A simple method for in vitro culture of all-stage embryos of Neocaridina sutsugamushi

Abstract

The invention discloses a simple method for in vitro culture of embryos of new rice shrimps at all stages, comprising or consisting of the following steps: step S1: preparing shrimp culture water; step S2: preparing embryo hatching culture water; step S3: assembling an in vitro hatching device; step S4: stripping embryos of new rice shrimps carrying eggs; step S5: incubating the new rice shrimps in vitro embryos at all stages in an incubator; step S6: treating the new rice shrimps in vitro embryos infected with bacteria with a trypsin solution. The in vitro culture method of new rice shrimp embryos of the invention is not limited by the embryo development period, has a wide range of applicability, can be directly applied to various stages of in vitro embryo development of sawtooth new rice shrimps, and is also applicable to in vitro culture of embryos of other new rice shrimps. At the same time, when the embryos are infected with bacteria, the in vitro embryos of new rice shrimps infected with bacteria can be rescued by blowing with a trypsin solution, effectively reducing hypha adhesion, so that the in vitro embryos of new rice shrimps can develop normally and hatch shrimplets.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and in particular relates to a simple method for in vitro culture of all-stage embryos of new rice shrimp. Background Art

[0002] Neocaridina is a genus of freshwater shrimp in the subfamily Spathinae of the order Decapoda of the subphylum Crustacea. It mainly includes Neocaridina denticulata, Neocaridina palmata, Neocaridina gracilipoda, etc. They mainly live among the aquatic plants in ponds. They are small freshwater shrimps. Adults are generally stable at about 2 cm. They have the habit of holding eggs. Fertilized eggs are large, with a long diameter of about 1.1 mm and a short diameter of about 0.65 mm. The fertilized eggs adhere to the mother and develop directly. Neocaridina has the characteristics of short generation cycle, strong reproductive ability, clear genetic background, easy embryo manipulation, low environmental requirements, and can be raised in large quantities in the laboratory. It can be used for large-scale genetic analysis and molecular breeding research based on embryo manipulation of decapods, and as a model animal, it provides a powerful tool and platform for various aspects of crustacean research.

[0003] Embryo in vitro hatching is a low-cost, high-efficiency technology for breeding seedlings. This technology is applied to the breeding of shrimps and crabs. It can not only reduce the cost of feeding and management of shrimps and crabs with eggs, but also reduce the chance of eggs falling or being damaged by collisions during the egg-holding process, which helps to improve the hatching rate and has a high degree of hatching synchronization. However, in most of the breeding processes of new rice shrimp, the egg-holding and hatching technology of new rice shrimp is not mature enough. The number of eggs held by the mother of new rice shrimp is small, and there are many bacteria and parasites in the hatching water, which can easily lead to the death of shrimp eggs or the synchronization cannot be guaranteed. Therefore, the requirements for in vitro hatching of new rice shrimp embryos are relatively strict. It is necessary to improve the disinfection and sterilization measures of the embryo water, ensure sufficient oxygen, and continuously explore a suitable culture environment to improve the in vitro hatching rate of new rice shrimp embryos.

[0004] At present, there are many in vitro hatching methods for new rice shrimp embryos, usually using formaldehyde, 75% ethanol, potassium permanganate, bronopol, hydrogen peroxide and other solutions to disinfect the water body, and using ultraviolet irradiation or veterinary antibiotics to inhibit the growth of water bacteria. However, the above methods are relatively single, the breeding equipment used is complicated to operate, the repeatability is poor, and antibiotic substances are prone to cause microecological environment deterioration or aquatic animal digestion and absorption disorders and cause new diseases. At the same time, there is a lack of rescue measures for infected embryos in existing culture methods. The in vitro hatching of new rice shrimp is relatively strict. Therefore, it is necessary to improve water disinfection measures, ensure sufficient oxygen supply, further inhibit the growth of water bacteria, reduce the risk of embryo infection, and rescue the infected new rice shrimp fetuses, thereby improving the hatching rate of new rice shrimp in vitro embryos. Summary of the invention

[0005] The purpose of the present invention is to improve the efficiency of in vitro hatching of embryos of new rice shrimp at all stages, in order to solve the technical problem:

[0006] The present invention provides a simple method for in vitro culture of all-stage embryos of new rice shrimp, which comprises or consists of the following steps:

[0007] Step S1: preparing adult shrimp breeding water, breeding adult New Rice Prawns in the adult shrimp breeding water, and obtaining adult New Rice Prawns carrying eggs;

[0008] Step S2: preparing embryo incubation culture water;

[0009] Step S3: assembling an in vitro incubation device, the in vitro incubation device comprising an incubation tank and an incubator with a water-permeable bottom, the incubator with a water-permeable bottom being suspended on the inner wall of the incubation tank; adding embryo incubation culture water into the in vitro incubation device;

[0010] Step S4: peeling off the embryos of the adult shrimps carrying eggs to obtain in vitro embryos of the shrimps;

[0011] Step S5: incubating the in vitro embryos of the new rice shrimp in an incubator for the entire period;

[0012] Step S6: treating the infected shrimp in vitro embryos, wherein the treatment of the infected shrimp in vitro embryos comprises blowing with 0.2-0.22%, 0.22-0.24%, 0.24-0.26%, 0.26-0.28% or 0.28-0.3% trypsin solution.

