Micro-injection device and method for hucho taimen eggs

Abstract

According to the hucho taimen roe micro-injection device and method, an injection pipe and a discharge pipe are arranged side by side and integrally manufactured, needle tips are arranged at the lower ends of the injection pipe and the discharge pipe, and a liquid storage tank is communicated with the discharge pipe through a connecting pipe. The method comprises the following steps: 1, semen taking; 2, healthy roes are taken, and a container containing the roes is placed on the ice-water mixture; 3, mixing 1000 + / -10 eggs with the seminal fluid, gently and uniformly stirring, and slowly adding water until the eggs are submerged; 4, standing for 2 minutes, washing the eggs with water at 10 DEG C, and removing redundant sperms; 5, injecting clean water again; 6, pouring 1.2% sepharose gel into a culture dish, and after the sepharose gel is coagulated, poking out a semicircular groove with the diameter of 4-5mm; seventhly, the fertilized eggs absorb water to expand, within 0-15 minutes after meeting water, the fertilized eggs are fixed in the semicircular grooves, and injection is conducted through a micro-injection device; 8, timely picking out the eggs which are damaged, severely leaked, not fertilized or have water mildew infection symptoms; and 9, incubating the fertilized eggs after injection. The microinjection device is used for microinjection of hucho taimen fertilized eggs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of aquaculture, and particularly relates to a salmon egg microinjection device and method. BACKGROUND

[0002] With the continuous maturity and perfection of gene editing technology, the microinjection technology relied on by the gene editing technology in aquatic animals has gradually become a routine operation. However, the diameters of the eggs of aquatic animals, the water absorption expansion time, the hardness, and the characteristics of the substances in the eggs are different, and in addition to a few fish such as zebrafish and cyprinid fish which have been studied for many years, injection failures caused by injection devices, time and methods are common, especially large cold-water fish, and there is no report on successful microinjection so far. The eggs of salmon and other large cold-water fish are full of particles, have great elasticity and hardness, and are filled with a large amount of viscous nutrients such as sugars, and are extremely easy to swell and burst during injection, or the liquid in the eggs seeps out to cause water mold infection in the later stage, and then cause injection failure or egg death. These problems make it extremely difficult to successfully operate the microinjection technology on the fertilized eggs. In view of such a situation, after fully understanding the characteristics of the fertilized eggs of salmon, a set of strict microinjection process is developed, and the designed microinjection device can effectively improve the success rate of microinjection. SUMMARY

[0003] The present application is to solve the problem that the fertilized eggs of salmon are full of particles, have great elasticity and hardness, and are filled with a large amount of viscous nutrients such as sugars, and are extremely easy to swell and burst during injection, or the liquid in the eggs seeps out to cause water mold infection in the later stage, and then cause injection failure or egg death, and the microinjection technology is extremely difficult to operate on the fertilized eggs, and provides a salmon egg microinjection device and method.

[0004] The technical solution adopted by the present application to solve the above problems is:

[0005] A salmon egg microinjection device, the microinjection device comprises an injection tube, an outer discharge tube, a connecting tube and a liquid storage pool, the injection tube and the outer discharge tube are arranged side by side and integrally formed, the upper end of the injection tube is an injection inlet, the lower end of the injection tube is an injection outlet, the upper end of the outer discharge tube is a closed end, the lower end of the outer discharge tube is a liquid suction port, the lower ends of the injection tube and the outer discharge tube are provided with needle tips, the length of the needle tip is 5mm, and the liquid storage pool is communicated with the outer discharge tube through the connecting tube.

[0006] Further, the cross section of the injection tube and the outer discharge tube is circular.

[0007] Further, the microinjection device further comprises a bacteria filtering and air permeable net, a side air permeable hole is arranged on the wall of the outer discharge tube, and a bacteria filtering and air permeable net is arranged at the side air permeable hole.

[0008] Further, an upper air permeable hole is arranged on the liquid storage pool, and a bacteria filtering and air permeable net is arranged at the upper air permeable hole. Further, an upper air permeable hole is arranged on the liquid storage pool, and a bacteria filtering and air permeable net is arranged at the upper air permeable hole.

