It can be seen from Table 25 that the contents of hydroxyproline (7.82±0.42 μg / g), EGF (52.21±2.03 pg / mL), PDGF (132.7±4.1 pg / mL) and TGF-β (222.7±8.8 pg / mL) in Component Twenty-two are the highest; Component Twenty-one and Component Twenty-three are the second; the repair ability of Component Twenty-four and Component Twenty-five is significantly weakened, and Control Group Five is the worst.

[0345] Principle analysis: The composite components of Component Twenty-two promote tissue repair through multi-pathway synergy: rosmarinic acid activates the TGF-β / Smad pathway, carnosic acid promotes the secretion of EGF, oleuropein enhances the recruitment ability of PDGF, and terpenol regulates cell proliferation and differentiation. The secretion of repair factors in Component Twenty-one and Component Twenty-three does not reach the optimal level due to the deviation of some component concentrations. The imbalance of concentrations in Component Twenty-four and Component Twenty-five leads to limited secretion of repair factors. There are no active components in Control Group Five to initiate the repair mechanism, and the indexes lag behind comprehensively.

[0346] Example 6 Breeding method 1 of mud crabs after parturition

[0347] The method for improving the microcirculation function of female mud crabs after parturition in this example includes the following steps:

[0348] I. Microcirculation preparation for mud crabs after parturition

[0349] Terpenoids, phenols and flavonoids are extracted from natural plants such as 27.5 g of rosemary petals, 22.5 g of sage leaves, 22.5 g of olive leaves, 17.5 g of perilla leaves, 12.5 g of green tea leaves, 12.5 g of red onions, 7.5 g of blueberry leaves and 7.5 g of grape seeds. Among them, the terpenoids and phenols contain rosmarinic acid at a content of 350 mg / L, carnosic acid at a content of 300 mg / L, oleuropein at a content of 300 mg / L, terpenol at a content of 250 mg / L, catechin at a content of 150 mg / L, quercetin at a content of 150 mg / L, ursolic acid at a content of 75 mg / L, and procyanidin at a content of 75 mg / L. The extraction and purification are carried out by ultrasonic-assisted 70% ethanol extraction method to obtain a post-partum microcirculation preparation for mud crabs.

[0350] In the post-partum microcirculation preparation of mud crabs, the terpenoids and phenols 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, specifically including active ingredients such as rosmarinic acid, carnosic acid, oleuropein, terpenol, catechin, quercetin, ursolic acid, and procyanidin, can effectively promote the process of nutrient transport in female mud crabs after parturition by affecting the hepatopancreas index, gonad index and other related indicators, and relieve physiological disorders caused by the accumulation of metabolic wastes, the lack of fat and energy reserves, and the weakening of tissue repair functions.

[0351] The specific steps of the preparation method of the post-partum microcirculation preparation for mud crabs are as follows:

[0352] Step 1: Preparation of plant materials

[0353] (1) Material collection: Weigh 27.5 parts of rosemary petals, 22.5 parts of sage leaves, 22.5 parts of olive leaves, 17.5 parts of perilla leaves, 17.5 parts of green tea leaves, 12.5 parts of red onions, 7.5 parts of blueberry leaves and 7.5 parts of grape seeds. (The plant materials can be obtained by purchasing in the market)

[0354] (2) Crushing: Use a crusher (model: LD-1300A high-speed universal crusher, LinDa Machinery Co., Ltd., Wenling, Zhejiang) to coarsely crush the above plant materials, and control the particle size to be 0.5 - 1 mm. The crushed materials are filled into a dry 100 ml airtight glass beaker (100 ml round-bottom glass beaker, Shanghai Heqi Glass Instrument Co., Ltd.) for standby.

[0355] Step 2: Ultrasonic-assisted ethanol extraction

[0356] (3) Solvent preparation: Use 70% ethanol solution (purchased in the market) as the extraction solvent. Divide 100 ml of 70% ethanol solution into 4 500 ml round-bottom glass flasks (500 ml round-bottom glass flasks, Shanghai Heqi Glass Instrument Co., Ltd.).

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

[0358] (5) Ultrasonic extraction: Place the 4 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 °C, extraction time: 40 minutes. After the extraction is completed, cool the extract to room temperature.

[0359] Step 3: Filtration and centrifugation

[0360] (6) Coarse filtration: Use a filter (model: MS-0.45μm polyethersulfone (PES) membrane filter, Shanghai Mosu Scientific Equipment Co., Ltd.) to preliminarily filter the extract to remove larger particles.

[0361] (7) Centrifugal separation: Transfer the coarsely filtered extract to a centrifuge (model: TG16-WS tabletop high-speed centrifuge, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.), and set the following parameters for centrifugation: rotation speed: 10,000 rpm, time: 15 minutes. After centrifugation, collect the supernatant and discard the remaining impurities.

