Procambarus clarkii in-vitro incubation device and in-vitro breeding method

Abstract

The invention discloses a procambarus clarkii in-vitro incubation device and an in-vitro breeding method, and belongs to the technical field of shrimp in-vitro breeding. The device comprises a transparent bucket body, a 90-degree elbow joint, a three-way pipe, an aeration pipe, a water outlet pipe, an air valve, a water outlet valve, a mesh and a filter screen. The in-vitro cultivation method comprises the following steps of: placing an egg-carrying lobster in clean water for temporary cultivation, stripping an embryo from the abdominal limb of the lobster by using a Michi comb after a lobster egg develops to a protointestinal stage, cleaning the embryo, removing residues, transferring the embryo into the device for incubation, pouring a self-made culture solution into the transparent bucket body, opening an air valve, and aerating the bucket through an aeration pipe to suspend the embryo. The procambarus clarkii in-vitro hatching device has the advantages of being unique in design, low in cost, visual, large in capacity, high in efficiency and easy to operate, a large number of shrimp eggs can be contained at the same time, the requirement for large-scale hatching is met, the shrimp eggs obtain appropriate water flow, oxygen and nutrition supply in the hatching process, and therefore the hatching success rate and the fry quality are improved.

Description

Technical Field

[0001] The invention relates to the technical field of shrimp in vitro breeding, in particular to an in vitro hatching device for Procambarus clarkii and an in vitro breeding method. Background Art

[0002] Procambarus clarkii, commonly known as crayfish, is a key aquaculture species with high economic value and market demand. Currently, the crayfish industry has become one of the most comprehensive and highest-valued sectors in my country's fishery industry chain. However, the development of seedling propagation technology for crayfish lags behind, and the production capacity of high-quality seedlings is significantly insufficient, becoming a major obstacle to the sustainable development of the industry. Intensive seedling propagation is an effective means to address these issues, and efficient hatching of fertilized eggs is a core component of this intensive breeding system. However, traditional crayfish aquaculture relies on broodstock incubation, which is plagued by unstable seedling survival rates, difficulty tracking developmental asynchrony, high labor investment, large space requirements for seedling rearing, and limited seedling yield per unit water body. These issues severely hinder the scale-up and intensive development of the industry. To address these issues, in vitro hatching technology has gradually attracted attention as a potential solution. In vitro hatching technology separates fertilized eggs from the mother and incubates them in a controlled environment, effectively improving hatching efficiency and seedling quality.

[0003] In recent years, with advances in aquaculture technology, in vitro incubation has become a research hotspot. However, high rates of moldy eggs and low spawning rates remain major challenges. Existing in vitro incubation equipment and culture media still have some shortcomings. For example, in vitro incubation equipment often has limited capacity, making efficient batch processing difficult. The lack of precise control over environmental factors during in vitro incubation is also a major factor restricting the development of in vitro incubation technology.

[0004] At present, in order to reduce the moldy egg rate, the existing hatching technology uses formaldehyde as the most widely used disinfectant in disinfection and antibacterial aspects. It is believed that it can replace the banned malachite green. However, due to the possibility that formaldehyde may be carcinogenic or have potential adverse effects on the aquatic environment if used improperly, its negative impact on the environment and shrimp seedlings cannot be ignored. Finding green and pollution-free biological agents that can improve hatching efficiency and seedling survival rate has important application prospects.

[0005] Chitosan, a natural polysaccharide, boasts excellent biocompatibility, broad-spectrum antimicrobial properties, and biodegradability. In recent years, it has been widely used in agriculture, food, medicine, and other fields. Studies have shown that chitosan not only effectively inhibits the growth of various pathogenic microorganisms but also promotes the growth and development of aquatic animals and enhances immune function. However, there are currently no reports on the use of chitosan in in vitro hatching media for Procambarus clarkii.

[0006] Therefore, how to provide an in vitro hatching device and in vitro seedling rearing method for Procambarus clarkii that can improve the hatching success rate and seedling quality is a problem that those skilled in the art are in urgent need of solving. Summary of the Invention

[0007] In view of this, the present invention provides an in vitro hatching device and an in vitro seedling breeding method for Procambarus clarkii.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] An in vitro hatching device for Procambarus clarkii comprises a transparent bucket body, a 90-degree elbow joint, a tee pipe, an aeration pipe, a water outlet pipe, an air valve, a water outlet valve, a mesh and a filter screen;

[0010] The narrow-necked mouth of the transparent water bucket body is threadedly connected to the water inlet end of the 90-degree elbow joint, the water outlet end of the 90-degree elbow joint is connected to the water inlet end of the tee pipe via a pipeline, the water outlet end of the tee pipe is connected to the water outlet pipe via a water outlet valve, and the air inlet end of the tee pipe is connected to the aeration pipe via an air valve. An opening is provided at the bottom of the transparent water bucket body, the mesh is placed in the transparent water bucket body, and the filter is fixedly installed at the narrow-necked mouth of the transparent water bucket body.