[0009] A method for microinjection of Oncorhynchus keta eggs, the method being achieved by the following steps:

[0010] Step one, after taking the sperm, examine the activity under the microscope, and store in a centrifuge tube at 4°C for standby;

[0011] Step two, take healthy fish eggs, add egg washing liquid until the eggs are immersed, and after the egg particles are fully separated, place the container containing the fish eggs on an ice-water mixture for storage standby;

[0012] Step three, take 1000±10 eggs and mix with the sperm in step one, gently stir evenly, and slowly add water until the eggs are immersed;

[0013] Step four, after standing for 2 minutes, wash the eggs with 10±0.5°C water to remove excess sperm;

[0014] Step five, re-inject clean water until the fish eggs are immersed;

[0015] Step six, pour 1.2% agarose gel into a culture dish, and after coagulation, use a disposable rubber head dropper to punch a semicircular groove with a diameter of 4-5mm on the surface of the gel;

[0016] Step seven, the Oncorhynchus keta fertilized eggs in step five swell after absorbing water, within 0-15 minutes after encountering water, fix the fertilized eggs in the semicircular groove on the surface of the gel in step six, and use the Oncorhynchus keta egg microinjection device under a body microscope to perform injection; vertically pierce the injection needle into the fertilized egg, push in 8 nL / egg of injection solution at a uniform speed, and after no more liquid flows into the reservoir, gently pull out the injection device, which completes the microinjection of one fertilized egg. Inject 60-80 eggs per minute, and process 1000±10 fertilized eggs at a time;

[0017] Step eight, immediately observe the fertilized eggs after microinjection, and remove the eggs with damaged, severe leakage, unfertilized, or water mold infection symptoms in time;

[0018] Step nine, incubate the injected fertilized eggs, and perform the whole process under flowing water. The water temperature during the incubation period of the fertilized eggs should be

[0019]

[0020]

[0021]

[0022]

[0023] ​​​​Keep between 7-9 ℃, eye lens appears period hatching water temperature should be increased to 10 ± 0.5 ℃, water quality requirements surface water or groundwater, hatching needs to be filtered impurities and aeration, ensure that the oxygen content is ≥7 mg / L, total ammonia nitrogen should be ≤0.02 mg / L, hatching, observe once in the morning and evening, pick out water mould infection or not development dead eggs, until the fish eggs hatching is completed.

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] 1. The injection device of the present application is used for injecting the fertilized eggs of Amur pike, and at the same time, the excess nutrients in the eggs are discharged through the discharge pipe and safely removed, reducing the pressure in the eggs, which not only avoids the explosion of the eggs, but also effectively avoids the pollution and infection risk caused by the residual substances in the water or on the surface of the eggs, reducing the risk of egg death.

[0026] 2. Since the Amur pike eggs are large in size, with a diameter of 5-6 mm, the egg membrane is thin, the content is rich and viscous, and the fertilized eggs swell rapidly after absorbing water, and the hardness of the swollen eggs gradually increases, and after reaching a certain hardness, they cannot be injected. At present, there is no successful case of using conventional microinjection method to operate Amur pike eggs, and after a large number of experiments and data accumulation, the present inventors use the injection device according to the method, and the hatching success rate after injection can reach 50-60%. The present application can accurately grasp the best injection period of Amur pike fertilized eggs, quantify the injection process, and constant hatching conditions, providing a solid technical guarantee for completing the microinjection of Amur pike fertilized eggs and successful hatching. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a structural schematic diagram of the microinjection device for Amur pike eggs;

[0028] Figure 2 is Figure 1 the B-B sectional view of DETAILED DESCRIPTION

[0029] Specific implementation one: combining Figure 1 and Figure 2This embodiment describes a device comprising an injection tube 1, an external discharge tube 2, a connecting pipe 3, a liquid reservoir 4, and a bacterial filter air permeable mesh 6. The injection tube 1 and external discharge tube 2 are arranged side by side and are integrally formed. The upper end of the injection tube 1 is an injection inlet 11, and the lower end of the injection tube 1 is an injection outlet 12. The upper end of the external discharge tube 2 is closed, and the lower end of the external discharge tube 2 is a liquid suction port 21. The lower ends of the injection tube 1 and external discharge tube 2 are provided with a needle tip 5. The length w of the needle tip 5 is 5 mm, which prevents the injection liquid from directly flowing back into the external discharge tube 2. The liquid reservoir 4 is connected to the external discharge tube 2 via the connecting pipe 3. The injection tube 1 is used to inject liquid, the external discharge tube 2 is responsible for collecting overflow liquid, and the liquid reservoir 4 is used to collect discharged liquid. The cross-sections of the injection tube 1 and external discharge tube 2 are circular. The wall of the external discharge tube 2 is provided with side air vents 22, and the bacterial filter air permeable mesh 6 is installed in place of the side air vents 22. The liquid reservoir 4 is provided with an upper vent hole 41, and a bacteria-filtering air-permeable net 6 is provided at the upper vent hole 41. The bacteria-filtering air-permeable net 6 is used to ensure that the internal and external air pressures are balanced during the injection process, and that excess nutrients in the egg flow into the liquid reservoir without hindrance. At the same time, it has the effect of isolating external bacteria from entering the egg and causing infection of the fertilized egg.