[0013] Furthermore, the shrimp culture water is fresh water that is boiled and then cooled to room temperature.

[0014] Furthermore, the embryo incubation culture water is a methylene blue solution with a concentration of 2 mg / L.

[0015] Furthermore, the in vitro incubation device also includes a filter, a constant temperature heating rod and an air stone. The filter and the constant temperature heating rod are fixed in the incubation tank; the air stone is placed at the bottom of the incubation tank to control water fluctuations so that the in vitro embryos of the new rice shrimp are constantly turned over.

[0016] Furthermore, an embryo stripping tool is used to strip the embryos of adult shrimps carrying eggs to obtain in vitro embryos of the shrimps; the embryo stripping tool has a bending arc, and the embryo stripping tool includes a sterile syringe and a sterile easy-to-bend material; the sterile easy-to-bend material includes one or more of sterile iron wire, stainless steel, tungsten steel, and platinum materials; the bending arc is 30°, 30-36°, 36-40°, 40-44°, 44-48°, 48-52°, 52-56°, 56-58°, 58-61°, 61-64°, 64-68°, 68-72°, 72-74°, 74-78°, 78-80°, 80°, 100°, 100-106°, 106-111°, 111-116°, 116-121°, 121-126°, 126-131°, 131-136°, 136-141°, 141-146°, 146-150° or 150°.

[0017] Furthermore, step S4 also includes placing the new rice shrimp in vitro embryos in 0.2-0.22%, 0.22-0.24%, 0.24-0.26%, 0.26-0.28% or 0.28-0.3% trypsin solution for cleaning; the cleaning conditions are uniformly blowing within 105-135 seconds, and the blowing frequency is 50-54 times / minute, 54-58 times / minute, 58-62 times / minute or 62-66 times / minute.

[0018] Furthermore, the full-period incubation of the in vitro embryos of the new rice shrimp includes from one-cell, two-cell, four-cell, eight-cell, cleavage stage, blastocyst stage, gastrula, pre-nauplius, post-nauplius, pre-zoa larvae and post-zoa larvae to hatching of shrimp larvae.

[0019] Further, the first object of the treatment of the infected new rice shrimp in vitro embryos is the infection of the early stage of the zoanthid larvae. The treatment method of the early stage of the zoanthid larvae is to use 0.2-0.22%, 0.22-0.24%, 0.24-0.26% or 0.26-0.28% trypsin solution for blowing, and the blowing time is 300 seconds, 300-335 seconds, 335-365 seconds, 365-395 seconds, 395-425 seconds, 425-455 seconds, 455-485 seconds, 485-515 seconds, 515-545 seconds, 545-575 seconds or 575-605 seconds per day, and the blowing is carried out continuously for 4 days, 5 days, 6 days or 7 days, and the frequency of blowing is 84-88 times / minute, 88-92 times / minute or 92-96 times / minute.

[0020] Furthermore, the second object of treatment of the infected new rice shrimp in vitro embryos is the infection of the late zoanthid larvae; the treatment method of the late zoanthid larvae is to use 0.2-0.22%, 0.22-0.24%, 0.24-0.26% or 0.26-0.28% trypsin solution for blowing, the blowing time is 300 seconds, 300-335 seconds, 335-365 seconds, 365-395 seconds, 395-425 seconds, 425-455 seconds or 455-485 seconds per day, and the blowing frequency is 36-40 times / minute, 40-44 times / minute, 44-48 times / minute, 48-52 times / minute, 52-56 times / minute or 56-59 times / minute.

[0021] Further, after the treatment of the in vitro embryos of the infected new rice shrimp, the embryo hatching rate H of the in vitro embryos of the infected new rice shrimp satisfies:

[0022] H ≥ h + 85% × (100% - h),

[0023] Among them, h is the highest embryo hatching rate of in vitro embryos of new rice shrimp that were not infected with trypsin solution.

[0024] The present invention uses a method for treating embryo incubation and culture water of serrated shrimp with a methylene blue solution of appropriate concentration, so that the in vitro embryo disinfection and water body antibacterial effect of serrated shrimp are better; the method of using trypsin solution to blow the in vitro embryo of serrated shrimp reduces the adhesion of hyphae on the surface of the in vitro embryo of serrated shrimp, so that the embryo of serrated shrimp can develop normally and finally hatch shrimplets. In addition, the embryo stripping tool used in the present invention is easy to obtain, the operation method is simple and efficient, the maternal stress time is short, and the damage to the mother and embryo is small. The in vitro embryo is controlled by a simple incubation device for water flow-the combination of a water body filter device, an air stone and an incubator air permeable mesh screen can effectively reduce the adhesion between embryos, prevent pathogen infection and transmission, and avoid damage to the in vitro embryo due to uneven fluctuation force or excessive impact.