[0011] The present invention has the following beneficial effects: The in vitro incubation device for Procambarus clarkii, featuring a unique design and a transparent water bucket, offers low cost, visual visualization, large capacity, high efficiency, and ease of operation. It can simultaneously accommodate large numbers of shrimp eggs, meeting the needs of large-scale incubation. The shrimp eggs receive optimal water flow, oxygen, and nutrient supply during the incubation process, thereby improving hatching success rates and hatching quality. An air valve connected to an aeration pipe controls the air volume, directing it upward from the bottom of the transparent water bucket. This not only aerates the water but also keeps the shrimp eggs suspended, preventing them from sinking to the bottom. The controlled airflow creates a weak circulation, simulating the oscillation of the mother shrimp's abdominal feet, promoting oxygen exchange within the eggs. This allows all the shrimp eggs to float slightly, preventing them from sinking to the bottom and preventing strong water flow from rupturing their membranes. The bottom of the transparent water bucket features an opening that serves as both a water inlet and an egg release port. A mesh serves as an artificial attachment matrix for the shrimp eggs, reducing the risk of cross-contamination with mold. A filter prevents the shrimp eggs from falling outside the transparent water bucket, minimizing egg loss.

[0012] Furthermore, it also includes a bracket, the bracket includes an upper square frame, a middle square frame and a lower square frame, and the frames of each layer are fixedly connected by vertical rods;

[0013] The bottom of the transparent bucket body is placed upside down between the upper square frame and the middle square frame. The bucket body is tangent to the four sides of the upper square frame. A U-shaped frame is fixedly installed in the middle of the middle rectangular frame. The narrow neck of the transparent bucket body passes through the middle opening of the U-shaped frame, and the U-shaped frame supports the bucket body.

[0014] The beneficial effects of adopting the above further technical solution are: the setting of the bracket can make the transparent bucket body installed firmly and away from the ground, which is convenient for aeration and drainage operations and easy to observe.

[0015] Furthermore, the upper square frame, the middle square frame and the lower square frame have the same size, and the vertices of each layer of the frame are fixedly connected by vertical rods of the same height. The height of the vertical rods is less than the height of the barrel body, and the bottom of the transparent bucket body is completely cut into an opening.

[0016] The beneficial effects of adopting the above further technical solution are: the in vitro hatching device for Procambarus clarkii is more neat and beautiful, and is firmly installed; the bottom of the transparent water bucket body is completely cut into an opening, and the water inlet and egg placement are more convenient.

[0017] Furthermore, the mesh has a pore size of 1 mm and an area of ​​256-324 cm 2 The material of the above-mentioned mesh is a nylon mesh woven with fine iron wires.

[0018] The beneficial effect of adopting the above further technical solution is that the material of the mesh enables the mesh to be suspended in the culture solution in the transparent bucket body, and gravity is given to prevent it from floating on the water surface.

[0019] Furthermore, the pore size of the filter is 0.5-1 mm.

[0020] The beneficial effect of adopting the above further technical solution is: preventing the embryos from falling outside the transparent water bucket body, thereby avoiding the loss of the embryos.

[0021] Furthermore, the bottom opening edge of the transparent bucket body is wrapped with black leather.

[0022] The beneficial effect of adopting the above-mentioned further technical solution is that the incision on the bottom of the barrel is wrapped with a black leather card to prevent damage to the eggs when placing the eggs, and the cut edge of the part can also be wrapped with a black leather card as a barrel cover.

[0023] Furthermore, the transparent bucket body is made of a transparent plastic bucket.

[0024] The beneficial effect of adopting the above further technical solution is that the transparent bucket body can be visualized and the development of the embryo can be observed at any time.

[0025] The present invention also provides an in vitro culture method for Procambarus clarkii, comprising the following steps: placing brooding crayfish in clean water for temporary culture; after the crayfish eggs develop to the gastrula stage, separating the embryos from the crayfish ventral limbs with a fine-gauge comb; cleaning the embryos to remove debris and then transferring them to the above-mentioned in vitro incubation device for incubation; pouring culture fluid into a transparent water bucket body; opening a water outlet valve; and simultaneously injecting culture fluid into the transparent water bucket body through an opening at the bottom of the bucket; adjusting the flow rate so that the water inlet volume is equal to the water outlet volume; opening an air valve; aerating the bucket through an aeration pipe to suspend the crayfish eggs; using a mesh sheet placed in the transparent water bucket body for attachment of shrimp eggs in the seedling I stage; and stopping injecting culture fluid into the transparent water bucket body until the eggs develop to the seedling III stage. The water outlet valve and the air valve are closed, and the in vitro culture of Procambarus clarkii is completed.