[0362] Step 4: Rotary evaporation and concentration

[0363] (8) Concentration operation: Transfer the centrifuged supernatant to a rotary evaporator (model: RE-52AA rotary evaporator, Shanghai Yarong Biochemical Instrument Factory) for concentration treatment. Set the following parameters: water bath temperature: 45 °C, rotation speed: 80 rpm. Evaporate and concentrate to approximately 50 ml of concentrated solution to ensure the retention of active ingredients in the liquid.

[0364] Step 5: Preparation of liquid preparation

[0365] (9) Dilution: Dilute 50 ml of the concentrated solution with pure water to 1000 ml, and stir well to prepare the post-partum microcirculation preparation for mud crabs.

[0366] (10) Storage: Pour the prepared post-partum microcirculation preparation for mud crabs into 2 L transparent glass containers and seal them for storage. The containers need to be light-proof and placed in a 4 °C refrigerator to prevent the degradation of active ingredients.

[0367] II. Auxiliary liquid

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

[0369] Experimental materials: Clostridium butyricum: 7 g, L-arginine: 5 g, vitamin C: 3 g, phosphate buffer solution (PBS): 4 - 6 ml; 500 mL glass beakers (model: GG-17, Shanghai Guanghua Glass Instrument Co., Ltd.), put the glass beakers into a high-pressure sterilizer (DXL-100S automatic high-pressure steam sterilizer, Shandong Dexiang Instrument Co., Ltd.) for high-temperature and high-pressure sterilization treatment, and wait for standby.

[0370] Step 2: Preparation of the auxiliary solution

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

[0372] (12) Add L-arginine and vitamin C: Dissolve 5 g of L-arginine in 12.5 ml of sterile water and stir for 4 minutes until completely dissolved, then dissolve 3 g of vitamin C in 10 ml of sterile water and stir for 3 minutes to ensure that the solution is transparent and free of precipitation. Finally, add the above two solutions to the Clostridium butyricum suspension respectively, and stir slowly while adding to mix evenly.

[0373] (13) Add PBS buffer solution: Take 5 ml of PBS buffer solution and slowly add it to the above mixture, and continue to stir 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 treatment

[0375] (14) Filtration: Sterile filter the mixed solution through a 0.22 μm needle filter (model: Syringe Filters 0.22 μm, Zhejiang Sepax Technologies Co., Ltd.) to remove bacteria, fungal spores, and other microbial contaminants.

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

[0377] III. Mixed Preparation

[0378] The ratio of the post - partum microcirculation preparation of mud crab to the auxiliary liquid is 5:1. That is, for every 5 ml of the microcirculation preparation, 1 ml of the auxiliary liquid needs to be added for mixing to obtain 6 ml of the mixed preparation.

[0379] IV. Steps for Administering the Mixed Preparation in Stages

[0380] The combined dosage and administration method of the post - partum microcirculation preparation of mud crab and the auxiliary liquid need to be adjusted adaptively according to the breeding stage of mud crab. The specific adjustment plan is as follows:

[0381] The First Stage (3 to 7 days after parturition)

[0382] Administration Time: 6:00 p.m. (18:00) every day;

[0383] Administration Frequency: Once a day;

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

[0385] The Second Stage (8 to 14 days after parturition)

[0386] Administration Time: 8:00 p.m. (20:00) and 5:00 a.m. (05:00) every day;

[0387] Administration Frequency: Twice a day;

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

[0389] The Third Stage (15 to 20 days after parturition)

[0390] Administration Time: 8:00 p.m. (20:00) and 5:00 a.m. (05:00) every day;

[0391] Administration Frequency: Twice a day;

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

[0393] Example 7 Breeding Method of Mud Crab after Parturition 2

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

[0395] I. Post - partum Microcirculation Preparation of Mud Crab

[0396] Terpenoids, phenols and flavonoids are extracted from natural plants such as 25 g of rosemary petals, 20 g of sage leaves, 20 g of olive leaves, 15 g of perilla leaves, 10 g of green tea leaves, 10 g of red onions, 5 g of blueberry leaves and 5 g of grape seeds. Among them, the terpenoids, phenols and flavonoids include rosmarinic acid at a content of 300 mg / L, carnosic acid at a content of 250 mg / L, oleuropein at a content of 250 mg / L, terpineol at a content of 200 mg / L, catechin at a content of 100 mg / L, quercetin at a content of 100 mg / L, ursolic acid at a content of 50 mg / L, proanthocyanidin at a content of 50 mg / L, etc. The extraction and purification are carried out by ultrasonic-assisted 70% ethanol extraction method to obtain the post-partum microcirculation preparation for mud crabs.

