CN111304145B - A method for improving hatching rate of Macrobrachium rosenbergii embryos in vitro - Google Patents

Abstract

The invention relates to a method for improving the in vitro culture hatchability of macrobrachium rosenbergii embryos, which comprises the following steps: preparing finished shrimp culture water; preparing a parent shrimp feed; culturing parent shrimps of the macrobrachium rosenbergii; preparing an egg washing liquid; fifthly, carrying out an in-vitro method of the embryo; sixthly, a hatching mode. The method of the invention carries out in vitro incubation on the fertilized embryos of the macrobrachium rosenbergii, reduces the defects caused by the direct oviposition incubation of the macrobrachium rosenbergii, increases the incubation rate of the in vitro incubation of the macrobrachium rosenbergii, improves the survival rate of the rupture of membranes of the macrobrachium rosenbergii and provides a good application technology for the industry.

Description

A method for improving hatching rate of Macrobrachium rosenbergii embryos in vitro

technical field

The invention belongs to the technical field of aquaculture, in particular to a method for improving the hatching rate of Macrobrachium rosenbergii embryos in vitro.

Background technique

Macrobrachium rosenbergii, also known as Malaysian prawn, freshwater long-armed prawn, is a large freshwater prawn native to Southeast Asia. It grows fast, has a wide range of food, good nutrient content in meat, and has a short breeding cycle. Since the artificial cultivation of Macrobrachium rosenbergii began in the 1960s, it has developed rapidly and is now more common in Southeast Asian countries and some other regions.

As the quality of life improves, so does the consumer demand for protein. Because of its high protein and low fat, aquatic products have gradually become the first choice of people. The price of high-quality aquatic products on the market is high and the supply capacity is limited, so the quantity and quality of aquatic products need to be improved to a certain extent. An important means of improving quality is molecular breeding. Molecular breeding of animals basically requires embryo manipulation, and directional breeding based on gene editing technology requires a large number of in vitro embryos for experimental manipulation. less.

Some existing in vitro culture technologies are not mature enough, the hatching rate of in vitro embryos is low, and the embryo development rate after in vitro operation is almost zero.

Through the search, the following journals and patent publications related to the patent application of the present invention were found:

1. Research on the embryonic development of Macrobrachium rosenbergii: Ⅰ. Morphology of the external structure of the embryo (classification number: Q954.4), including: The embryonic development process of Macrobrachium rosenbergii can be accurately divided into cleavage stage, blastocyst stage, gastrula There are seven stages: pre-nauplii stage, post-nauplii stage, pre-nauplii stage and flea larval stage. Under the condition of 28 ℃ water temperature, the whole process of embryonic development takes about 480 hours.

2. An in vitro hatching method of Australian freshwater lobster (CN105557590B), comprising: (1) after obtaining fertilized eggs, first cleaning the fertilized eggs, and then placing them in an in vitro hatching tray for artificial incubation. (2) The in vitro incubation tray is equipped with a pre-filter bucket, an ozone sterilizer, a heating tube, an aerator and an isolation net that match the water body. The fertilized eggs are placed on the isolation mesh. The fertilized eggs maintain a slight movement, and the ozone machine is regularly sterilized by time control. The fertilized eggs hatch after about 40-50 days to hatch the larvae. By adopting the in vitro hatching method of the present invention, the infection rate of fertilized eggs can be reduced to less than 10%, the hatching survival rate can reach more than 90%, the hatching survival rate of fertilized eggs can be greatly improved, and the yield of shrimp fry can be effectively improved. Has significant economic benefits.

By comparison, the patent application of the present invention is substantially different from the above-mentioned publications.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the deficiencies of the prior art, and to provide a method for improving the hatching rate of Macrobrachium rosenbergii embryos in vitro culture. The method hatches the fertilized embryos of Macrobrachium rosenbergii in vitro, so that the mother shrimp directly holds the eggs before. The disadvantages of hatching are reduced, and the hatching rate of in vitro culture of Macrobrachium rosenbergii is increased.

