Method to significantly improve the survival rate of fertilized eggs of mandarin fish after micromanipulation

Abstract

The present invention provides a method for significantly improving the survival rate of fertilized eggs of mandarin fish after micromanipulation, and its main technologies include: artificial insemination of mandarin fish, microinjection, hatching of fertilized eggs, and maintenance of culture medium during hatching. The present invention adopts an improved artificial insemination method and fertilized egg culture medium formula, which significantly improves the hatching rate and survival rate of fertilized eggs after microinjection, and provides technical support for genome editing breeding of mandarin fish.

Description

Technical Field

[0001] The invention relates to the technical field of genetic breeding of mandarin fish, and in particular to a method for significantly improving the survival rate of mandarin fish fertilized eggs after micromanipulation. Background Art

[0002] Fish fertilized egg microinjection technology is an important technology in fish developmental biology and fish genetic breeding. Researchers can use microinjection technology and micromanipulation technology to perform transgenic, gene knockdown, gene knockout, cell transplantation, nuclear transplantation and drug injection. Fish fertilized eggs are covered by egg membranes, which maintain a relatively stable environment for fertilized eggs and embryos until they mature and shed the membranes, and then they can directly enter the water body and move freely. However, the consequences of egg membrane damage in freshwater fish are different. Our subject group found that zebrafish, carp, crucian carp and other carp fish are basically not affected, but the damage to the egg membrane of mandarin fish fertilized eggs seriously affects the fertilized egg's emergence rate and feeding rate, and the survival rate is close to zero.

[0003] Liang Xufang and others found through genome sequencing and analysis that mandarin fish is a relatively recent fish that moved from seawater to freshwater, so it may have adaptability in osmotic pressure regulation during the embryonic stage. When the egg membrane is destroyed by micromanipulation, it will lead to osmotic pressure regulation disorder, resulting in a decrease in embryonic survival rate, which will cause the micromanipulated fish eggs to be unable to hatch or ingest food and die.

[0004] After the applicant conducted extensive research and eliminated multiple factors such as water quality, dissolved oxygen, and stillness, we found that appropriate osmotic pressure and ion support are necessary conditions for the survival of fertilized eggs of mandarin fish after micromanipulation.

[0005] In view of this, it is necessary to design an improved method for improving the survival rate of fertilized eggs of mandarin fish after micromanipulation to solve the above problems. Summary of the invention

[0006] In view of the above-mentioned defects of the prior art, the object of the present invention is to provide a method for significantly improving the survival rate of mandarin fish fertilized eggs after micromanipulation, so as to solve the problem of too low survival rate of mandarin fish fertilized eggs after micromanipulation.

[0007] To achieve the above object, the present invention provides a method for significantly improving the survival rate of fertilized eggs of mandarin fish after micromanipulation, comprising the following steps:

[0008] Step 1: After the injection of oxytocin to the mandarin fish parent reaches the effect time, gently squeeze the abdomen of the female fish and the male fish to collect eggs and sperm, absorb the eggs and sperm into a container and mix them evenly, add sea salt solution, stir and suspend the sperm-egg mixture, let it stand, add aerated water, let it stand, pour out the excess water, add aerated water to submerge the fertilized eggs, inflate the container, and let it stand for use;

[0009] Step 2, dissolving NaCl, KCl, CaCl2 and HEPES in water to prepare a culture medium stock solution;

[0010] Step 3, using agarose and 1X concentration culture medium to make a fertilized egg holding groove;

[0011] Step 4, suck the fertilized eggs into the fertilized egg holding tank, arrange the fertilized eggs neatly, add 1X concentration culture medium, and use a microinjector to inject the gene knockout system into the animal pole of the fertilized eggs;

[0012] Step 5: prepare a container, inject 1X concentration culture medium, place the injected fertilized eggs in an incubator, fix the incubator below the liquid level of the container, ventilate, and incubate with running water;

[0013] Step 6: Continuously culture the incubator with culture solutions of different concentrations. After the culture is completed, dilute the culture solution gradually within five hours and finally add fresh water.

[0014] As a further improvement of the present invention, in step 1, the volume ratio of the sucked eggs, sperm and the added sea salt solution is 10:0.2:10; and the standing time is 30 to 35 seconds.

[0015] Furthermore, the concentration of the sea salt solution is 0.4-0.6%.

[0016] In step 2, the mass ratio of NaCl, KCl, CaCl2 and HEPES is (30-35):(0.9-1.3):(0.8-1.2):(5.5-6.5).

[0017] Furthermore, the concentration of the prepared culture medium stock solution is five times that of the standard working solution, and is prepared to the required concentration before use.