[0397] The specific steps of the preparation method are as follows:

[0398] Step 1: Preparation of plant materials

[0399] (1) Material collection: Weigh 25 g of rosemary petals, 20 g of sage leaves, 20 g of olive leaves, 15 g of perilla leaves, 10 g of green tea leaves, 10 g of red onions, 5 g of blueberry leaves and 5 g of grape seeds. (The plant materials are obtained by purchasing in the market)

[0400] II. Auxiliary liquid

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

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

[0403] Step 2: Preparation of the auxiliary liquid

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

[0405] (12) Addition of L-arginine and vitamin C: Dissolve 4 g of L-arginine in 10 ml of sterile water, stir for 3 - 5 minutes until completely dissolved. Then dissolve 2 g of vitamin C in 8 ml of sterile water, stir for 2 - 4 minutes to ensure the solution is clear and free of precipitation. Finally, add the above two solutions separately to the Clostridium butyricum suspension, adding while slowly stirring to mix evenly.

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

[0407] Example 8: Cultivation method 3 for female mud crabs after spawning

[0408] It is basically the same as the cultivation method in Example 1, with the differences being:

[0409] I. Microcirculation preparation for female mud crabs after spawning

[0410] Extract terpenoids, phenols, and flavonoids from natural plants such as 30 g of rosemary petals, 25 g of sage leaves, 25 g of olive leaves, 20 g of perilla leaves, 15 g of green tea leaves, 15 g of red onions, 10 g of blueberry leaves, and 10 g of grape seeds. Among them, the terpenoids, phenols, and flavonoids contain rosmarinic acid at a content of 400 mg / L, carnosic acid at a content of 350 mg / L, oleuropein at a content of 350 mg / L, terpineol at a content of 300 mg / L, catechin at a content of 200 mg / L, quercetin at a content of 200 mg / L, ursolic acid at a content of 100 mg / L, procyanidin at a content of 100 mg / L, etc. Extract and refine through ultrasonic-assisted 70% ethanol extraction method to obtain the microcirculation preparation for female mud crabs after spawning.

[0411] The specific steps of the preparation method are as follows:

[0412] Step 1: Preparation of plant materials

[0413] (1) Material collection: Weigh 30 g of rosemary petals, 25 g of sage leaves, 25 g of olive leaves, 20 g of perilla leaves, 15 g of green tea leaves, 15 g of red onions, 10 g of blueberry leaves, and 10 g of grape seeds. (The plant materials are obtained by purchasing in the market)

[0414] II. Auxiliary liquid

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

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

[0417] Step 2: Preparation of the auxiliary solution

[0418] (11) Dissolve Clostridium butyricum: In a sterile laminar flow hood (model: BSC-1100IIA2-X, developed by Guangdong Keyipu Laboratory Equipment Co., Ltd.), take 8 g of Clostridium butyricum powder and add 25 ml of sterile pure water; use an electric stirrer (model: IKARW20 Digital Dissolver, Beijing Labtech Instruments Co., Ltd.) to stir at medium speed for 3 - 5 minutes to ensure complete dissolution of Clostridium butyricum and form a uniform suspension.

[0419] (12) Add L-arginine and vitamin C: Dissolve 6 g of L-arginine in 10 ml of sterile water and stir for 3 - 5 minutes until completely dissolved. Then dissolve 4 g of vitamin C in 8 ml of sterile water and stir for 2 - 4 minutes to ensure the solution is transparent and has no precipitate. Finally, add the above two solutions to the Clostridium butyricum suspension respectively, and stir slowly while adding to mix evenly.

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

[0421] Example 9: Experiment on the full-component concentration of the postpartum microcirculation preparation for mud crabs

[0422] The purpose of this example is to explore the component ratio of the postpartum microcirculation preparation for mud crabs that is beneficial to the postpartum rejuvenation of mud crabs and the concentration with the best effect. The experimental materials and sources in this example are the same as those in Example 1.

[0423] I. Preparation of the postpartum microcirculation preparation for mud crabs with different component ratios

[0424] 1.1 Twenty-six groups of components: In Example 7, ultrasonic-assisted 70% ethanol extraction method was used for extraction and purification to obtain the plant content of the postpartum microcirculation preparation for mud crabs, including 25 g of rosemary petals, 20 g of sage leaves, 20 g of olive leaves, 15 g of perilla leaves, 10 g of green tea leaves, 10 g of red onion, 5 g of blueberry leaves, and 5 g of grape seeds. The concentrations of the various components of the compounds finally extracted according to the method in Example 7 are: rosmarinic acid 300 mg / L, carnosic acid 250 mg / L, oleuropein 250 mg / L, terpenol 200 mg / L, catechin 100 mg / L, quercetin 100 mg / L, ursolic acid 50 mg / L, and procyanidin 50 mg / L.