[0025] Therefore, the in vitro culture method of the embryo of the sawtooth shrimp is not limited by the embryonic development period, has a wide range of applicability, can be directly applied to each period of the in vitro embryonic development of the sawtooth shrimp, and the culture method is also applicable to the embryo in vitro culture of other shrimps. At the same time, when the in vitro embryo of the sawtooth shrimp is contaminated with bacteria, 0.25% trypsin solution is used for blowing, and the time of blowing is 5 minutes or more, preferably 5-8 minutes or 5-10 minutes per day, and the frequency of blowing is 45-90 times / minute, which can save the infected sawtooth shrimp embryo, effectively reduce hypha adhesion, so that the embryo can develop normally and hatch shrimp, and the hatching rate is 85% or more, and even the hatching rate reaches 100%. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The above content and the following specific embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. It should be noted that the accompanying drawings are only examples of the technical solutions claimed for protection.

[0027] Figure 1 This is a schematic diagram of the new shrimp hatching device (A is the hatching tank; a is the filter; b is the incubator; c is the air stone; d is the constant temperature heating rod);

[0028] Figure 2 This is a schematic diagram of the bending arc of the front section of the new shrimp embryo peeling tool being 30-80°;

[0029] Figure 3 This is a schematic diagram of the bending arc of the front section of the new shrimp embryo peeling tool being 100-150°;

[0030] Figure 4 The full-stage development diagram of the in vitro embryo of the serrated shrimp in Test Example 1 of the present invention (A is one-cell; B is two-cell; C is four-cell; D is eight-cell; E is the cleavage stage; F is the blastocyst stage; G is the gastrula; H is the early nauplius; I is the late nauplius; J is the early zoanthid; K is the late zoanthid; L is the shrimp larva; the scale bar of the in vitro embryo is 100 μm, and the scale bar of the shrimp larva is 1 mm);

[0031] Figure 5 The in vitro embryonic development diagram of the serrated shrimp in Test Example 2 of the present invention (A is the gastrula; B is the early nauplius; C is the late nauplius; D is the early zoanthid; E is the late zoanthid; F is the shrimp larva; the scale bar for the in vitro embryo is 100 μm, and the scale bar for the shrimp larva is 1 mm);

[0032] Figure 6 The comparison results of the in vitro embryo infection treatment of the serrated shrimp in the early stage of the zoanthid larvae in Test Example 3 of the present invention (A is the development result of the whole infection on the first day; B is the development result of the embryo on the third day without any treatment after infection; C is the development result of the embryo on the third day after infection with only 0.25% trypsin solution for 5 minutes but without blowing; D is the development result of the embryo on the third day after infection with ultrapure water for 5 minutes every day, the blowing frequency is 90 times / minute, and the blowing frequency is continuously blown for 5 minutes every day; E is the development result of the embryo on the third day after infection with 0.25% trypsin solution The embryonic development results after 2 days of stopping blowing after 5 minutes of blowing and 3 consecutive days with a blowing frequency of 90 times / minute; F is the embryonic development results after 4 days of continuous blowing with 0.25% trypsin solution after infection with bacteria and 4 minutes of blowing and 90 times / minute; G is the embryonic development results after 4 days of continuous blowing with 0.25% trypsin solution after infection with bacteria and 5 minutes or more, with a blowing frequency of 90 times / minute; H is the result of the development into shrimp larvae after processing in G; the scale bar for in vitro embryos is 100 μm, and the scale bar for shrimp larvae is 1 mm);

[0033] Figure 7 This is a comparative diagram of the infection results of the in vitro embryos of the serrated shrimp in the late zoanthid stage in Test Example 4 of the present invention (A is a serrated shrimp embryo infected with bacteria in the late zoanthid stage with a heartbeat of 150 times / minute; B is the embryo development result of blowing with 0.25% trypsin solution for 5 minutes at a frequency of 90 times / minute; C is the embryo development result of blowing with 0.25% trypsin solution for 5 minutes or more at a frequency of 45 times / minute; the scale of the in vitro embryo is 100 μm, and the scale of the shrimp larvae is 1 mm). DETAILED DESCRIPTION

[0034] The detailed features and advantages of the present invention are described in detail in the specific implementation modes below, and the contents are sufficient to enable any person skilled in the art to understand the technical contents of the present invention and implement them accordingly. Moreover, according to the description, claims and drawings disclosed in this specification, those skilled in the art can easily understand the relevant objects and advantages of the present invention.

[0035] It should be noted that in this specification, similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined and explained in the subsequent drawings.

[0036] In order to make the purpose, technical scheme and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0037] (1) Source of sample materials

[0038] In the present embodiment, the serrated shrimp was purchased from Qinghai famous and high-quality aquatic product breeding factory in Luoshan County, Xinyang, Henan Province.