[0030] Specific implementation method 2: Combination Figure 1 This embodiment is described, and is implemented by the following steps:

[0031] Step 1: After collecting semen, examine the activity under a microscope and temporarily store it in a centrifuge tube at 4°C for later use;

[0032] Step 2: Take healthy fish eggs, add egg washing solution until the fertilized eggs are immersed, and after the eggs are fully separated, place the fish eggs in the container.

[0033] The container is placed on an ice-water mixture for storage. The purpose of placing the container on an ice-water mixture is to preserve the container at a low temperature.

[0034] Step 3: Take 1000±10 eggs and mix them with the semen from step 1, stir gently, and slowly add water until the eggs are covered;

[0035] Step 4: After standing for 2 minutes, wash the eggs with 10±0.5℃ water to remove excess sperm;

[0036] Step 5: Refill with clean water until the fish eggs are submerged;

[0037] Step 6: Pour 1.2% agarose gel into the culture dish. After solidification, use a disposable rubber-tipped dropper to

[0038] The surface is neatly punched with semicircular grooves with a diameter of about 4-5 mm to fix the eggs to be injected;

[0039] Step 7: The Taimen salmon fertilized eggs in step 5 absorb water and swell. Within 0-15 minutes after contacting water, fix the fertilized eggs in the

[0040] The gel surface semicircular groove in step six is injected by using the Amur salmon egg microinjection device under the stereomicroscope; the injection needle tip 5 is vertically inserted into the fertilized egg, 8 nL / egg of injection solution is pushed in at a uniform speed, and the injection device is gently pulled out after no more liquid flows into the reservoir 4, which completes the microinjection of one fertilized egg. 60-80 eggs are injected per minute, and it is most suitable to process 1000±10 fertilized eggs at a time.

[0041] Step eight, the fertilized eggs are observed immediately after microinjection, and the eggs with damage, serious liquid leakage, unfertilization or water mold infection are picked out in time.

[0042] Step nine, the injected fertilized eggs are hatched, and the water temperature during the hatching period should be kept at 7-9°C, and the water temperature during the ocular lens appearance period should be increased to 10±0.5°C. The water quality requirements are that surface water or underground water can be used, and underground water is better, which can keep the temperature constant and the water quality stable. After filtration and aeration, the oxygen content should be ≥7 mg / L, and the total ammonia nitrogen should be ≤0.02 mg / L. The dead eggs with water mold infection or no development are picked out in the morning and evening, until the fish eggs are hatched, which marks the successful completion of the microinjection process.

[0043] In the experiment, it is found that the Amur salmon fertilized egg absorbs water and swells, and within 0-3 minutes after water is encountered, it is soft, elastic, smooth and has no stickiness; within 4-15 minutes, the fertilized egg gradually becomes hard and has greater elasticity, and 1-2 μL of point-shaped liquid is overflowed after injection; within 16-22 minutes, the fertilized egg becomes further hardened, and it is difficult to inject, and 4-5 μL of liquid is rapidly overflowed after injection; within 23-30 minutes, the fertilized egg is hard and full, and it is impossible to inject. Therefore, it is most suitable to microinject the Amur salmon fertilized egg within 0-15 minutes after water is encountered in step seven.