[0018] In step 3, the preparation method of the fertilized egg clamping groove is: mix 1X concentration culture medium and agarose, heat to boiling, shake well, cool, introduce into a flat dish, then tilt and place the clamping groove mold so that the mold floats on the surface of the agarose solution, let it stand, wait for the agarose solution to solidify, demold, and obtain the fertilized egg clamping groove.

[0019] Furthermore, the mass fraction of agarose in the fertilized egg holding groove is 2%.

[0020] Furthermore, the temperature reduction range is 45-55°C; and the thickness of the plate is 10 cm.

[0021] In step 4, the droplet size of microinjection is 0.5 to 1‰ of the size of the fertilized egg.

[0022] In step 6, the continuous culture method is: after culturing the incubator in 1X concentration culture medium for 24 hours, the culture medium is replaced with 0.7X concentration culture medium, and the culture is continued for 24 hours, and then replaced with 0.5X concentration culture medium and continued to be cultured until the fertilized eggs hatch out of the membrane.

[0023] The beneficial effects of the present invention are:

[0024] (1) The present invention provides a method for significantly improving the survival rate of fertilized eggs of mandarin fish after micromanipulation, and its main technologies include: artificial insemination of mandarin fish, microinjection, hatching of fertilized eggs, and maintenance of culture medium during hatching. The present invention adopts an improved artificial insemination method and fertilized egg culture medium formula, which greatly improves the hatching rate and survival rate of fertilized eggs after microinjection, and provides technical support for genome editing breeding of mandarin fish.

[0025] (2) The present invention adds a sea salt solution of a specific concentration during the artificial insemination process to activate the egg and sperm to complete the fertilization process in a short time, thereby improving the success rate of fertilization.

[0026] (3) The present invention reduces the dramatic change in osmotic pressure after egg membrane damage by adjusting the culture medium, greatly improves the survival rate of fertilized eggs after micromanipulation or microinjection, and significantly increases the success rate of precise molecular design genetic breeding. In addition, the present method may also be applicable to the key technical field of micromanipulation or microinjection of freshwater Perciformes and marine Perciformes fish, laying a foundation for precise molecular design genetic breeding of fish in my country.

[0027] (4) The method of the present invention is used for microinjection technology and micromanipulation technology, which can significantly improve the membrane emergence rate and survival rate in operations such as transgenic, gene knockdown, gene knockout, cell transplantation, nuclear transplantation and drug injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a structural schematic diagram of the fertilized egg clamping groove mold. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0030] It should also be noted that, in order to avoid obscuring the present invention due to unnecessary details, only structures and / or processing steps closely related to the scheme of the present invention are shown in the drawings, while other details that are not closely related to the present invention are omitted.

[0031] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0032] The invention provides a method for improving the survival rate of fertilized eggs of mandarin fish including mandarin fish chuatsi, big-eyed mandarin fish and spotted mandarin fish after micromanipulation. The main techniques include artificial insemination of mandarin fish, microinjection, hatching of fertilized eggs and maintenance of culture medium during hatching.

[0033] Specifically, a method for significantly improving the survival rate of fertilized eggs of mandarin fish after micromanipulation comprises the following steps:

[0034] Step 1: After the injection of oxytocin reaches the effective time, gently squeeze the abdomen of the female and male fish to collect eggs and sperm, absorb the eggs and sperm into a container and mix them evenly, add sea salt solution, stir and suspend the sperm-egg mixture, let it stand, add aerated water, let it stand, pour out excess water, add aerated water to submerge the fertilized eggs, inflate the container, and let it stand for use.

[0035] Specifically, the parent of mandarin fish is a well-developed female fish, characterized by a swollen abdomen, substantial and soft to the touch. You can also use an egg digger to collect some eggs, preferably stage IV eggs. For male fish, a light squeeze of the abdomen will produce milky white semen with a certain viscosity, which will quickly disperse when it comes into contact with water. You can also use a microscope to observe sperm motility, take 1 to 2 microliters and place them on a slide, add 50 microliters of water, and observe the rapid movement of sperm, preferably for more than 30 seconds.