[0425] 1. Component twenty-seven group: Extracted and refined by ultrasonic-assisted 70% ethanol extraction method in Example 6 to obtain the plant content of the postpartum microcirculation preparation of mud crab, including 27.5 g of rosemary petals, 22.5 g of sage leaves, 22.5 g of olive leaves, 17.5 g of perilla leaves, 12.5 g of green tea leaves, 12.5 g of red onions, 7.5 g of blueberry leaves and 7.5 g of grape seeds. The concentrations of the compounds in each component finally extracted according to the method of Example 6 are: rosmarinic acid 350 mg / L, carnosic acid 300 mg / L, oleuropein 300 mg / L, terpenol 250 mg / L, catechin 150 mg / L, quercetin 150 mg / L, ursolic acid 75 mg / L, procyanidin 75 mg / L.

[0426] 1. Component twenty-eight group: Extracted and refined by ultrasonic-assisted 70% ethanol extraction method in Example 8 to obtain the plant content of the postpartum microcirculation preparation of mud crab, including 30 g of rosemary petals, 25 g of sage leaves, 25 g of olive leaves, 20 g of perilla leaves, 15 g of green tea leaves, 15 g of red onions, 10 g of blueberry leaves and 10 g of grape seeds. The concentrations of the compounds in each component finally extracted according to the method of Example 8 are: rosmarinic acid 400 mg / L, carnosic acid 350 mg / L, oleuropein 350 mg / L, terpenol 300 mg / L, catechin 200 mg / L, quercetin 200 mg / L, ursolic acid 100 mg / L, procyanidin 100 mg / L.

[0427] II. Comparative experiments on postpartum microcirculation preparations of each component of mud crab

[0428] It was carried out in the indoor aquaculture ponds of Dongying Kenuo Aquaculture Co., Ltd. from May to June 2023. 75 female mud crabs within 1 to 3 days after parturition were collected from the southern coastal areas, transported by air to the experimental site, and then centrally disinfected. After that, they were put into two indoor cement ponds of 30 m2 for temporary cultivation for 1 to 2 days. After temporary cultivation, every 5 female mud crabs with roe were respectively put into 12 laboratory ponds of 20 m2 for experiments. 5 postpartum female crabs were put into one pond, and at the same time, 5 tiles were put into each pond. Among them, ponds 1 to 3 were group twenty-six of components, ponds 4 to 6 were group twenty-seven of components, ponds 7 to 9 were group twenty-eight of components, and ponds 10 to 12 were control group six (without using the postpartum microcirculation preparation and auxiliary liquid for mud crabs). The specific usage methods of the postpartum microcirculation preparation and auxiliary liquid for mud crabs in each experimental group were as follows: The first stage (3 to 7 days after parturition): At 6 pm every day, 12.5 ml of the mixed preparation mixed in a ratio of 5:1 was added to each cubic meter of water body, and the application frequency was once a day. The second stage (8 to 14 days after parturition): At 8 pm every day and 5 am the next morning, 17.5 ml of the mixed preparation mixed in a ratio of 5:1 was respectively added to each cubic meter of water body, and the application frequency was twice a day. The third stage (15 to 20 days after parturition): At 8 pm every day and 5 am the next morning, 25 ml of the mixed preparation mixed in a ratio of 5:1 was respectively added to each cubic meter of water body, and the application frequency was twice a day. During the breeding period, baits such as hard clams and razor clams were fed, and all the residual baits were cleaned up 3 hours after feeding. One-third of the water was changed every three days, and the water change was carried out 4 to 5 hours after the postpartum microcirculation preparation and auxiliary liquid for mud crabs were splashed. The experimental period was 40 days. After the end of the experimental period, the survival rate, weight gain rate, condition factor and recovery time of female mud crabs in each group were detected and calculated, and comparative analysis was carried out for each group. The recovery time was mainly measured by the states of the following three aspects: activity level, feeding situation and growth rate. First, in terms of the activity level, we would observe the activity degree of the mud crabs after receiving the treatment. Generally speaking, the mud crabs with a faster recovery process would show higher activity and foraging behavior. Secondly, in terms of the feeding situation, we would record in detail the time required for the mud crabs to resume feeding from the start of receiving the treatment. Generally speaking, if the mud crabs recovered faster, they could resume feeding in a shorter time. Finally, the growth rate was also one of the important indicators to measure the recovery time. We would evaluate whether they had resumed the normal growth state by regularly measuring the growth parameters such as the weight or condition factor of the mud crabs.

[0429] Sampling and analysis were carried out on the mud crabs fished from each pond. First, an electronic balance (FA1204, Shanghai Precision Scientific Instruments Co., Ltd.) was used to weigh them (accurate to 0.01 g). Before weighing, the surface moisture of the crabs was gently wiped off with a dry towel. Then, a vernier caliper (LG-150, Shanghai Precision Measuring Tools & Cutting Tools Co., Ltd.) was used to measure their growth indicators. After the growth indicators were measured, the mud crabs were anesthetized on ice. A 2.0 mL disposable sterile syringe rinsed with anticoagulant was used to draw 1.5 mL of hemolymph sample from the base of the back of the carapace into the heart. The hemolymph was mixed with anticoagulant at a ratio of 1:1 (volume ratio) and centrifuged at 8000 r / min for 10 min at 4 °C. The supernatant was taken for the determination of hemolymph-related indicators.