[0039] (2) Sources of reagents and consumables

[0040] Table 1 Sources and item numbers of reagents and consumables required for the experiment

[0041] name Manufacturer Part Number Methylene blue Maclean M859248 0.25% Trypsin Solution Bioengineering E607001 1× PBS buffer Biosharp BL302A Sterile syringe Shanghai Bio-Tech Co., Ltd. FS801 tweezers LABMIK KSI-0014-01 Air Stone Zhejiang Zhoushan Sensen Group Co., Ltd. ZY-0040

[0042] (3) Source of instruments and equipment

[0043] Table 2 Sources and models of instruments and equipment required for the experiment

[0044] name Manufacturer model Filters Zhejiang Zhoushan Sensen Group Co., Ltd. JF120B Incubator Guangzhou Yangyunze Trading Co., Ltd. 19471003127367 Hatching tank Zhejiang Zhoushan Sensen Group Co., Ltd. HWK-500B Heating rod Yongcheng Aquarium Co., Ltd. MX-1027C Screen Yantai Shangshai Wire Mesh Products Co., Ltd. 1843 High-resolution fully automated stereo fluorescence microscope (stereoscope for short) Leica Thunder M205

[0045] Example

[0046] A simple method for in vitro culture of all-stage embryos of Neocaridina sutsugamushi comprises the following steps:

[0047] S1. Prepare shrimp culture water. The specific steps are as follows:

[0048] New rice shrimp is a freshwater shrimp. The water used for adult shrimp breeding is fresh water that has been boiled and then cooled to room temperature. The adult new rice shrimp can be put into the water for breeding to obtain adult new rice shrimp carrying eggs.

[0049] S2. Prepare embryo incubation culture water. The specific steps are as follows:

[0050] (1) Filter the shrimp culture water through a 200-300 mesh sieve for later use;

[0051] (2) Preparation of methylene blue stock solution: weigh 2 g of methylene blue powder and dissolve it in 100 mL of ultrapure water to prepare a 20 g / L methylene blue stock solution;

[0052] (3) Take 0.1 mL of methylene blue stock solution and dilute it to 1 L of fresh water filtered through a 200-300 mesh sieve. Mix well to obtain embryo incubation water. The concentration of methylene blue solution in embryo incubation water is 2 mg / L. Embryo incubation water requires fewer reagents and is simple to prepare. It can be prepared immediately before use.

[0053] S3. Assemble the in vitro incubation device. The specific steps are as follows:

[0054] Add an appropriate amount of embryo incubation water to the incubation tank, and place a sufficient amount of air stones at the bottom of the incubation tank to promote water fluctuations, so that the embryos can be constantly turned over, reduce adhesion between embryos, and prevent infection and spread of pathogens; hang multiple incubators with permeable bottoms on the wall of the incubation tank; place a filter in the incubation tank to maintain uniform water flow and uniform oxygen diffusion; and provide a constant temperature heating rod. The in vitro incubation device in this application is easy to assemble and simple to operate, and can be used for laboratory breeding or commercial breeding. The specific new rice shrimp incubation device is as follows Figure 1 shown.

[0055] S4. The embryos of adult shrimps of Neospermum serrulate, which are carrying eggs, are stripped. The specific steps are as follows:

[0056] (1) If Figure 2 , Figure 3As shown, the front end of the sterile syringe is bent at an arc of 30-80° or 100-150°, or a suitable sterile iron wire, stainless steel, tungsten steel, platinum or other materials are selected to be bent at an arc of 30-80° or 100-150°, a mature and healthy adult serrated shrimp with eggs is placed under a stereoscope, one hand gently clamps the head of the adult serrated shrimp with eggs through tweezers, and the other hand uses the bending part of the sterile syringe or the bending part of the sterile iron wire, stainless steel, tungsten steel, platinum or other materials to gently peel off the embryo, and the embryos in the abdomen of the adult serrated shrimp with eggs are taken out one by one, and the whole operation is performed under a stereoscope to avoid damage to the mother, so as to obtain the in vitro embryos of the serrated shrimp.

[0057] (2) Immediately collect the in vitro embryos of C. serrulate and place them in 1× PBS buffer or ultrapure water for 15 min. Use the bend of a sterile syringe or the bend of sterile iron wire, stainless steel, tungsten steel, platinum and other materials to separate the adhered in vitro embryos of C. serrulate. Then, use 0.25% trypsin solution to blow and clean the separated in vitro embryos of C. serrulate, blowing and washing evenly for about 120 times within 2 minutes. Place the embryos under a stereoscope to observe the various stages of development of the in vitro embryos of C. serrulate.

[0058] S5. Incubate the in vitro embryos of the new shrimp in an incubator for the entire period. The specific steps are as follows:

[0059] (1) Take the in vitro embryos of the serrated shrimp observed under the stereoscope in S4 and place them in a sterilized incubator with a water-permeable bottom and an 80-mesh screen (8 cm×8 cm×10 cm). The number of in vitro embryos of the serrated shrimp in each incubator is controlled to be 30 or less, and the incubator is hung on the inner wall of the incubation tank. The water height in the incubator is maintained at about 1 / 3 of the incubator height.