[0026] The culture medium comprises well water, calcium chloride, magnesium chloride, zinc chloride, chitosan and methylene blue solution, wherein the concentration of calcium chloride in the culture medium is 80-140 mg / L, the concentration of magnesium chloride is 60-90 mg / L, the concentration of zinc chloride is 2-8 mg / L, the concentration of chitosan is 20 mg / L, and the concentration of methylene blue is 0.2-0.4 mg / L; or, the culture medium comprises well water, calcium chloride, magnesium chloride, zinc chloride, chitosan and liquid allicin Type VI, wherein the concentration of calcium chloride in the culture medium is 80-140 mg / L, the concentration of magnesium chloride is 60-90 mg / L, the concentration of zinc chloride is 2-8 mg / L, the concentration of chitosan is 20 mg / L, and the concentration of liquid allicin Type VI is 0.02-0.05 mg / L.

[0027] The beneficial effects of the present invention are as follows: well water is easy to obtain, low-cost, and more suitable for large-scale incubation.

[0028] The present invention adopts chitosan as the antibacterial component of the culture medium, which can effectively replace traditional chemical disinfectants and reduce the mold egg rate.

[0029] The present invention uses chitosan as the main antibacterial ingredient, combined with low-dose methylene blue, to ensure antibacterial effectiveness while reducing the dosage of traditional highly toxic disinfectants (such as formaldehyde). Chitosan (a natural polymer) and methylene blue (a low-toxic chemical agent) work synergistically to reduce toxicity while maintaining a mildew-proof effect. Traditional chemical disinfectants include highly toxic, high-residue formaldehyde and chlorine preparations; methylene blue is a low-toxic auxiliary ingredient, and its dosage is far lower than conventional disinfection concentrations. Liquid allicin Type VI is a natural biological antibacterial agent that works synergistically with chitosan to reduce toxicity while maintaining a mildew-proof effect.

[0030] Furthermore, the water quality indicators of the above-mentioned well water are:

[0031] The concentrations of each metal ion are: sodium ion 10-15 mg / L, magnesium ion 8-10 mg / L, aluminum ion 0.005-0.01 mg / L, potassium ion 1.0-2.0 mg / L, calcium ion 12-15 mg / L, cobalt ion 0.0005-0.002 mg / L, zinc ion 0-0.001 mg / L, silver ion 0-0.001 mg / L, the pH of the well water is 7.0-7.5, the salinity is 0.005-0.015%, and the ORP is 330-350 mv.

[0032] Furthermore, the aeration controls the dissolved oxygen in the culture medium to 6±0.5 mg / L.

[0033] The beneficial effects of adopting the above further technical solution are: controlling the airflow to create a weak circulation, simulating the swinging of the mother shrimp's abdominal feet, promoting oxygen exchange in the eggs, and making all embryos float slightly without sinking to the bottom, avoiding the rupture of the egg membrane caused by the impact of strong water flow.

[0034] Furthermore, the temperature of the culture solution is 27±1°C.

[0035] Furthermore, the pitch of the dense-tooth comb is 1-2 mm. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a schematic diagram of the structure of the in vitro hatching device for Procambarus clarkii.

[0037] Figure 2 This is a schematic diagram of the in vitro hatching device for Procambarus clarkii without the support structure installed.

[0038] Figure 3 This is a schematic diagram of the support structure of the in vitro hatching device for Procambarus clarkii.

[0039] Figure 4 This is a physical picture of the in vitro hatching device for Procambarus clarkii.

[0040] Figure 5 This is a physical picture of the 90-degree elbow joint.

[0041] Figure 6 This is a real picture of the thin-necked mouth of a transparent bucket.

[0042] Figure 7 This is a real picture of the bottom of the transparent bucket.

[0043] Figure 8 This is a picture of the steps for in vitro hatching of Procambarus clarkii.