The technical scheme adopted by the present invention to solve its technical problems is:

A method for improving the hatching rate of Macrobrachium rosenbergii embryos in vitro, the steps are as follows:

(1) Preparation of adult shrimp aquaculture water: Since Macrobrachium rosenbergii is freshwater shrimp, ordinary adult Macrobrachium rosenbergii only needs to aerate the sterilized water for more than three days before being put into adult shrimp breeding;

(2) Preparation of broodstock feed: its components and parts by weight are as follows:

10-15 parts of seaweed powder, 10-15 parts of chlorella, 10-15 parts of shrimp meal, 10-15 parts of fish meal, 5-10 parts of probiotics, 1-2 parts of chitin, 1-2 parts of complex minerals, 1-2 parts of multivitamins, 10-15 parts of wheat flour, 10-15 parts of spinach, 10-20 parts of fermented corn gluten, mix the raw materials and get it;

(3) Breeding of the broodstock of Macrobrachium rosenbergii: put the adult shrimp in the culture tank according to the number of each m ³ /10 tails, culture with the aquaculture prepared in step (1), feed the adult shrimp with the broodstock feed prepared in step (2), wait for the female shrimp brood;

(4) Preparation of egg washing liquid: in a sterile environment, mix neutral protease, trypsin and collagenase in a mass ratio of 1:1.5:1.2, and then add water to make an aqueous solution with a mass concentration of 1%, that is, egg washing The egg wash solution should be stored in a refrigerator at 4°C;

⑸In vitro method of embryos: Wet clean absorbent cotton with egg washing liquid, place the moistened absorbent cotton on the part of the existing broodstock that needs to be shedding eggs for no more than 10 hours, stay for 1 minute, and use the hatching water to remove the broodstock. The eggs on the body are washed down, disinfected with a low-concentration potassium permanganate solution for 10 seconds, and stored at 4°C for future use. The equipment used in the whole process is disinfected and sterilized;

(6) Incubation method: take out the preserved fertilized eggs, observe the stage of the cells under a stereoscope, and take pictures to record; pour the prepared low-concentration potassium permanganate solution into a sterilized glass dish, and put the Put the fertilized eggs to be hatched into a low-concentration potassium permanganate solution in a glass dish and let stand for 10 to 15 seconds, then use a sterilized plastic straw to suck out all the eggs, put them into a sterilized container, and pour into the prepared Good hatching water, the amount of hatching water is slowly added according to the number of 5-10ml/egg, and after 24 hours of incubation at room temperature 25 ± 1 ℃, re-sterilize and change the water and remove bad eggs regularly.

And, the preparation steps of hatching water in described step (5) are as follows:

Accurately measure the filtered seawater with a salinity of 10‰, sterilize it, take it out, and cool it to room temperature; accurately weigh EDTA, PBS buffer, and DMEM, respectively, pour them into the sterilized seawater, and add 5g per 1L of sterilized seawater. EDTA, 3ml PBS buffer, 2gDMEM, 0.22μm filter membrane filtration sterilization to obtain incubation water; sealed and stored, and placed at room temperature for use.

Moreover, the sterilization is sterilized in a high-temperature and high-pressure sterilizer for 2 hours.

Also, each 1 L of PBS buffer is prepared as follows:

Weigh 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4, dissolve them in 800ml filtered seawater, adjust the pH of the solution to 7.4 with HCl, and finally add filtered seawater to 1L.

Moreover, the incubation water prepared in step ⑸ should not be used for more than one month.

And, the preparation step of described low concentration potassium permanganate solution is as follows:

Weigh the potassium permanganate reagent, then add distilled water, add 0.1 mg of potassium permanganate reagent per 1L of distilled water, mix well, and store in a sealed container, that is, the prepared low-concentration potassium permanganate solution.

The advantages and positive effects obtained by the present invention are:

1. In the method of the present invention, the fertilized embryos of Macrobrachium rosenbergii are incubated in vitro, so that the disadvantages caused by the direct egg hatching of the mother shrimp are reduced, the hatching rate of the in vitro culture of Macrobrachium rosenbergii is increased, and the membrane rupture of Macrobrachium rosenbergii is improved. The survival rate of larvae provides a good application technology for the industry.

2. In modern biotechnology, cross-breeding is very mature. To better improve the yield and quality of animals and plants, molecular breeding has become a necessary technology. Aquatic species that can be cultured with embryos in vitro can be candidates for molecular breeding of crustaceans.

