Ovum removing liquid, egg removing device, egg removing method and shrimp egg in-vitro culture method

Abstract

The invention discloses an egg-carrying shrimp egg-removing liquid, an egg-removing device, an egg-removing method and a shrimp egg in-vitro culture method, and belongs to the technical field of aquaculture. The egg-carrying lobster egg-removing liquid is obtained by dissolving protease and sodium chloride in water and adding acid to adjust the pH value; the mass ratio of the protease to the sodium chloride to the water is (0.006-0.010): (0.006-0.009): 1. According to the invention, through the combined work of the egg-carrying shrimp egg-removing liquid, the egg-removing device and the fertilized egg in-vitro culture, the egg-removing and culture conditions are optimized. While the emergence rate of the fertilized eggs is guaranteed, intensive treatment of the shrimp eggs is carried out, and a new thought is provided for industrialization and industrialized breeding of the procambarus clarkii.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, and in particular to an egg-removing liquid for egg-bearing shrimps, an egg-removing device, an egg-removing method and an in vitro culture method for shrimp eggs. Background Art

[0002] Procambarus clarkii, also known as crayfish, is native to North America and was introduced to my country in the first half of the last century. The shrimp has the characteristics of delicious meat, fast growth rate and strong environmental adaptability, which makes it deeply loved by fishermen and consumers. Since the 21st century, China's Procambarus clarkii breeding technology has become increasingly mature and perfect, breeding methods have become more diversified, and breeding output has also increased. According to the China Fisheries Statistical Yearbook: my country's Procambarus clarkii production reached 1.6387 million tons in 2018 and 2.0896 million tons in 2019, exceeding 2 million tons for the first time. At present, Procambarus clarkii has become the sixth largest freshwater economic aquaculture species in my country.

[0003] However, under the production model of reverse selection and catching the big and keeping the small, the farmed offspring are frequently plagued by diseases and have uneven specifications, which seriously restricts the sustainable and healthy development of the Procambarus clarkii industry. It is urgent to improve the germplasm performance through breeding of good varieties and form large-scale production of good varieties.

[0004] Although the in vitro culture and egg removal methods of Procambarus clarkii are theoretically feasible, they still face many challenges in practical applications, such as low hatching rate, susceptibility to infection, and high environmental requirements. These problems need to be solved through further technical improvements and optimization to improve the production efficiency and quality of Procambarus clarkii seedlings.

[0005] Therefore, it is urgent to study how to establish a rapid and non-destructive method for removing eggs from egg-bearing female shrimps in order to increase the production of Procambarus clarkii on a large scale. At the same time, it is necessary to explore how to improve the survival rate of shrimp eggs. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides an egg-removing liquid for egg-bearing shrimp, an egg-removing device, an egg-removing method and an in vitro culture method for shrimp eggs. The present invention optimizes the egg-removing and culture conditions by combining the egg-removing liquid for egg-bearing shrimp, the egg-removing device and the in vitro culture of fertilized eggs. While ensuring the emergence rate of fertilized eggs, the shrimp eggs are intensively processed, which provides a new idea for the industrialization and factory-based seedling cultivation of Procambarus clarkii.

[0007] The technical solution of the present invention is as follows:

[0008] The first aspect of the present invention provides an egg-removing liquid for lobsters with eggs, wherein the egg-removing liquid for lobsters with eggs is obtained by dissolving protease and sodium chloride in water, and adding acid to adjust the pH;

[0009] The mass ratio of the protease, sodium chloride and water is 0.006-0.010:0.006-0.009:1.

[0010] Preferably, the enzyme includes pepsin; it may also include trypsin and alkaline protease, and when the three proteases are included, the mass ratio of trypsin: pepsin: alkaline protease is 3:1:1,

[0011] And / or, the acid includes at least one of hydrochloric acid, acetic acid, and citric acid;

[0012] And / or, the pH adjustment refers to adjusting the pH to 7;

[0013] And / or, the temperature of the water is 22-26°C.