[0044] Among them, 1-transparent bucket body, 2-90 degree elbow joint, 3-tee pipe, 4-aeration pipe, 5-outlet pipe, 6-air valve, 7-outlet valve, 8-mesh, 9-filter, 10-narrow neck bucket mouth, 11-bracket, 12-upper square frame, 13-middle square frame, 14-lower square frame, 15-vertical rod, 16-mesh-shaped frame, 17-black pickup. DETAILED DESCRIPTION

[0045] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0046] The in vitro hatching device of Procambarus clarkii comprises a transparent bucket body 1, a 90-degree elbow joint 2, a tee pipe 3, an aeration pipe 4, a water outlet pipe 5, an air valve 6, a water outlet valve 7, a mesh 8 and a filter 9;

[0047] The narrow-necked opening 10 of the transparent bucket body 1 is threadedly connected to the water inlet end of the 90-degree elbow joint 2. The water outlet end of the 90-degree elbow joint 2 is connected to the water inlet end of the tee pipe 3 via a pipeline. The water outlet end of the tee pipe 3 is connected to the narrow-necked opening 10 of the transparent bucket body 1 via a water outlet valve 7 and a water outlet pipe 5. The air inlet end of the tee pipe 3 is connected to the aeration pipe 4 via an air valve 6. The bottom of the transparent bucket body 1 is provided with an opening, a mesh 8 is placed in the transparent bucket body 1, and a filter 9 is fixedly installed at the narrow-necked opening 10 of the transparent bucket body 1. The in vitro hatching device for Procambarus clarkii of the present invention is uniquely designed and has the characteristics of low cost, visualization, large capacity, high efficiency, and easy operation. It can simultaneously accommodate a large number of shrimp eggs to meet the needs of large-scale hatching. The shrimp eggs receive appropriate water flow, oxygen, and nutrient supply during the hatching process, thereby improving the hatching success rate and seedling quality. The air valve 6 is connected to the aeration pipe 4, and the air volume is controlled so that the air is blown upward from the bottom of the transparent water bucket body 1. It is used for water aeration and the embryo is suspended without sinking to the bottom. The air flow is controlled to create a weak circulation, simulate the swinging of the female shrimp's abdominal foot, promote the oxygen exchange of the egg body, and all the embryos float slightly without sinking to the bottom, avoiding the impact of strong water flow and causing the egg membrane to rupture. The bottom of the transparent water bucket body 1 is provided with an opening, which can be used as a water inlet and an egg-laying port. The mesh 8 simulates the mother's tail segment line and the appendage bristles as an attachment matrix for embryo attachment. The filter screen 9 prevents the embryo from falling into the outside of the transparent water bucket body 1, avoiding the loss of the embryo.

[0048] In one embodiment, the bracket 11 is further included. The bracket 11 includes an upper square frame 12, a middle square frame 13 and a lower square frame 14. The frames are fixedly connected via vertical rods 15.

[0049] The transparent water bucket body 1 is placed upside down, with its bottom facing upward, between an upper square frame 12 and a middle square frame 13. The body is tangential to the four sides of the upper square frame 12. A "M"-shaped frame is fixedly mounted in the middle of the middle rectangular frame. The narrow neck 10 of the transparent water bucket body 1 passes through the middle opening of the "M"-shaped frame 16, which supports the body. The provision of a bracket 11 allows the transparent water bucket body 1 to be securely mounted and off the ground, facilitating aeration and drainage operations and allowing for easy observation.

[0050] In one embodiment, the upper square frame 12, the middle square frame 13, and the lower square frame 14 are of the same size. The vertices of the frames are fixedly connected by vertical rods 15 of the same height. The height of the vertical rods 15 is less than the height of the barrel. The bottom of the transparent barrel body 1 is completely cut into an opening. This makes the in vitro hatching device for Procambarus clarkii more neat and aesthetically pleasing, and allows for secure installation. The completely cut-out bottom of the transparent barrel body 1 provides more convenient water inlet and egg placement.

[0051] In one embodiment, the mesh 8 has a pore size of 1 mm and an area of ​​256-324 cm 2 The length and width of the mesh 8 are smaller than the diameter of the bucket. The mesh 8 is made of a nylon mesh woven with fine iron wires to prevent it from floating on the water surface due to gravity. The material of the mesh 8 allows the mesh 8 to be suspended in the culture medium in the transparent bucket body 1.

[0052] In one embodiment, the pore size of the filter 9 is 0.5-1 mm, which prevents the embryos from falling outside the transparent bucket body 1 and avoids the loss of the embryos.

[0053] In one embodiment, the bottom opening edge of the transparent water bucket body 1 is wrapped with a black pickup 17 to prevent damage to the eggs when laying them.

[0054] In one embodiment, the transparent bucket body 1 is made of a transparent plastic bucket, so that the transparent bucket body 1 can be visualized and the development of the embryo can be observed at any time.