[0044]

[0045]

[0046] ​​​

Claims

1. A device for microinjection of Taimen salmon eggs, characterized by: The microinjection device comprises an injection tube (1), an effluent tube (2), a connecting pipe (3) and a liquid reservoir (4), wherein the injection tube (1) and the effluent tube (2) are arranged side by side and are integrally manufactured. The upper end of the injection tube (1) is an injection inlet (11), the lower end of the injection tube (1) is an injection outlet (12), the upper end of the external discharge tube (2) is a closed end, the lower end of the external discharge tube (2) is a liquid suction port (21), the lower ends of the injection tube (1) and the external discharge tube (2) are provided with a needle tip (5), the length (w) of the needle tip (5) is 5 mm, and the liquid storage tank (4) is connected to the external discharge tube (2) through the connecting pipe (3).

2. The device for microinjection of Taimen salmon eggs according to claim 1, characterized in that: The cross sections of the injection tube (1) and the external discharge tube (2) are circular.

3. A taimen salmon egg microinjection device according to claim 1 or 2, characterized in that: The microinjection device further comprises a bacteria-filtering air-permeable net (6), a side air-permeable hole (22) is provided on the wall of the external discharge tube (2), and a bacteria-filtering air-permeable net (6) is provided at the side air-permeable hole (22).

4. The device for microinjection of Taimen salmon eggs according to claim 3, characterized in that: The liquid storage tank (4) is provided with an upper air vent (41), and a bacteria filtering air permeable net (6) is provided at the upper air vent (41).

5. A method for microinjecting taimen eggs using the taimen egg microinjection device according to claim 1, 2 or 4, characterized in that: The method is achieved by the following steps: Step 1: After collecting semen, examine the activity under a microscope and temporarily store it in a centrifuge tube at 4°C for later use; Step 2: Take healthy fish eggs, add egg washing solution until the fertilized eggs are immersed, and after the eggs are fully separated, place the fish eggs in the container. Place the container on the ice-water mixture and store it for later use; Step 3: Take 1000±10 eggs and mix them with the semen from step 1, stir gently, and slowly add water until the eggs are covered; Step 4: After standing for 2 minutes, wash the eggs with 10±0.5℃ water to remove excess sperm; Step 5: Refill with clean water until the fish eggs are submerged; Step 6: Pour 1.2% agarose gel into the culture dish. After solidification, use a disposable rubber-tipped dropper to drop the gel on the surface. Neatly poke out semicircular grooves with a diameter of 4-5mm; Step 7: The Taimen salmon fertilized eggs in step 5 absorb water and swell. Within 0-15 minutes after contacting water, fix the fertilized eggs in step 5. In the semicircular groove on the surface of the gel in step 6, the injection is performed using a salmon egg microinjection device under a stereo microscope; the injection needle tip (5) is vertically inserted into the fertilized egg, and 8 nL / egg injection solution is pushed in at a constant speed. When no more liquid flows into the reservoir (4), the injection device is gently pulled out, and the microinjection of one fertilized egg is completed. 60-80 eggs are injected per minute, and 1000±10 fertilized eggs are processed at a time; Step 8: Immediately observe the fertilized eggs after microinjection and identify those that are damaged, leaking fluid, unfertilized, or have symptoms of Saprolegnia infection. Pick out the eggs in time; Step 9: Incubate the injected fertilized eggs in a continuous water flow state. The water temperature during the incubation period should be kept Maintained between 7℃ and 9℃, the hatching water temperature should be raised to 10±0.5℃ during the lens appearance period. Water quality requirements: surface water or groundwater. Hatching must be done after filtering impurities and aeration to ensure that the oxygen content is ≥7mg / L and the total ammonia nitrogen should be ≤0.02mg / L. Observe once in the morning and evening every day, pick out dead eggs infected with water mold or undeveloped eggs, until the eggs are hatched.

6. The device for microinjection of Taimen salmon eggs according to claim 5, characterized in that: In the seventh step, the fertilized Taimen salmon eggs absorb water and swell. Within 7 minutes after contacting water, 75 eggs are injected per minute using a microinjection device.

7. The device for microinjection of Taimen salmon eggs according to claim 5, characterized in that: In the seventh step, the fertilized Taimen salmon eggs absorb water and swell. Within 8-10 minutes after coming into contact with water, 70 eggs are injected per minute using a microinjection device.

8. The device for microinjection of Taimen salmon eggs according to claim 5, characterized in that: In step seven, the fertilized Taimen salmon eggs absorb water and swell. Within 11-14 minutes after coming into contact with water, 60 eggs are injected per minute using a microinjection device.

Images