[0060] (2) Replace 1 / 3-1 / 2 of the embryo incubation water in the incubation tank every day. Replace the embryo incubation water in the incubation tank once every 3 days. Keep the water temperature constant at 24°C.

[0061] (3) Regularly take out the in vitro embryos of the serrated shrimp and place them under a stereoscope to observe their growth and development period and development status. Care should be taken to prevent the in vitro embryos of the serrated shrimp from sticking together.

[0062] (4) When the in vitro embryos of the serrated shrimp develop from the one-cell stage to the late nauplius stage, it is necessary to ensure that there is sufficient oxygen in the incubation tank. The air output of the air stone can make the in vitro embryos of the serrated shrimp turn evenly in the incubator. When the in vitro embryos of the serrated shrimp enter the early zoanthid stage, the air output of the air stone should be reduced to prevent excessive water fluctuations from hindering the rupture of the membrane of the in vitro embryos of the serrated shrimp.

[0063] S6. Treat the infected shrimp embryos in vitro. The specific steps are as follows:

[0064] (1) To prevent the in vitro embryos of Neomyces serrulate from being contaminated by bacteria, the embryos were washed with 0.25% trypsin solution by repeated blowing for 2 minutes at a frequency of 60 times per minute to prevent the embryos from sticking.

[0065] (2) When the in vitro embryos of C. serrulate are in the early stage of Zona larvae, they need to be repeatedly blown with 0.25% trypsin solution for 5 minutes or more, preferably 5-8 minutes or 5-10 minutes, at a frequency of 90 times / minute, for 4 consecutive days or more;

[0066] (3) When the heartbeat of the in vitro embryo of the serrated shrimp is above 150 beats / minute or the embryo is in the late zoanthid stage and is contaminated with bacteria, use 0.25% trypsin solution to gently blow and clean it. The force should not be too strong at this time. Gently blow and clean for 5 minutes or more, preferably 5-8 minutes, and the frequency of blowing and cleaning is 45-89 times / minute.

[0067] Test Example 1

[0068] The in vitro embryos of the serrulate shrimp at the one-cell stage observed under a stereoscope in step S4 of Example are subjected to an in vitro hatching experiment, comprising the following steps:

[0069] (1) placing an in vitro embryo of a one-cell shrimp in an incubator;

[0070] (2) Regularly replace 1 / 3-1 / 2 of the embryo incubation water in the incubation tank every day. At the same time, replace the embryo incubation water in the incubation tank once every 3 days. The water temperature should be kept constant at 24°C.

[0071] (3) During the intervals between changes in the embryo incubation and culture water, the development period of the in vitro embryos of the C. serrulate was continuously observed through a stereoscope until the shrimp hatched, and the number of in vitro embryos of the C. serrulate was recorded;

[0072] (4) From the one-cell stage to the nauplius stage, the gas stone released a large amount of gas, and the in vitro embryos of Neomyces serrulate flipped up and down;

[0073] (5) After the in vitro embryos of the serrated shrimp enter the zoanthid stage, the gas output of the air stone is reduced to prevent the larvae of the serrated shrimp from breaking their membranes;

[0074] (6) After about 21 days, the larvae of Neocaridina serrulate are hatched, and the hatching rate of Neocaridina serrulate from the one-cell stage to the larvae is calculated.

[0075] Test Example 1: The results of stereomicroscope examination of the whole period of in vitro embryonic development of Neomyces serrulate were as follows: Figure 4As shown in the figure, there are obvious differences between different developmental stages. As the development period of the in vitro embryos of the serrated shrimp is closer to the hatching of the shrimp, the hatching rate of the in vitro embryos of the serrated shrimp is higher, and there are no malformed in vitro embryos of the serrated shrimp during the in vitro culture process. Among them, the hatching rate of the in vitro embryos of the serrated shrimp in the one-cell stage reached 35%, that is, the hatching rate from the one-cell stage to the shrimp.

[0076] Test Example 2

[0077] The in vitro embryos of any period of the scaly shrimp observed under a stereoscope in step S4 of Example are subjected to an in vitro hatching experiment, comprising the following steps:

[0078] (1) randomly selecting in vitro embryos of Neomycinus serrulate and observing them through a stereoscope, and finding that the in vitro embryos of Neomycinus serrulate are in the gastrula stage, and placing the in vitro embryos of Neomycinus serrulate in the gastrula stage in an incubation device;

[0079] (2) Regularly replace 1 / 3-1 / 2 of the embryo incubation water in the incubation tank every day. At the same time, replace the embryo incubation water in the incubation tank once every 3 days. The water temperature should be kept constant at 24°C.

[0080] (3) From the gastrula stage to the nauplius stage, the gas stone released a large amount of gas, and the embryos of Neomyces serrulate flipped up and down;

[0081] (4) After the in vitro embryos of Neomyces serrulate enter the zoanthid stage, the gas output of the air stone is reduced to prevent the larvae from breaking the membrane;

[0082] (5) Observe the shrimps under a stereomicroscope until their larvae are hatched, and calculate the hatching rate of the shrimps from the gastrula stage to the larvae.