[0055] Example 1

[0056] The in vitro culture method of Procambarus clarkii comprises the following steps: collecting healthy crayfish with eggs and placing them in clean water for temporary culture; after the eggs develop to the gastrula stage, using a comb with a 1mm pitch to separate the embryos from the crayfish ventral limbs and placing them in a culture dish; washing the embryos twice with clean water and removing the residue with a Pasteur pipette; and then transferring the embryos to an in vitro incubation device for incubation; pouring culture medium into a transparent water bucket body 1 made of a barreled water bucket; opening a water outlet valve 7; and injecting liquid into the transparent water bucket body 1 through an opening at the bottom of the bucket. The culture medium is at a temperature of 27±1°C, the flow rate is adjusted so that the water inlet is equal to the water outlet, and the daily water exchange volume is maintained at ≥2 / 3 to maintain a flowing water state. The air valve 6 is opened, and aeration is introduced into the bucket through the aeration pipe 4 to suspend the shrimp eggs. The aeration controls the dissolved oxygen in the culture medium to 6±0.5 mg / L. A mesh 8 is placed in the transparent water bucket body 1 for the attachment of shrimp eggs in the seedling I stage. Until the shrimp eggs develop to the seedling III stage, the injection of culture medium into the transparent water bucket body 1 is stopped, and the outlet valve 7 and the air valve 6 are closed. The in vitro cultivation of Procambarus clarkii is completed.

[0057] The culture medium included well water, calcium chloride, magnesium chloride, zinc chloride, chitosan, and methylene blue solution. The concentrations of calcium chloride, magnesium chloride, zinc chloride, chitosan, and methylene blue in the culture medium were 83 mg / L, 68.4 mg / L, 3 mg / L, 20 mg / L, and 0.2 mg / L. The number of moldy shrimp eggs and the number of emerged shrimp fry in each group were observed and recorded daily. The moldy egg rate and the emergence rate (seedling stage III) were calculated on days 2 and 5. The water quality was tested using an Agilent ICP-MS-7850 (HNYJ-03-11) inductively coupled plasma mass spectrometer. The pH, salinity, and redox potential (ORP) of the water were measured using a TE-1800 handheld multi-parameter high-precision water quality meter.

[0058] Table 1 Water quality test results of well water and culture fluid in Example 1

[0059]

[0060]

[0061] Table 2 Effect of culture medium of Example 1 on mold egg rate and germination rate

[0062] 2d mold egg rate 5d mold egg rate Seedling emergence rate culture medium 3 / 300＝0.33％ 9 / 300＝3％ 215 / 300＝71.67％

[0063] Example 2

[0064] The in vitro culture method of Procambarus clarkii comprises the following steps: collecting healthy crayfish with eggs and placing them in clean water for temporary culture; after the eggs develop to the gastrula stage, using a comb with a 1 mm pitch to separate the embryos from the crayfish ventral limbs and place them in a culture dish; washing the embryos twice with clean water, removing the residue with a Pasteur pipette, and then transferring the embryos to an in vitro incubation device for incubation; pouring culture medium into a transparent water bucket body 1, opening a water outlet valve 7, and simultaneously injecting culture medium into the transparent water bucket body 1 through an opening at the bottom of the bucket; and incubating the culture medium. The temperature of the nutrient solution is 27±1°C, the flow rate is adjusted so that the water inlet is equal to the water outlet, and the daily water exchange volume is maintained at ≥2 / 3 to maintain a flowing water state. The air valve 6 is opened, and aeration is introduced into the bucket through the aeration pipe 4 to suspend the shrimp eggs. The aeration controls the dissolved oxygen in the culture solution to 6±0.5 mg / L. A mesh 8 is placed in the transparent water bucket body 1 for the shrimp eggs in the seedling I stage to attach. Until the shrimp eggs develop to the seedling III stage, the injection of culture solution into the transparent water bucket body 1 is stopped, and the outlet valve 7 and the air valve 6 are closed. The in vitro cultivation of Procambarus clarkii is completed.

[0065] The culture medium includes well water, calcium chloride, magnesium chloride, zinc chloride, chitosan and liquid allicin type VI (product name: liquid allicin type VI, trade name: thioetherfloxacin, containing 15 wt% of allicin, liquid allicin type VI is an active ingredient extracted from garlic, mainly containing sulfur compounds such as allicin and ajoene). The concentration of calcium chloride in the culture medium is 83 mg / L, the concentration of magnesium chloride is 68.4 mg / L, the concentration of zinc chloride is 3 mg / L, the concentration of chitosan is 20 mg / L, and the concentration of liquid allicin type VI is 0.045 mg / L. The water quality is tested using an Agilent ICP-MS-7850 (HNYJ-03-11) inductively coupled plasma mass spectrometer; and the pH, salinity, and oxidation-reduction potential (ORP) of the water are measured using a TE-1800 handheld multi-parameter high-precision water quality meter.