3. The method of the present invention provides a technical platform for in vitro fertilization and embryo manipulation of Macrobrachium rosenbergii, and provides necessary auxiliary technology for gene editing of economical crustaceans.

4. The method of the present invention can provide preliminary necessary technologies for obtaining embryonic stem cells in different stages of crustaceans.

5. The method of the present invention can provide preliminary necessary technologies for some omics technologies such as single-cell transcriptome sequencing proteome sequencing and metabolome sequencing in crustacean developmental research.

6. The broodstock feed used by the method of the present invention can increase the quantity and quality of the brood eggs of the female shrimp to a certain extent, and improve the physique of the broodstock to a certain extent.

7. The compound formula egg washing liquid used in the method of the present invention can prevent the fertilized eggs of the broodstock from being affected by artificial external forces while being peeled off one by one.

8. The hatching water used in the method of the present invention can prevent the fertilized eggs from being disturbed by aquatic microorganisms while hatching, thereby improving the in vitro hatching rate of Macrobrachium rosenbergii

9. The method of the present invention is simple and easy to use, and does not have too many conditions.

Description of drawings

Fig. 1 is the picture continuously recorded during the in vitro culture of Macrobrachium rosenbergii in the present invention; wherein, 1. blastocyst stage:; 2-3. gastrula stage; 4. pre-nauplii stage; 5-6. post-nauplius stage Larval stage; 7-8. Preliminary larval stage; 9-11. Intramembranous larval stage; 12. Membrane-breaking larvae.

Detailed ways

The embodiments of the present invention will be described in detail below. It should be noted that the embodiments are descriptive, not restrictive, and cannot limit the protection scope of the present invention.

The raw materials used in the present invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional methods in the art unless otherwise specified.

A method for improving the hatching rate of Macrobrachium rosenbergii embryos in vitro, the steps are as follows:

(1) Preparation of adult shrimp aquaculture water: Since Macrobrachium rosenbergii is freshwater shrimp, ordinary adult Macrobrachium rosenbergii only needs to aerate the sterilized water for more than three days before being put into adult shrimp breeding;

(2) Preparation of broodstock feed: its components and parts by weight are as follows:

10-15 parts of seaweed powder, 10-15 parts of chlorella, 10-15 parts of shrimp meal, 10-15 parts of fish meal, 5-10 parts of probiotics, 1-2 parts of chitin, 1-2 parts of complex minerals, 1-2 parts of multivitamins, 10-15 parts of wheat flour, 10-15 parts of spinach, 10-20 parts of fermented corn gluten, mix the raw materials and get it;

(3) Breeding of the broodstock of Macrobrachium rosenbergii: put the adult shrimp in the culture tank according to the number of each m ³ /10 tails, culture with the aquaculture prepared in step (1), feed the adult shrimp with the broodstock feed prepared in step (2), wait for the female shrimp brood;

(4) Preparation of egg washing liquid: in a sterile environment, mix neutral protease, trypsin and collagenase in a mass ratio of 1:1.5:1.2, and then add water to make an aqueous solution with a mass concentration of 1%, that is, egg washing The egg wash solution should be stored in a refrigerator at 4°C;

⑸In vitro method of embryos: Wet clean absorbent cotton with egg washing liquid, place the moistened absorbent cotton on the part of the existing broodstock that has just held eggs (no more than 10 hours) and need to shed eggs, stay for 1 minute, and use incubation The eggs of the broodstock are washed down with water, and the low-concentration potassium permanganate solution is sterilized for 10 seconds, and stored at 4 °C for future use. The equipment used in the whole process is disinfected and sterilized; The broodstock and the desired fertilized eggs are damaged, and the fertilized eggs are completely separated without harming the broodstock. Can reduce and prevent the infection of aquatic organisms and microorganisms.

(6) Incubation method: take out the preserved fertilized eggs, observe the stage of the cells under a stereoscope, and take pictures to record; pour the prepared low-concentration potassium permanganate solution into a sterilized glass dish, and put the Put the fertilized eggs to be hatched into a low-concentration potassium permanganate solution in a glass dish and let stand for 10 to 15 seconds, then use a sterilized plastic straw to suck out all the eggs, put them into a sterilized container, and pour into the prepared Good hatching water, the amount of hatching water is slowly added according to the number of 5-10ml/egg, and after 24 hours of incubation at room temperature 25 ± 1 ℃, re-sterilize and change the water and remove bad eggs regularly.