[0083] Test Example 2: The results of stereomicroscope examination of in vitro embryonic development of Neomyces serrulate Figure 5 As shown, the hatching rate of shrimplets hatched from in vitro embryos in the nauplii stage of the serrated shrimp is 80% or above, and the hatching rate of shrimplets hatched from in vitro embryos in the zoanthid stage of the serrated shrimp is 90% or above, and even reaches 100%.

[0084] Test Example 3

[0085] The in vitro embryos of the scaly shrimp infected with bacteria in step S5 of Example were subjected to a trypsin solution treatment experiment, comprising the following steps:

[0086] (1) The developmental period of the infected Neocaridina serrulate embryos was observed through a stereomicroscope, and it was found that the infected Neocaridina serrulate embryos were in the early stage of zoanthid larvae. The infected Neocaridina serrulate embryos in the early stage of zoanthid larvae (i.e., the first object) were placed separately in a new incubation device;

[0087] (2) Regularly replace 1 / 3-1 / 2 of the embryo incubation water in the incubation tank every day. At the same time, replace the embryo incubation water in the incubation tank once every 3 days. The water temperature should be kept constant at 24°C.

[0088] (3) The in vitro embryos of Neocaridina serrulate that were infected with bacteria in the early stage of zoanthid larvae were divided into 6 treatment groups: the first treatment group: the in vitro embryos were not treated with any treatment after infection; the second treatment group: the in vitro embryos were soaked in 0.25% trypsin solution for 5 minutes every day without blowing; the third treatment group: the in vitro embryos were blown with ultrapure water for 5 minutes or more, preferably 5-8 minutes or 5-10 minutes, and the frequency of blowing was 90 times / minute every day; the fourth treatment group: the in vitro embryos were soaked in 0.25% trypsin solution every day The solution was blown on the infected in vitro embryos for 5 minutes, continuously for 3 days, and then the blowing was stopped for 2 days, with a frequency of 90 times / minute; the fifth treatment group: the infected in vitro embryos were blown on with 0.25% trypsin solution for 4 minutes every day, continuously for 4 days, with a frequency of 90 times / minute; the sixth treatment group: the infected in vitro embryos were blown on with 0.25% trypsin solution for 5 minutes or more, preferably 5-8 minutes or 5-10 minutes, continuously for 4 days, with a frequency of 90 times / minute.

[0089] (4) The hatching status of the embryos of Neocaridina serrulate that were infected with bacteria in the early stage of zoa larvae after being treated in the above step (3) was observed under a stereoscope, and the developmental results of the embryos of Neocaridina serrulate that were infected with bacteria in the early stage of zoa larvae on the first day of infection were observed under a stereoscope as a control. The hatching rates of the shrimp hatched from the in vitro embryos after infection in the six treatment groups were calculated.

[0090] Test case 3 results are as follows Figure 6As shown: the first treatment group: the growth and development of the in vitro embryos of the serrated new rice shrimp infected in the early stage of zoanthids has stagnated, they are damaged and dead, and the hatching rate of the hatched shrimps is 0. The second treatment group: the hyphae of the in vitro embryos of the serrated new rice shrimp infected in the early stage of zoanthids spread and adhered, the growth and development has stagnated, the embryos died, and the hatching rate of the hatched shrimps was 0. The third treatment group: the hyphae adhesion of the in vitro embryos of the serrated new rice shrimp infected in the early stage of zoanthids continued to expand, resulting in stagnation of embryo development, death due to lack of oxygen, and the hatching rate of the hatched shrimps was 0. The fourth treatment group: after the in vitro embryos of the serrated new rice shrimp infected in the early stage of zoanthids were blown with 0.25% trypsin solution for 3 days, the blowing was stopped for 2 days and it was observed that the in vitro embryos of the serrated new rice shrimp infected in the early stage of zoanthids were already in the late stage of zoanthids, but due to severe infection, the shrimps could not be successfully hatched, and the hatching rate was 0. The fifth treatment group: the mycelial adhesion of the in vitro embryos of the serrated shrimp infected with bacteria in the early stage of zoanthids was reduced, and the in vitro embryos of the serrated shrimp infected with bacteria in the early stage of zoanthids were already in the late stage of zoanthids, but due to the short daily blowing time, the mycelium spread slightly, hindering the membrane breaking process, and the shrimp larvae could not be successfully hatched, and the hatching rate was 0. The sixth treatment group: the mycelial adhesion of the in vitro embryos of the serrated shrimp infected with bacteria in the early stage of zoanthids was significantly reduced, and the in vitro embryos of the serrated shrimp grew and developed normally and the hatching rate of shrimp larvae was 100%.