[0066] Table 3 Water quality test results of well water and culture fluid in Example 2

[0067]

[0068] Table 4 Effect of culture medium of Example 2 on mold egg rate and germination rate

[0069] 2d mold egg rate 5d mold egg rate Seedling emergence rate culture medium 2 / 300＝0.66％ 3 / 300＝2.33％ 216 / 300＝72％

[0070] The emergence rates of Example 1 and Example 2 both reached over 70%.

[0071] Effect experiment:

[0072] Experiment 1

[0073] The in vitro culture method of Procambarus clarkii comprises the following steps: collecting healthy crayfish with eggs and placing them in clean water for temporary culture; after the eggs develop to the gastrula stage, using a comb with a 1 mm pitch to separate the embryos from the crayfish ventral limbs and place them in a culture dish; washing the embryos twice with clean water, removing the residue with a Pasteur pipette, and then transferring the embryos to an in vitro incubation device for incubation; pouring ultrapure water, tap water or well water into a transparent water bucket body 1; opening a water outlet valve 7; and simultaneously injecting ultrapure water, tap water or well water into the transparent water bucket body 1 through an opening at the bottom of the bucket. The temperature of well water, ultrapure water, tap water or well water is 27±1°C. The flow rate is adjusted so that the water inlet is equal to the water outlet, and the daily water exchange volume is maintained at ≥2 / 3 to maintain a flowing water state. The air valve 6 is opened, and aeration is introduced into the bucket through the aeration pipe 4 to suspend the shrimp eggs. The aeration controls the dissolved oxygen in the culture medium to 6±0.5 mg / L. A mesh 8 is placed in the transparent water bucket body 1 for the shrimp eggs in the seedling I stage to attach. Until the shrimp eggs develop to the seedling III stage, the injection of culture medium into the transparent water bucket body 1 is stopped, and the water outlet valve 7 and air valve 6 are closed. The in vitro culture of Procambarus clarkii is completed.

[0074] The number of moldy shrimp eggs and the number of shrimp seedlings in each group were observed and recorded every day. The moldy egg rate and seedling emergence rate (seedling stage III) were calculated on days 2 and 5. The water quality was tested using an Agilent ICP-MS-7850 (HNYJ-03-11) inductively coupled plasma mass spectrometer. The pH, salinity, and oxidation-reduction potential (ORP) of the water were measured using a TE-1800 handheld multi-parameter high-precision water quality meter.

[0075] Table 5 Water quality test results of ultrapure water, tap water or well water in Experiment 1

[0076]

[0077] Table 6 Effects of different water qualities on mold egg rate and germination rate in Experiment 1

[0078] 2d mold egg rate 5d mold egg rate Seedling emergence rate Tap water 3 / 300＝1％ 258 / 300＝86％ 28 / 300＝9.3％ well water 2 / 300＝0.6％ 237 / 300＝79％ 41 / 300＝13.7％ Ultrapure water 0 / 300＝0 3 / 300＝1％ 42 / 300＝14％

[0079] Conclusion: By comparing the effects of ultrapure water, tap water, and well water on shrimp egg hatching, we found that ultrapure water had a lower rate of mold infestation and a relatively higher larval emergence rate. Ultrapure water has limitations due to its high cost and limited practical applications, making it more suitable for laboratory use and less suitable for industrial-scale seedling production. Well water, on the other hand, is more suitable for industrial-scale seedling production. We sought to improve well water as a primary water source and identify an efficient and ecological medium for industrial-scale seedling production. Further exploration was conducted using ultrapure water as a primary water source. The three water qualities were tested and compared with well and tap water to identify key nutrients lacking in ultrapure water. Further exploration, based on ultrapure water, which has a lower incidence of water mold, was conducted by adding appropriate nutrients and biological disinfectants to identify a culture medium suitable for efficient seedling production in Procambarus clarkii.