[0036] Place the selected mature parents in a cage in a cement pool with a water depth of 1 to 1.2 meters. The cage is made of nylon with a mesh size of 3 to 5 mm to prevent scratching the parents. Use nanotube aeration to maintain dissolved oxygen above 7 mg / L. Female fish are injected with two injections, and the drug is prepared according to 40 micrograms per kilogram of LHRH A2 plus 20 micrograms per kilogram of dioxin maleate. The first injection is 10% of the dose, and the remaining dose is injected 10 hours later. The estrus period is reached about 20 hours after the second injection at a water temperature of 25 degrees Celsius. Gently press the abdomen of the female fish to move the eggs from the abdominal cavity to the cloaca, rupture the ovarian membrane, and discharge the egg cells. Place the egg cells in a clean, water-free container, absorb the eggs and sperm and mix them evenly, add sea salt solution, stir and suspend the sperm-egg mixture, let it stand for 30 to 35 seconds, and add aerated water with a volume of 8 to 10 times the added sea salt solution. Let it stand for 30 to 35 seconds and pour out the excess water. Add an appropriate amount of aerated water to submerge the fertilized eggs, inflate the container, and let it stand for later use.

[0037] In the above steps, the volume ratio of the sucked eggs, sperm and added sea salt solution is preferably 10:0.2:10.

[0038] The concentration of the sea salt solution is preferably 0.4-0.6%, and more preferably, the concentration of the sea salt solution is 0.6%.

[0039] Step 2: Dissolve NaCl, KCl, CaCl2 and HEPES in water to prepare a culture medium stock solution.

[0040] Specifically, NaCl, KCl, CaCl2 and HEPES were dissolved in water, fixed to volume, and a 5X concentration culture medium stock solution was prepared, which was previously prepared to the required concentration.

[0041] In the above steps, the mass ratio of NaCl, KCl, CaCl2 and HEPES is preferably (30-35):(0.9-1.3):(0.8-1.2):(5.5-6.5).

[0042] Step 3: Use agarose and 1X concentration culture medium to make a fertilized egg holding groove.

[0043] Specifically, the fertilized egg holding groove mold can be purchased or customized in the shape of Figure 1 As shown, you can also use a zebrafish-specific mold. Mix 1X culture medium and agarose, heat to boiling, shake, cool, and introduce into a flat dish. Then tilt and put the clamping groove mold into it. Be careful to avoid bubbles. Let the mold float on the surface of the agarose solution. Let it stand until the agarose solution solidifies. Use pointed tweezers to carefully lift the mold and demould it to get the fertilized egg clamping groove.

[0044] In the above steps, the mass fraction of agarose in the fertilized egg holding groove is preferably 2%.

[0045] In the above steps, the temperature reduction range is preferably 45 to 55°C.

[0046] In the above steps, the thickness of the dish is preferably 10 cm.

[0047] Step 4: aspirate the fertilized eggs into the fertilized egg holding tank, arrange the fertilized eggs neatly, add 1X concentration culture medium, and use a microinjector to inject the gene knockout system into the animal pole of the fertilized eggs.

[0048] Specifically, the glass fiber injection needle was drawn using a glass fiber drawing instrument from Sutter instruments, using high borosilicate glass fiber with an inner diameter of 0.8 mm and an outer diameter of 1.2 mm, with a fiber guide wire inside. The specific parameters of the two-step needle drawing procedure are: the shape of the glass fiber tip should have a slightly larger slope to increase the strength of the needle tip, the needle mouth should not be too large, and the droplet size is preferably 0.5 to 1‰ of the size of the fertilized egg under a pressure of 20 kPa.

[0049] Use a pipette to suck the fertilized eggs before the 2-cell stage into the fertilized egg holding tank, arrange them side by side in the holding tank, and add a small amount of 1X culture medium to the holding tank to prevent the water from drying out. Use a glass fiber injection needle to inject the microinjection droplet into the animal pole of the fertilized egg. The micromanipulation steps are arranged according to the experimental requirements.

[0050] Step 5: Prepare a container, inject 1X concentration culture medium, place the injected fertilized eggs in an incubator, fix the incubator below the liquid level of the container, ventilate, and incubate with running water.

[0051] Specifically, prepare a 50-100L square glass fish tank, inject a suitable volume of 1X culture medium, place the injected fertilized eggs in an incubator, place the incubator below the liquid level in the glass tank, fix it with a suction cup, ventilate it, and make the fertilized eggs roll up and down for incubation in running water.

[0052] Step 6: Continuously culture the incubator with culture solutions of different concentrations. After the culture is completed, dilute the culture solution gradually within five hours and finally add fresh water.

[0053] Specifically, after culturing in the incubator with 1X culture medium for 24 hours, the culture medium is replaced with 0.7X concentration culture medium, and then cultured for 24 hours, and then replaced with 0.5X concentration culture medium and continued to culture until the fertilized eggs hatched out of the membrane. The culture medium was gradually diluted within five hours and finally added with fresh water.