[0430] III. Analysis of the results of the comparative experiment

[0431] After the 40-day breeding experiment, it is necessary to detect and analyze the growth indicators, nutrition transport-related detection indicators, metabolic waste accumulation-related indicators, fat and energy reserve-related indicators, and tissue repair-related indicators of female mud crabs after parturition.

[0432] 1. Analysis of the results of the growth indicator detection

[0433] Table 26 Effects of mud crab postpartum microcirculation preparations with different composition doses on the postpartum growth indicators of mud crabs

[0434] Component group 26 Component group 27 Component group 28 Control group 6 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] It can be seen from Table 26 that there are significant differences in the survival rate, weight gain rate, condition factor, and recovery time between Group 26, Group 27, and Group 28 of the components and Group 6 of the control. This shows that within the range of the content parameters of the active ingredients in each component (rosmarinic acid content is 300 - 400 mg / L, carnosic acid content is 250 - 350 mg / L, oleuropein content is 250 - 350 mg / L, terpenol content is 200 - 300 mg / L, catechin content is 100 - 200 mg / L, quercetin content is 100 - 200 mg / L, ursolic acid content is 50 - 100 mg / L, and procyanidin content is 50 - 100 mg / L), it has an obvious promoting effect on the growth state of female mud crabs after parturition;

[0437] In addition, in terms of the survival rate, Group 27 of the components (91.00% ± 3.08%) is the highest and significantly better than other groups; in terms of the weight gain rate, Group 27 of the components (46.8% ± 3.12%) is also the highest; in terms of the condition factor, Group 27 of the components (61.0 ± 2.45 g / cm 3)Performed the best; in terms of recovery time, the twenty-seventh component group (18.83 ± 2.05 days) was the shortest; overall, the twenty-seventh component group showed the best overall performance in growth indicators, and the specific advantages are as follows:

[0438] 1) Appropriate content of plant extracts: The active ingredients in the postpartum microcirculation preparation of mud crabs come from plant extracts such as rosmarinic acid, carnosic acid, oleuropein, terpenol, catechin, quercetin, ursolic acid, and procyanidin. The twenty-seventh component group uses 8 - 10 grams of plant raw materials, and the content of plant extracts is more abundant compared to the twenty-sixth component group (5 - 7 grams), which can better play the roles of improving microcirculation and promoting growth; while the twenty-eighth component group (11 - 13 grams) has more plant raw materials, but there may be problems such as too high extract concentration or imbalance in other component ratios, affecting the effect. For example, components such as catechin and procyanidin in plant extracts can regulate metabolism and promote growth, and appropriate content helps the growth and development of mud crabs, while too much or too little is not conducive to their growth.

[0439] 2) Matched with the physiological needs of mud crabs: After giving birth, mud crabs need to restore their physical functions. Appropriate doses of plant extracts can better meet their nutritional requirements, promote metabolism, increase the weight gain rate and fullness, enhance immunity, improve survival rate, and shorten the recovery time; the amount of plant raw materials in the twenty-seventh component group is more in line with the physiological recovery needs of mud crabs after giving birth, while other groups cannot achieve such ideal effects.

[0440] 2. Analysis of the test results of nutritional transport indicators

[0441] Table 27 Effects of postpartum microcirculation preparations of mud crabs with different composition doses on nutritional transport-related indicators after mud crab giving birth

[0442]

[0443]

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

[0445] It can be seen from Table 27 that there are significant differences in the hepatopancreas index, gonad index, triglyceride, and total cholesterol between the twenty-sixth component group, the twenty-seventh component group, the twenty-eighth component group and the sixth control group, indicating that within the range of the effective ingredient content parameters of each component, it has an obvious promoting effect on the nutritional transport of mud crabs after giving birth;

[0446] In addition, in terms of the hepatopancreas index and gonad index, the twenty-seventh component group (8.4% ± 0.25%, 7.3% ± 0.30%) was significantly higher than other groups; in terms of triglyceride and total cholesterol levels, the twenty-seventh component group (0.82 ± 0.04 mmol / L, 1.52 ± 0.05 mmol / L) was the lowest and significantly lower than other groups. This indicates that the twenty-seventh component group performed best in the nutritional transport-related indicators, with obvious effects on promoting the functions of the hepatopancreas and gonads and optimizing blood lipid metabolism. The specific advantages are as follows:

[0447] 3) Good synergistic effect of active ingredients: Multiple components in the plant extract, such as rosmarinic acid and procyanidins, have the effects of regulating blood lipid metabolism, promoting the absorption of nutrients, and restoring organ functions; the amount of plant raw materials in the twenty-seventh component group makes the proportion of these active ingredients relatively appropriate, with a better synergistic effect, thus more effectively promoting the digestion, absorption, and storage of nutrients by the hepatopancreas, and increasing the hepatopancreas index; at the same time, it also helps the development of the gonads and improves the gonad index. In other groups, the coordination of component proportions may affect the exertion of this synergistic effect;

[0448] 4) Reducing the metabolic burden: An appropriate content of plant extract helps to reduce triglyceride and total cholesterol levels, relieve the metabolic burden of the mud crabs, enable nutrients to be transported and utilized more efficiently, and promote the recovery of overall physiological functions. The twenty-seventh component group is outstanding in this regard, and other groups may not achieve the same metabolic regulation effect due to inappropriate amounts of plant raw materials.