[0080] Experiment 2

[0081] The in vitro culture method of Procambarus clarkii comprises the following steps: collecting healthy crayfish with eggs and placing them in clean water for temporary culture; after the eggs develop to the gastrula stage, using a comb with a 1 mm pitch to separate the embryos from the crayfish ventral limbs and place them in a culture dish; washing the embryos twice with clean water, removing the residue with a Pasteur pipette, and then transferring the embryos to an in vitro incubation device for incubation; pouring culture medium into a transparent water bucket body 1, opening a water outlet valve 7, and simultaneously injecting culture medium into the transparent water bucket body 1 through an opening at the bottom of the bucket; and incubating the culture medium. The temperature of the nutrient solution is 27±1°C, the flow rate is adjusted so that the water inlet is equal to the water outlet, and the daily water exchange volume is maintained at ≥2 / 3 to maintain a flowing water state. The air valve 6 is opened, and aeration is introduced into the bucket through the aeration pipe 4 to suspend the shrimp eggs. The aeration controls the dissolved oxygen in the culture solution to 6±0.5 mg / L. A mesh 8 is placed in the transparent water bucket body 1 for the shrimp eggs in the seedling I stage to attach. Until the shrimp eggs develop to the seedling III stage, the injection of culture solution into the transparent water bucket body 1 is stopped, and the outlet valve 7 and the air valve 6 are closed. The in vitro cultivation of Procambarus clarkii is completed.

[0082] The culture medium included ultrapure water, calcium chloride, magnesium chloride, and zinc chloride. The concentrations of calcium chloride, magnesium chloride, and zinc chloride in the culture medium were 83 mg / L, 68.4 mg / L, and 3 mg / L, respectively. The number of moldy shrimp eggs and the number of emerged shrimp fry in each group were observed and recorded daily. The moldy egg rate and the emergence rate (seedling stage III) were calculated on days 2 and 5. The water quality was tested using an Agilent ICP-MS-7850 (HNYJ-03-11) inductively coupled plasma mass spectrometer. The pH, salinity, and oxidation-reduction potential (ORP) of the water were measured using a TE-1800 handheld multi-parameter high-precision water quality meter.

[0083] Experiment 3

[0084] The in vitro culture method of Procambarus clarkii comprises the following steps: collecting healthy crayfish with eggs and placing them in clean water for temporary culture; after the eggs develop to the gastrula stage, using a comb with a 1 mm pitch to separate the embryos from the crayfish ventral limbs and place them in a culture dish; washing the embryos twice with clean water, removing the residue with a Pasteur pipette, and then transferring the embryos to an in vitro incubation device for incubation; pouring culture medium into a transparent water bucket body 1, opening a water outlet valve 7, and simultaneously injecting culture medium into the transparent water bucket body 1 through an opening at the bottom of the bucket; and incubating the culture medium. The temperature of the nutrient solution is 27±1°C, the flow rate is adjusted so that the water inlet is equal to the water outlet, and the daily water exchange volume is maintained at ≥2 / 3 to maintain a flowing water state. The air valve 6 is opened, and aeration is introduced into the bucket through the aeration pipe 4 to suspend the shrimp eggs. The aeration controls the dissolved oxygen in the culture solution to 6±0.5 mg / L. A mesh 8 is placed in the transparent water bucket body 1 for the shrimp eggs in the seedling I stage to attach. Until the shrimp eggs develop to the seedling III stage, the injection of culture solution into the transparent water bucket body 1 is stopped, and the outlet valve 7 and the air valve 6 are closed. The in vitro cultivation of Procambarus clarkii is completed.

[0085] The culture medium included ultrapure water, calcium chloride, magnesium chloride, zinc chloride, and chitosan. The concentrations of calcium chloride, magnesium chloride, zinc chloride, and chitosan in the culture medium were 83 mg / L, 68.4 mg / L, 3 mg / L, and 20 mg / L. The number of moldy shrimp eggs and the number of emerged shrimp fry in each group were observed and recorded daily. The moldy egg rate and the emergence rate (seedling stage III) were calculated on days 2 and 5. The water quality was tested using an Agilent ICP-MS-7850 (HNYJ-03-11) inductively coupled plasma mass spectrometer. The pH, salinity, and oxidation-reduction potential (ORP) of the water were measured using a TE-1800 handheld multi-parameter high-precision water quality meter.

[0086] Table 7 Effects of nutrients and chitosan on mold egg rate and germination rate

[0087] 2d mold egg rate 5d mold egg rate Seedling emergence rate Experiment 2 15 / 300＝5％ 10 / 300＝3.3％ 60 / 300＝20％ Experiment 3 3 / 300＝1％ 0 / 300＝0％ 107 / 300＝35.7％

[0088] Table 8 Water quality test results of nutrient substances and chitosan addition groups

[0089]

[0090]

[0091] Conclusion: The emergence rate was significantly improved after the three nutrients calcium chloride, magnesium chloride and zinc chloride were used as antibacterial ingredients, and chitosan was used as an antibacterial component.