Preferably, the preparation steps of the hatching water in the step (5) are as follows:

Accurately measure the filtered seawater with a salinity of 10‰, sterilize it, take it out, and cool it to room temperature; accurately weigh EDTA, PBS buffer, and DMEM, respectively, pour them into the sterilized seawater, and add 5g per 1L of sterilized seawater. EDTA, 3ml PBS buffer, 2gDMEM, 0.22μm filter membrane filtration sterilization to obtain incubation water; sealed and stored, and placed at room temperature for use.

Preferably, the sterilization is sterilized in a high temperature and high pressure sterilizer for 2 hours.

Preferably, each 1 L of PBS buffer is prepared as follows:

Weigh 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4, dissolve them in 800ml filtered seawater, adjust the pH of the solution to 7.4 with HCl, and finally add filtered seawater to 1L.

Preferably, the service life of the hatching water configured in step (5) is not more than one month.

Preferably, the preparation steps of the low-concentration potassium permanganate solution are as follows:

Weigh the potassium permanganate reagent, then add distilled water, add 0.1 mg of potassium permanganate reagent per 1L of distilled water, mix well, and store in a sealed container, that is, the prepared low-concentration potassium permanganate solution.

More specifically, relevant preparation and detection are as follows:

A method for improving the hatching rate of Macrobrachium rosenbergii embryos in vitro, the steps are as follows:

a. In vitro method of embryos: Wet clean absorbent cotton with egg washing liquid, place the moistened absorbent cotton on the part where the broodstock needs to be degreased, stay for 1 minute, and wash the eggs from the broodstock with hatching water, and the low Sterilize with potassium permanganate for 10 seconds and store at 4°C for later use.

b. Incubation method: take out the preserved fertilized eggs, observe the period in which the cells are under a stereoscope, and take pictures to record. Pour the prepared low-concentration potassium permanganate solution into a sterilized glass dish, put the fertilized eggs to be hatched into the low-concentration potassium permanganate solution in the glass dish and let stand for 10-15 seconds, then use the Use a sterilized plastic straw to suck out all the eggs, put them into a sterilized 100ml beaker, and pour the prepared hatching water. The amount of hatching water is slowly added in the amount of 10ml/(each egg). After 24 hours, the fertilized eggs were sucked out with a plastic straw, put into a certain amount of low-concentration potassium permanganate solution, and after standing for 10-15 seconds, sucked out. Rinse the beaker with a low-concentration potassium permanganate solution, and place the aspirated sterilized fertilized eggs into the rinsed beaker. Then pour the hatching water proportionally, and continue to cultivate. Observed once a day, and photographed under a stereo microscope, the results are shown in Figure 1.

Record the result:

a. Blastocyst stage: Day 1-2.

b. Gastrulation phase: Day 3-4.

c. Pre-nauplii stage: Days 5-8.

d. Post-nauplii stage: Days 9-10.

e. Pre-flea larval stage: Days 11-13.

f. Intramembranous flea larval stage: Day 14-19.

g. Membrane larvae: Day 20.

1. The hatching rate of the fertilized eggs of Macrobrachium rosenbergii operated in vitro in the present invention is about 75%, which is about 8% higher than that of the common fertilized eggs of Macrobrachium rosenbergii. The in vitro culture technology of the present invention can achieve a hatching rate of up to 100% in some culture environments, and does not produce some bad states of in vitro culture.

2. The days of membrane rupture of the fertilized eggs of Macrobrachium rosenbergii after the in vitro operation and culture of the patent of the present invention are similar to those of the fertilized eggs of Macrobrachium rosenbergii under normal reproduction conditions. method of cultivation.

3. The egg wash liquid used for separating the eggs before the in vitro operation of the present invention can ensure that the fertilized eggs are completely separated and the fertilized eggs are not affected by the chemical components in the egg wash liquid to the greatest extent.

4. The feeding formula of the broodstock of Macrobrachium rosenbergii operated by the present invention can increase the number of eggs of the broodstock of Macrobrachium rosenbergii to a certain extent, and enhance the physique of the broodstock of Macrobrachium rosenbergii.