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

[0450] Table 28 Effects of postpartum microcirculation preparations of mud crabs with different composition doses on the related indicators of postpartum metabolic waste accumulation in mud crabs

[0451] Component group 26 Component group 27 Component group 28 Control group 6 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 > Antibacterial peptide (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] It can be seen from Table 28 that the twenty-sixth component group, the twenty-seventh component group, and the twenty-eighth component group had significant differences from the sixth control group in urea nitrogen, ammonia, uric acid, antibacterial peptides, and lysozyme, indicating that within the range of the effective ingredient content parameters of each component, it had an obvious promoting effect on the metabolic waste clearance function of postpartum mud crabs;

[0454] In addition, among the indicators related to the accumulation of metabolic wastes, in terms of the contents of urea nitrogen, ammonia, and uric acid, the twenty-seventh component group (2.8 ± 0.1 mg / dL, 0.6 ± 0.1 mg / L, 4.1 ± 0.1 mg / dL) was the lowest and significantly lower than other groups; in terms of the contents of antimicrobial peptides and lysozyme, the twenty-seventh component group (195 ± 12 μg / mL, 180 ± 12 U / mL) was the highest and significantly higher than other groups; this indicates that the twenty-seventh component group can most effectively reduce the accumulation of metabolic wastes in the mud crab and enhance its immune function. The specific advantages are as follows:

[0455] 5) Regulate the balance of metabolism and immune function: Quercetin has the effects of antioxidation, anti-inflammation, and regulating immune function. The appropriate amount of plant raw materials in the twenty-seventh component group enables these components to better play the role of regulating the metabolic process in the mud crab, promoting the liver to remove metabolic wastes such as urea nitrogen, ammonia, and uric acid, thereby reducing their accumulation in the body. At the same time, it can also effectively stimulate the mud crab's immune system to produce more antimicrobial peptides and lysozyme, enhancing the immune function; due to the differences in the amount of plant raw materials in other groups, this balance of metabolic regulation and immune enhancement cannot be achieved;

[0456] 6) Maintain the stability of the internal environment: The lower accumulation of metabolic wastes helps to maintain the stability of the internal environment of the mud crab, reduce the damage of harmful substances to the body, and further promote the recovery of its physiological functions; the twenty-seventh component group has obvious advantages in this regard, providing a good internal environment for the health recovery of the mud crab.

[0457] 4. Analysis of the test results of fat and energy reserve indicators

[0458] Table 29 Effects of postpartum microcirculation preparations of mud crabs with different composition doses on fat and energy reserve-related indicators of postpartum mud crabs

[0459]

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

[0461] It can be seen from Table 29 that the twenty-sixth component group, the twenty-seventh component group, and the twenty-eighth component group had significant differences from the sixth control group in the HDL, LDL, FAS, and CPT indicators, indicating that within the range of the content parameters of the effective components of each component, it has an obvious promoting effect on the fat and energy reserves of postpartum mud crabs;

[0462] In addition, in terms of high-density lipoprotein (HDL) levels, the twenty-seventh component group (62 ± 3 mg / dL) was the highest and significantly higher than other groups; in terms of low-density lipoprotein (LDL) levels, the twenty-seventh component group (96 ± 4 mg / dL) was the lowest and significantly lower than other groups; in terms of the activities of fatty acid synthase and carnitine palmitoyltransferase, the twenty-seventh component group (1.7 ± 0.1 U / mg, 2.7 ± 0.1 U / mg) was the highest and significantly higher than other groups. This indicates that the twenty-seventh component group performed best in the indicators related to fat and energy reserves, and had a significant effect on improving lipid metabolism and energy reserves. The specific advantages are as follows:

[0463] 7) Optimize lipid metabolism pathways: Components such as terpenols and catechins in plant extracts can regulate lipid metabolism, affect HDL and LDL levels, and the activities of fatty acid synthase and carnitine palmitoyltransferase; the amount of plant raw materials in the twenty-seventh component group can enable these components to play their roles in appropriate proportions, promote the synthesis of HDL, reduce LDL levels, optimize the lipid metabolism balance, and thus contribute to fat synthesis and energy reserves; in other groups of this example, due to different amounts of plant raw materials, the regulation of lipid metabolism may be imbalanced, affecting the indicators related to fat and energy reserves;

[0464] 8) Meet energy requirements and reserves: After spawning, mud crabs need to restore energy reserves. The proportion of components in the plant extract of the twenty-seventh component group is more conducive to promoting the activities of enzymes related to fat synthesis and energy reserves, enabling mud crabs to accumulate energy more effectively and meet the energy requirements for their growth and recovery; the effects of other groups in this example are not as good as those of the twenty-seventh component group in this regard.