[0054] The following is a description of a method for significantly improving the survival rate of fertilized eggs of mandarin fish after micromanipulation provided by the present invention in conjunction with specific embodiments.

[0055] Example 1

[0056] This embodiment provides a method for significantly improving the survival rate of fertilized eggs of mandarin fish after micromanipulation, comprising the following steps:

[0057] Step 1. After the injection of oxytocin to the mandarin fish parent reaches the effect time, gently squeeze the abdomen of the female and male fish, collect the eggs and sperm and put them into a clean, water-free stainless steel basin, absorb 10 ml of eggs and 0.2 ml of sperm and mix them evenly, add 10 ml of 0.6% sea salt solution, stir and suspend the sperm-egg mixture, let it stand for 30 seconds, add 100 ml of ordinary aerated water, let it stand for 30 seconds, pour out the excess water, add an appropriate amount of aerated water to submerge the fertilized eggs, inflate the container, and let it stand for use.

[0058] Step 2: Take 33.9g NaCl, 1.1g KCl, 1.0g CaCl2 and 5.95g HEPES, dissolve them in water, make up to 1 liter, prepare 5X concentration culture medium stock solution, and use it to prepare the required concentration.

[0059] Step 3: Prepare a fertilized egg holding groove with agarose, and customize Figure 1 The fertilized egg clamping groove mold shown is mixed with 50 ml of 1X culture medium and 1 g of agarose, heated to boiling in a microwave oven, shaken, and when the temperature drops to about 50 degrees Celsius, the agarose solution is introduced into a 10 cm flat dish, and then the clamping groove mold is tilted and placed in, being careful to avoid bubbles, so that the mold floats on the surface of the agarose solution, and allowed to stand until the agarose solution solidifies. The mold is carefully lifted up with pointed tweezers to obtain the fertilized egg clamping groove.

[0060] Step 4: Use a pipette to suck the fertilized eggs before the 2-cell stage into the fertilized egg holding tank, arrange them side by side in the holding tank, and add a small amount of 1X culture medium to the holding tank to prevent the water from drying out. Inject a 0.5‰ microinjection droplet of the size of the fertilized egg into the animal pole of the fertilized egg. Perform micromanipulation steps according to experimental requirements.

[0061] Step 5: prepare a 100L square glass fish tank, inject an appropriate volume of 1X culture medium, place the injected fertilized eggs into a commercially available sparid fish incubator, place the incubator below the liquid level in the glass tank, fix it with a suction cup, and ventilate it so that the fertilized eggs roll up and down and incubate in running water.

[0062] Step 6: After culturing in the incubator in 1X concentration culture medium for 24 hours, change the culture medium to 0.7X concentration culture medium, culture for 24 hours, change to 0.5X concentration culture medium and continue culturing until the fertilized eggs hatch out of the membrane, dilute the culture medium gradually within five hours, and finally add fresh water.

[0063] In Example 1, the hatching rate of the fertilized eggs of mandarin fish after micromanipulation was 85%; and the survival rate was 70%.

[0064] Comparative Example 1-2

[0065] Comparative Examples 1-2 respectively provide a method for significantly improving the survival rate of fertilized eggs of mandarin fish after micromanipulation. Compared with Example 1, the only difference is that in Comparative Example 1, 1X concentration culture medium is used for culturing in step 6 until the fertilized eggs hatch out of the membrane; in Comparative Example 2, in step 6, the incubator is cultured in 1X, 0.6X, and 0.3X concentration culture medium respectively until the fertilized eggs hatch out of the membrane; other experimental parameters and conditions are the same as those in Example 1 and are not repeated here.

[0066] In Comparative Example 1, the hatching rate of the fertilized eggs of mandarin fish after micromanipulation was 40%; and the survival rate was 15%.

[0067] In Comparative Example 2, the hatching rate of the fertilized eggs of mandarin fish after micromanipulation was 70%; and the survival rate was 61%.

[0068] From the test results, it can be found that the change of culture medium concentration has a great influence on the hatching rate and survival rate of fertilized eggs of mandarin fish after micromanipulation. The culture medium with 1X, 0.7X and 0.5X concentrations used in Example 1 is the most suitable.

[0069] Comparative Examples 3-4

[0070] Comparative Examples 3-4 respectively provide a method for significantly improving the survival rate of fertilized eggs of mandarin fish after micromanipulation. Compared with Example 1, the only difference is that in Comparative Example 3, no sea salt solution is used in step 1; Comparative Example 4 uses a sea salt solution with a concentration of 0.8% in step 1; other experimental parameters and conditions are the same as in Example 1 and are not repeated here.