5. Using the in vitro culture technology of Macrobrachium rosenbergii of the present invention can prevent the fertilized eggs of Macrobrachium rosenbergii from being infected by aquatic microorganisms during the hatching process, thereby preventing the fertilized eggs from becoming bad eggs.

6. The in vitro culture technology of the present invention is simple to operate, and does not have too high requirements on the conditions of the surrounding environment.

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, therefore , the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims (3)

1. a method for improving the hatching rate of Macrobrachium rosenbergii embryos in vitro, is characterized in that: step is as follows: (1) Preparation of adult shrimp aquaculture water: Since Macrobrachium rosenbergii is freshwater shrimp, ordinary adult Macrobrachium rosenbergii only needs to aerate the sterilized water for more than three days before being put into adult shrimp breeding; (2) Preparation of broodstock feed: its components and parts by weight are as follows: 10-15 parts of seaweed powder, 10-15 parts of chlorella, 10-15 parts of shrimp meal, 10-15 parts of fish meal, 5-10 parts of probiotics, 1-2 parts of chitin, 1-2 parts of complex minerals, 1-2 parts of multivitamins, 10-15 parts of wheat flour, 10-15 parts of spinach, 10-20 parts of fermented corn gluten, mix the raw materials and get it; (3) Breeding of the broodstock of Macrobrachium rosenbergii: put the adult shrimp in the culture tank according to the number of each m ³ /10 tails, culture with the aquaculture prepared in step (1), feed the adult shrimp with the broodstock feed prepared in step (2), wait for the female shrimp brood; (4) Preparation of egg washing liquid: in a sterile environment, mix neutral protease, trypsin and collagenase in a mass ratio of 1:1.5:1.2, and then add water to make an aqueous solution with a mass concentration of 1%, that is, egg washing The egg wash solution should be stored in a refrigerator at 4°C; ⑸In vitro method of embryos: Wet clean absorbent cotton with egg washing liquid, place the moistened absorbent cotton on the part of the existing broodstock that needs to be shedding eggs for no more than 10 hours, stay for 1 minute, and use the hatching water to remove the broodstock. The eggs on the body are washed down, disinfected with a low-concentration potassium permanganate solution for 10 seconds, and stored at 4°C for future use. The equipment used in the whole process is disinfected and sterilized; (6) Incubation method: take out the preserved fertilized eggs, observe the stage of the cells under a stereoscope, and take pictures to record; pour the prepared low-concentration potassium permanganate solution into a sterilized glass dish, and put the Put the fertilized eggs to be hatched into a low-concentration potassium permanganate solution in a glass dish and let stand for 10 to 15 seconds, then use a sterilized plastic straw to suck out all the eggs, put them into a sterilized container, and pour into the prepared Good hatching water, the amount of hatching water is slowly added according to the number of 5-10ml/egg, after 24 hours of incubation at room temperature 25 ± 1 ℃, re-sterilize and change the water and remove bad eggs regularly; The preparation steps of the hatching water in the step (5) are as follows: Accurately measure the filtered seawater with a salinity of 10‰, sterilize it, take it out, and cool it to room temperature; accurately weigh EDTA, PBS buffer, and DMEM, respectively, pour them into the sterilized seawater, and add 5g per 1L of sterilized seawater. EDTA, 3ml PBS buffer, 2g DMEM, 0.22μm filter membrane filter sterilization to obtain incubation water; sealed and stored at room temperature for later use; Prepare each 1 L of PBS buffer as follows: Weigh 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4, dissolve them in 800ml filtered seawater, adjust the pH of the solution to 7.4 with HCl, and finally add filtered seawater to 1L; The preparation steps of the low-concentration potassium permanganate solution are as follows: Weigh the potassium permanganate reagent, then add distilled water, add 0.1 mg of potassium permanganate reagent per 1L of distilled water, mix well, and store in a sealed container, that is, the prepared low-concentration potassium permanganate solution. 2. The method for improving the hatching rate of Macrobrachium rosenbergii embryos in vitro according to claim 1, wherein the sterilization is to put into a high temperature and high pressure sterilizer for sterilization for 2h. 3. The method for improving the hatching rate of Macrobrachium rosenbergii embryos in vitro according to claim 1, characterized in that: the hatching water service life configured in step (5) is not more than one month.

Images