[0465] 5. Analysis of tissue repair index detection results

[0466] Table 30 Effects of postpartum microcirculation preparations of mud crabs with different composition doses on tissue repair-related indicators after mud crab spawning

[0467]

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

[0469] The data in Table 30 show that the twenty-sixth component group, the twenty-seventh component group, and the twenty-eighth component group had significant differences from the sixth control group in terms of hydroxyproline, epidermal growth factor, platelet-derived growth factor, and transforming growth factor-β indicators, indicating that within the range of effective ingredient content parameters of each component, it had an obvious promoting effect on the tissue repair of mud crabs after spawning;

[0470] In addition, in terms of hydroxyproline content, Group 27 (8.2 ± 0.2 μg / g) was the highest and significantly higher than other groups; in terms of the contents of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-β (TGF-β), Group 27 (56 ± 2 pg / mL, 127 ± 4 pg / mL, 214 ± 10 pg / mL) were all significantly higher than other groups. This indicates that Group 27 showed the best performance in the indicators related to tissue repair and could effectively accelerate the repair and regeneration of the postpartum tissues of mud crabs. The specific advantages are as follows:

[0471] 9) Promote cell proliferation and repair: Components such as carnosic acid and ursolic acid in plant extracts can promote collagen synthesis and stimulate the secretion of growth factors, thus accelerating tissue repair. The appropriate amount of plant raw materials in Group 27 enables these components to play a better role, promote the synthesis of hydroxyproline, increase the collagen content, and provide a basis for tissue repair. At the same time, it stimulates the secretion of growth factors such as EGF, PDGF, and TGF-β, enhancing cell proliferation and regeneration ability. The inappropriate amount of plant raw materials in other groups may affect the effect of these components in promoting tissue repair.

[0472] 10) Improvement of the overall physiological state promotes repair: By improving the overall physiological state of mud crabs, such as blood circulation and excretion of metabolic wastes (as shown by the good performance of Group 27 in other indicators in the previous table), favorable conditions are created for tissue repair, enabling the relevant processes of tissue repair to proceed more smoothly. The advantages of Group 27 in improving the overall physiological state indirectly promote the improvement of the indicators related to tissue repair, while the comprehensive effect of other groups in this regard is not as good as that of Group 27.

[0473] 3.6 Summary of Results

[0474] In summary, the specific detection parameters of Component Group 26 to Component Group 28 in this embodiment for growth indicators, nutrient transport indicators, metabolic waste accumulation indicators, fat and energy reserve indicators, and tissue repair indicators are all significantly better than those of Control Group 6; in particular, the specific detection parameters of Component Group 27 for growth indicators, nutrient transport indicators, metabolic waste accumulation indicators, fat and energy reserve indicators, and tissue repair indicators are all significantly better than those of Component Group 26 and Component Group 28; therefore, it can be concluded that when the content of active ingredients in the postpartum microcirculation preparation for mud crabs is within the range of rosmarinic acid content of 300 - 400 mg / L, carnosic acid content of 250 - 350 mg / L, oleuropein content of 250 - 350 mg / L, terpenol content of 200 - 300 mg / L, catechin content of 100 - 200 mg / L, quercetin content of 100 - 200 mg / L, ursolic acid content of 50 - 100 mg / L, and proanthocyanidin content of 50 - 100 mg / L, and in combination with the auxiliary liquid for application to postpartum female crabs, it is beneficial to the fattening and rejuvenation of female crabs after parturition; especially when the content of active ingredients in the postpartum microcirculation preparation for mud crabs is rosmarinic acid content of 350 mg / L, carnosic acid content of 300 mg / L, oleuropein content of 300 mg / L, terpenol content of 250 mg / L, catechin content of 150 mg / L, quercetin content of 150 mg / L, ursolic acid content of 75 mg / L, and proanthocyanidin content of 75 mg / L, the effect is better.

Claims

1. A method for culturing blue crabs after birth, characterized in that: The invention comprises a blue crab postpartum microcirculation preparation, which contains at least one of the following components and contents: 300-400 mg / L of rosmarinic acid, 250-350 mg / L of carnosic acid, 250-350 mg / L of oleuropein, and 200-300 mg / L of terpene alcohol.

2. The method for culturing blue crabs after spawning according to claim 2, characterized in that: The blue crab postpartum microcirculation preparation also includes the following components: rosmarinic acid, carnosic acid, oleuropein, and terpene alcohol, wherein the content of rosmarinic acid is 300-400 mg / L, the content of carnosic 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.