[0092] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An in vitro hatching device for Procambarus clarkii, characterized in that: Including transparent bucket body, 90-degree elbow joint, tee pipe, aeration pipe, outlet pipe, air valve, outlet valve, mesh and filter; The narrow-necked mouth of the transparent water bucket body is threadedly connected to the water inlet end of the 90-degree elbow joint, the water outlet end of the 90-degree elbow joint is connected to the water inlet end of the tee pipe via a pipeline, the water outlet end of the tee pipe is connected to the water outlet pipe via a water outlet valve, and the air inlet end of the tee pipe is connected to the aeration pipe via an air valve. An opening is provided at the bottom of the transparent water bucket body, the mesh is placed in the transparent water bucket body, and the filter is fixedly installed at the narrow-necked mouth of the transparent water bucket body.

2. The in vitro hatching device for Procambarus clarkii according to claim 1, characterized in that: It also includes a bracket, which includes an upper square frame, a middle square frame and a lower square frame, and each layer of the frame is fixedly connected by a vertical rod; The bottom of the transparent water bucket body is upside down between the upper square frame and the middle square frame, the barrel body is tangent to the four sides of the upper square frame, and a U-shaped frame is fixedly installed in the middle of the middle rectangular frame. The narrow neck of the transparent water bucket body passes through the middle opening of the U-shaped frame, and the U-shaped frame supports the barrel body.

3. The in vitro hatching device for Procambarus clarkii according to claim 1, characterized in that: The mesh has a pore size of 1 mm and an area of ​​256-324 cm 2 The mesh is made of nylon mesh woven with thin iron wires.

4. The in vitro hatching device for Procambarus clarkii according to claim 1, characterized in that: The pore size of the filter is 0.5-1 mm.

5. The in vitro hatching device for Procambarus clarkii according to claim 1, characterized in that: The bottom opening edge of the transparent water bucket body is wrapped with black pickup.

6. A method for in vitro cultivation of Procambarus clarkii, characterized in that: The following steps are involved: The method comprises placing the egg-carrying shrimp in clean water for temporary rearing, and after the shrimp eggs develop to the gastrula stage, peeling the embryos from the crayfish ventral limbs with a fine-gauge comb, cleaning the embryos to remove residues, and then transferring them to the in vitro crayfish incubation device according to any one of claims 1 to 5 for incubation, pouring culture fluid into a transparent water bucket body, opening a water outlet valve, and simultaneously injecting culture fluid into the transparent water bucket body through an opening at the bottom of the bucket, adjusting the flow rate so that the water inlet is equal to the water outlet, opening an air valve, and aerating the bucket through an aeration pipe to suspend the shrimp eggs, and placing a mesh sheet in the transparent water bucket body for attachment of shrimp eggs in the seedling I stage until the shrimp eggs develop to the seedling III stage, stopping injecting culture fluid into the transparent water bucket body, closing the water outlet valve and the air valve, and completing the in vitro crayfish incubation. The culture solution comprises well water, calcium chloride, magnesium chloride, zinc chloride, chitosan and methylene blue solution, wherein the concentration of calcium chloride in the culture solution is 80-140 mg / L, the concentration of magnesium chloride is 60-90 mg / L, the concentration of zinc chloride is 2-8 mg / L, the concentration of chitosan is 20 mg / L, and the concentration of methylene blue is 0.2-0.4 mg / L; or, the culture solution comprises well water, calcium chloride, magnesium chloride, zinc chloride, chitosan and liquid allicin type VI, wherein the concentration of calcium chloride in the culture solution is 80-140 mg / L, the concentration of magnesium chloride is 60-90 mg / L, the concentration of zinc chloride is 2-8 mg / L, the concentration of chitosan is 20 mg / L, and the concentration of liquid allicin type VI is 0.02-0.05 mg / L.

7. The method for in vitro cultivation of Procambarus clarkii according to claim 6, characterized in that: The water quality indicators of the well water are: The concentrations of each metal ion are: sodium ion 10-15 mg / L, magnesium ion 8-10 mg / L, aluminum ion 0.005-0.01 mg / L, potassium ion 1.0-2.0 mg / L, calcium ion 12-15 mg / L, cobalt ion 0.0005-0.002 mg / L, zinc ion 0-0.001 mg / L, silver ion 0-0.001 mg / L, the pH of the well water is 7.0-7.5, the salinity is 0.005-0.015%, and the ORP is 330-350 mv.

8. The method for in vitro cultivation of Procambarus clarkii according to claim 6, characterized in that: The aeration controls the dissolved oxygen in the culture solution to be 6±0.5 mg / L.

9. The method for in vitro cultivation of Procambarus clarkii according to claim 6, characterized in that: The temperature of the culture solution is 27±1°C.

10. The method for in vitro cultivation of Procambarus clarkii according to claim 6, characterized in that: The close-tooth comb has a tooth pitch of 1-2 mm.

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