[0071] In order to explore the effect of sea salt solution on fertilization rate, the fertilization rate of eggs and sperm after treatment in step 1 of Example 1 and Comparative Examples 3-4 was tested, wherein the fertilization rate in Example 1 was 89%, the fertilization rate in Comparative Example 3 was 74%, and the fertilization rate in Comparative Example 4 was 25%.

[0072] From the test results, it can be found that the concentration of sea salt solution has a great influence on the fertilization rate of eggs and sperm. When the concentration of sea salt solution is 0.6%, the fertilization rate is the highest. When it exceeds 0.6%, the fertilization rate drops sharply.

[0073] In summary, the method provided by the present invention for significantly improving the survival rate of fertilized eggs of mandarin fish after micromanipulation adopts an improved artificial insemination method and a fertilized egg culture fluid formula, which significantly improves the fertilization rate of eggs and sperm as well as the hatching rate and survival rate of fertilized eggs after microinjection.

[0074] The above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for significantly improving the survival rate of fertilized eggs of mandarin fish after micromanipulation, It is characterized in that The following steps are involved: Step 1, after the oxytocin injection reaches the effect time, the abdomen of the female and male fish is gently squeezed to collect eggs and sperm, the eggs and sperm are sucked into a container and mixed evenly, a sea salt solution is added, the concentration of the sea salt solution is 0.6%, the sperm-egg mixture is stirred and suspended, and allowed to stand, aerated water is added, and allowed to stand, excess water is poured out, aerated water is added to submerge the fertilized eggs, the container is inflated, and allowed to stand for use; Step 2: NaCl, KCl, CaCl 2 and HEPES were dissolved in water to prepare the culture medium stock solution; Step 3, using agarose and 1X concentration culture medium to make a fertilized egg holding groove; Step 4, suck the fertilized eggs into the fertilized egg holding tank, arrange the fertilized eggs neatly, add 1X concentration culture medium, and use a microinjector to inject the gene knockout system into the animal pole of the fertilized eggs; Step 5: prepare a container, inject 1X concentration culture medium, place the injected fertilized eggs in an incubator, fix the incubator below the liquid level of the container, ventilate, and incubate with running water; Step 6, continuously culturing the incubator with culture solutions of different concentrations, and after the culturing is completed, diluting the culture solution gradually within five hours, and finally adding fresh water; the continuous culturing method is: after culturing the incubator with 1X concentration culture solution for 24 hours, changing the culture solution to 0.7X concentration culture solution, continuing to culture for 24 hours, changing to 0.5X concentration culture solution and continuing to culture until the fertilized eggs hatch out of the membrane.

2. The method for significantly improving the survival rate of fertilized eggs of mandarin fish after micromanipulation according to claim 1, It is characterized in that In step 1, the volume ratio of the sucked eggs, sperm and added sea salt solution is 10:0.2:10; the standing time is 30 to 35 seconds.

3. The method for significantly improving the survival rate of fertilized eggs of mandarin fish after micromanipulation according to claim 1, It is characterized in that In step 2, NaCl, KCl, CaCl 2 The mass ratio of HEPES is (30~35):(0.9~1.3):(0.8~1.2):(5.5~6.5).

4. The method for significantly improving the survival rate of fertilized eggs of mandarin fish after micromanipulation according to claim 1, It is characterized in that In step 2, the concentration of the prepared culture medium stock solution is five times that of the standard working solution, and it is prepared to the required concentration before use.

5. The method for significantly improving the survival rate of fertilized eggs of mandarin fish after micromanipulation according to claim 1, It is characterized in that In step 3, the preparation method of the fertilized egg clamping groove is: mix 1X concentration culture medium and agarose, heat to boiling, shake well, cool, introduce into a flat dish, then tilt and place the clamping groove mold so that the mold floats on the surface of the agarose solution, let it stand, wait for the agarose solution to solidify, demold, and obtain the fertilized egg clamping groove.

6. The method for significantly improving the survival rate of fertilized eggs of mandarin fish after micromanipulation according to claim 5, It is characterized in that The mass fraction of agarose in the fertilized egg holding groove is 2%.

7. The method for significantly improving the survival rate of fertilized eggs of mandarin fish after micromanipulation according to claim 5, It is characterized in that The cooling range is 45~55℃; the thickness of the plate is 10 cm.

8. The method for significantly improving the survival rate of fertilized eggs of mandarin fish after micromanipulation according to claim 1, It is characterized in that In step 4, the droplet size for microinjection is 0.5~1‰ of the size of the fertilized egg.

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