3. The method for culturing blue crabs after spawning according to claim 2, characterized in that: The blue crab postpartum microcirculation preparation also includes the following components: catechin, quercetin, ursolic acid, and proanthocyanidins, wherein the catechin content is 100-200 mg / L, the quercetin content is 100-200 mg / L, the ursolic acid content is 50-100 mg / L, and the proanthocyanidins content is 50-100 mg / L.

4. The method for culturing blue crabs after spawning according to claim 3, characterized in that: The contents of the components in the blue crab postpartum microcirculation preparation are as follows: rosmarinic acid content is 350 mg / L, carnosic acid 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.

5. The method for culturing blue crabs after spawning according to claim 3, characterized in that: The method for preparing the blue crab postpartum microcirculation preparation comprises 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 basil leaves, 10-15 parts of green tea leaves, 10-15 parts of red onions, 5-10 parts of blueberry leaves and 5-10 parts of grape seeds, and mix them to obtain a raw material mixture; 2) Ultrasonic extraction: 60 to 80 portions of the raw material mixture obtained in step 1) are crushed, and 70% ethanol solution is used as an extractant to perform ultrasonic extraction on the crushed powder to obtain an extraction solution; 3) Filtration and centrifugation: The extraction solution obtained in step 2) is preliminarily filtered to obtain a primary filtration solution, and the primary filtration solution is centrifuged to discard the lower layer impurities to obtain a post-centrifugation solution; 4) Concentration: Evaporating and concentrating the centrifuged solution obtained in step 3) to 1 / 2 volume to obtain a concentrated solution; 5) Dilution and storage: The concentrated solution obtained in step 4) is diluted with water to obtain the blue crab postpartum microcirculation preparation, wherein the volume ratio of the concentrated solution to water during dilution is 1:

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

6. The method for culturing blue crabs after spawning according to any one of claims 1 to 4, characterized in that: The blue crab postpartum microcirculation preparation is mixed with an auxiliary liquid for use, and the components and contents of each 4 to 6 mL of the auxiliary liquid are: 6 to 8 g of Clostridium butyricum, 4 to 6 g of L-arginine, 2 to 4 g of vitamin C, and 4 to 6 ml of 1× phosphate buffer. The blue crab postpartum microcirculation preparation and the auxiliary liquid are mixed in a volume ratio of 5:1 to obtain a mixed preparation.

7. The method for culturing blue crabs after spawning according to claim 6, characterized in that: The auxiliary liquid configuration method comprises the following steps: 6) Dissolving raw materials: In a sterile clean bench, weigh 6 to 8 parts by weight of Clostridium butyricum powder, add 8 to 25 parts by weight of sterile water and stir thoroughly to obtain a Clostridium butyricum suspension; weigh 4 to 6 parts by weight of L-arginine powder, dissolve it in 10 to 15 parts by weight of sterile water and stir thoroughly to obtain an L-arginine solution; weigh 2 to 4 parts by weight of vitamin C powder, dissolve it in 8 to 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 solution in a volume ratio of 8 to 25:10 to 15:8 to 12:4 to 6 to obtain a raw material mixed solution; 7) Filtration sterilization: The raw material mixed solution obtained in step 6) is sterile filtered through a 0.22 μm syringe filter to obtain the auxiliary solution; 8) Storage: The auxiliary liquid obtained in step 8) is placed in a sealed container and stored at 4° C. away from light.

8. The method for culturing blue crabs after spawning according to claim 6 or 7, characterized in that: The method includes the steps of phased application. Specifically, the method includes three phases of application: During the period of 3 to 7 days after the blue crabs are laid: spray once a day at 18:00, with the amount of each spray being 10 to 15 ml of the mixed preparation per cubic meter of water; During the period of 8 to 14 days after the blue crabs are laid: spray once at 5:00 and 20:00 every day, with the spraying amount each time being 15 to 20 ml of the mixed preparation per cubic meter of water; During the 15-20 days after the blue crabs are laid: spray once at 5:00 and 20:00 daily, with each application amount being 20-30 ml of the mixed preparation per cubic meter of water.

9. The method for culturing blue crabs after spawning according to claim 8, characterized in that: The three-stage administration methods in the step of staged administration are respectively: 3-7 days after the blue crabs are born: spray once a day at 18:00, with the amount of each spray being 12.5 ml of the mixed preparation per cubic meter of water; During the period of 8 to 14 days after the blue crabs are laid: spray once at 5:00 and 20:00 every day, with the spraying amount each time being 17.5 ml of the mixed preparation per cubic meter of water; During the period of 15 to 20 days after the blue crabs are laid: spray once at 5:00 and 20:00 every day, with each spraying amount being 25 ml of the mixed preparation per cubic meter of water.

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