[0091] Test Example 4

[0092] The in vitro embryos of the scaly shrimp infected with bacteria in step S5 of Example were subjected to a trypsin solution treatment experiment, comprising the following steps:

[0093] (1) The developmental period of the infected Neocaridina serrulate embryos was observed through a stereomicroscope, and it was found that the infected Neocaridina serrulate embryos were in the late zoanthid stage. The infected Neocaridina serrulate embryos in the late zoanthid stage (i.e., the second object) were placed separately in a new incubation device;

[0094] (2) Regularly replace 1 / 3-1 / 2 of the embryo incubation water in the incubation tank every day. At the same time, replace the embryo incubation water in the incubation tank once every 3 days. The water temperature should be kept constant at 24°C.

[0095] (3) In the intervals between changing the embryo incubation and culture water, the infection of the in vitro embryos of Neomycin serrulate shrimp in the late zoanthid stage was observed through a stereoscope. After the heartbeat reached 150 beats / minute, they were divided into two experimental groups. The first experimental group: the infected in vitro embryos were gently blown with 0.25% trypsin solution for 5 minutes or more, preferably 5-8 minutes, and the frequency of blowing was 90 times / minute; the second experimental group: the infected in vitro embryos were gently blown with 0.25% trypsin solution for 5 minutes or more, preferably 5-8 minutes, and the frequency of blowing was 45 times / minute.

[0096] (4) The hatching status of the in vitro embryos of Neocaridina serrulate in the first and second experimental groups after being infected with bacteria in the late zoanthid stage and treated with 0.25% trypsin solution was observed under a stereoscope. The in vitro embryos of Neocaridina serrulate in the late zoanthid stage with a heartbeat of 150 beats / min observed under a stereoscope were used as a control, and the hatching rate was calculated.

[0097] Test case 4 results are as follows Figure 7 As shown, the first experimental group was gently blown by 0.25% trypsin solution for 5 minutes or more, preferably 5-8 minutes, and the blowing frequency was 90 times / minute. The in vitro embryos of the serrated shrimps in the late stage of the zoanthid larvae were damaged, broken and died, and the hatching rate of the hatched shrimps was 0. The second experimental group was gently blown by 0.25% trypsin solution for 5 minutes or more, preferably 5-8 minutes, and the blowing frequency was 45 times / minute. The in vitro embryos of the serrated shrimps in the late stage of the zoanthid larvae were able to develop normally, and the hatching rate of the normally hatched shrimps was 100%.

[0098] It can be concluded that the simple and efficient hatching device disclosed in the present invention is a combination of a water flow control-water filtering device, an air stone and an incubator air-permeable mesh screen, which can reduce adhesion between embryos, prevent infection and spread of pathogens, and avoid damage to the in vitro embryos of the new rice shrimp due to uneven water fluctuation or excessive impact; the embryo stripping tool used in the present invention is easy to obtain, the operation method is simple and efficient, the maternal stress time is short, and the damage to the new rice shrimp mother and the new rice shrimp in vitro embryo is small; the method of treating embryo incubation and culture water with a methylene blue solution provided by the present invention makes the disinfection of the serrated new rice shrimp embryo and the antibacterial effect of the water better; the method of blowing the in vitro embryos of the new rice shrimp with a trypsin solution reduces the adhesion of hyphae on the embryo surface, so that the in vitro embryos of the new rice shrimp can develop normally, and the hatching rate of the finally hatched shrimplets is 85% or more, or even 100%. Therefore, the simple method for in vitro culture of embryos of the whole period of the new rice shrimp of the present invention is not limited by the development period of the in vitro embryos of the new rice shrimp, has a wide range of applicability, and can be directly applied to various periods of in vitro embryo development of the sawtooth new rice shrimp. The culture method is also applicable to the in vitro culture of embryos of other new rice shrimps. At the same time, when the in vitro embryos of the new rice shrimp of the present invention are contaminated with bacteria, 0.25% trypsin solution is used for blowing, and the blowing time is 5 minutes or more, preferably 5-8 minutes or 5-10 minutes, and the blowing frequency is 45-90 times / minute, which can save the in vitro embryos of the sawtooth new rice shrimp contaminated with bacteria, effectively reduce hypha adhesion, and enable the in vitro embryos of the new rice shrimp to develop normally and hatch shrimplets with a hatching rate of 100%. After the treatment of the in vitro embryos of the new rice shrimp contaminated with bacteria, the embryo hatching rate H of the new rice shrimp embryos satisfies:

[0099] H ≥ h + 85% × (100% - h),

[0100] Among them, h is the highest embryo hatching rate of in vitro embryos of new rice shrimp that were not infected with trypsin solution.

[0101] The terms and expressions used herein are for descriptive purposes only and the present invention should not be limited to these terms and expressions. The use of these terms and expressions does not mean to exclude any equivalent features of the illustrations and descriptions (or parts thereof), and it should be recognized that various modifications that may exist should also be included in the scope of the claims. Other modifications, changes and substitutions may also exist. Accordingly, the claims should be deemed to cover all such equivalents.

[0102] Similarly, it should be pointed out that although the present invention has been described with reference to the current specific embodiments, ordinary technicians in this technical field should realize that the above embodiments are only used to illustrate the present invention, and various equivalent changes or substitutions may be made without departing from the spirit of the present invention. Therefore, as long as the changes and modifications to the above embodiments are within the scope of the essential spirit of the present invention, they will fall within the scope of the claims of the present invention.

Claims

1. A simple method for in vitro culture of all-stage embryos of new rice shrimp, characterized in that: The method comprises or consists of the following steps: Step S1: preparing adult shrimp breeding water, breeding adult New Rice Prawns in the adult shrimp breeding water, and obtaining adult New Rice Prawns carrying eggs; Step S2: preparing embryo incubation culture water, wherein the embryo incubation culture water is a methylene blue solution with a concentration of 2 mg / L; Step S3: assembling an in vitro incubation device, the in vitro incubation device comprising an incubation tank and an incubator with a water-permeable bottom, the incubator being suspended on the inner wall of the incubation tank; adding the embryo incubation culture water into the in vitro incubation device; Step S4: peeling off the embryos of the adult shrimps carrying eggs to obtain in vitro embryos of the shrimps, and placing the in vitro embryos of the shrimps in a 0.2-0.22%, 0.22-0.24%, 0.24-0.26%, 0.26-0.28% or 0.28-0.3% trypsin solution for cleaning; the cleaning condition is uniformly blowing within 105-135 seconds, and the blowing frequency is 50-54 times / minute, 54-58 times / minute, 58-62 times / minute or 62-66 times / minute; Step S5: performing full-period incubation of the in vitro embryos of the new rice shrimp in the incubator; Step S6: treating the infected shrimp in vitro embryos, wherein the treatment of the infected shrimp in vitro embryos comprises blowing with the 0.2-0.22%, 0.22-0.24%, 0.24-0.26% or 0.26-0.28% trypsin solution, wherein the first object of the treatment of the infected shrimp in vitro embryos is the early stage of the zoanthid larvae, and the blowing time is 300-335 seconds, 335-365 seconds, 365-395 seconds, 395-425 seconds, 4 25-455 seconds, 455-485 seconds, 485-515 seconds, 515-545 seconds, 545-575 seconds or 575-605 seconds, continuously blowing and beating for 4 days, 5 days, 6 days or 7 days, the frequency of the blowing and beating is 84-88 times / minute, 88-92 times / minute or 92-96 times / minute, so as to rescue the in vitro embryos of the infected new rice shrimp, so that the in vitro embryos of the infected new rice shrimp develop normally and hatch shrimplet, and the hatching rate of the in vitro embryos of the infected new rice shrimp is 85-100%.

2. The simple method according to claim 1, characterized in that: The shrimp culture water is fresh water that is boiled and then cooled to room temperature.

3. The simple method according to claim 1, characterized in that: The in vitro hatching device also includes a filter, a constant temperature heating rod and an air stone. The filter and the constant temperature heating rod are fixed in the hatching tank. The air stone is placed at the bottom of the hatching tank to control water fluctuations so that the in vitro embryos of the new rice shrimp are constantly turned over.

4. The simple method according to claim 1, characterized in that: The embryos of the adult shrimps carrying eggs are peeled off by using an embryo peeling tool to obtain the in vitro embryos of the shrimps; the embryo peeling tool has a bending arc, and the embryo peeling tool comprises a sterile syringe and a sterile easy-to-bend material; the sterile easy-to-bend material comprises one or more of sterile iron wire, stainless steel, tungsten steel, and platinum material; the bending arc is 30-36°, 36-40°, 40-44°, 44-48°, 48-5 2°, 52-56°, 56-58°, 58-61°, 61-64°, 64-68°, 68-72°, 72-74°, 74-78°, 78-80°, 100-106°, 106-111°, 111-116°, 116-121°, 121-126°, 126-131°, 131-136°, 136-141°, 141-146° or 146-150°.

5. The simple method according to claim 1, characterized in that: The whole period of incubation of the in vitro embryo of the new rice shrimp includes from one cell, two cells, four cells, eight cells, cleavage stage, blastocyst stage, gastrula, early nauplius, late nauplius, early zoanthid and late zoanthid to hatching shrimplets.

6. The simplified method according to claim 1, characterized in that: In the step S6, the second object of the treatment of the infected new rice shrimp in vitro embryos is the infection of the late zoanthid larvae; the treatment method of the late zoanthid larvae is to use the 0.2-0.22%, 0.22-0.24%, 0.24-0.26% or 0.26-0.28% trypsin solution for blowing, the blowing time is 300-335 seconds, 335-365 seconds, 365-395 seconds, 395-425 seconds, 425-455 seconds or 455-485 seconds per day, and the blowing frequency is 36-40 times / minute, 40-44 times / minute, 44-48 times / minute, 48-52 times / minute, 52-56 times / minute or 56-59 times / minute.

7. The simplified method according to any one of claims 1 and 6, characterized in that: After the treatment of the in vitro embryos of the infected new rice shrimp, the embryo hatching rate H of the in vitro embryos of the infected new rice shrimp satisfies: H ≥ h + 85% × (100% - h) Wherein, h is the maximum embryo hatching rate of the in vitro embryos of the infected shrimp without using the trypsin solution.

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