[0014] A second aspect of the present invention provides an egg removal method, which comprises the following steps:

[0015] S1: prepare the shrimps with eggs, pre-process them and set them aside;

[0016] S2: After the egg removal device is treated, an aeration stone connected to an aeration pump is added;

[0017] S3: adding water, sodium chloride, and protease to the egg removal device in sequence, stirring to dissolve, and adding acid to adjust the pH to obtain the egg removal solution according to any one of claims 1 to 2;

[0018] S4: Turn on the oxygen pump, add oxygen for 10 minutes, put the shrimp with eggs in step S1 into the de-oviposition solution, keep the temperature constant, continue to add oxygen, and de-oviposition under continuous light for 100-150 minutes;

[0019] S5: Collect female shrimp and shrimp eggs separately.

[0020] Preferably, in step S1, the size of the egg-bearing shrimp is 50-70 g per tail.

[0021] Preferably, in step S2, the despawning device comprises a shrimp placing device (1) and an egg collector; the shrimp placing device (1) is arranged above the egg collector.

[0022] Preferably, the upper end area of ​​the shrimp placing device (1) is larger than the bottom area; the upper end of the shrimp placing device (1) is open, and the bottom is closed and provided with a through hole (6);

[0023] Preferably, the through hole (6) allows shrimp eggs to pass through but cannot pass through the shrimps carrying eggs and the separated female shrimps.

[0024] Preferably, the egg collector is composed of a shrimp egg collection chamber (2) and a bottom plate (3); the upper end of the shrimp egg collection chamber (2) is open, and the upper end area is larger than the bottom area of ​​the shrimp placement device (1) and smaller than the upper end area of ​​the shrimp placement device (1), thereby ensuring that the shrimp placement device (1) is fixedly placed above the egg collection chamber (2) and does not contact the bottom of the egg collection chamber (2).

[0025] Preferably, in step S1, the mass volume ratio of the egg-bearing shrimp to the egg-removing liquid is 3kg:4-5L;

[0026] The third aspect of the present invention provides a method for culturing lobster in vitro, comprising the following steps:

[0027] (1) obtaining shrimp eggs according to the despawning method of the second aspect, and grading and treating the eggs for later use;

[0028] (2) Prepare the shaker, culture medium and culture dishes;

[0029] (3) Add the shrimp eggs to the culture medium, seal it and place it in a shaker, add the culture medium every 3-4 days, and keep the amount of the culture medium at the initial state; after 12-15 days of culture, the shrimp eggs will become shrimp fry.

[0030] Preferably, in step (2), the preparation of the shaking table includes sterilizing the shaking table, setting the temperature of the shaking table to 25-28°C and the speed to 100-150 r / min;

[0031] In step S3, the culture solution includes sodium chloride solution, nutrient solution, and double antibody solution, and the pH value is 6-7;

[0032] The concentration of the sodium chloride solution is 25-45 mmol / L, the nutrient solution is DMEM nutrient solution; the dual antibody solution is HyClone penicillin-streptomycin 100 times solution, the common working concentration of penicillin is 100 U / mL; the common working concentration of streptomycin is: 100 μg / mL (SV30010 Nanjing Yuanqidun Biotechnology Co., Ltd.);

[0033] The volume ratio of the sodium chloride solution, the nutrient solution and the double antibody solution is 2:1:1.

[0034] The beneficial technical effects of the present invention are:

[0035] The present invention can specifically act on the connecting substances between the fertilized egg and the swimming feet of the brooding shrimp by using pepsin under a specific pH value environment (adjusted by hydrogen chloride, acetic acid or citric acid), and reduce the adhesion between the fertilized egg and the swimming feet by hydrolyzing the specific chemical bonds in these connecting substances, thereby achieving smooth shedding of the fertilized egg without damaging the structure and function of the fertilized egg. The ionic environment formed after the sodium chloride is dissolved forms a balance with the osmotic pressure inside the fertilized egg, thereby protecting the cell structure integrity of the fertilized egg.

[0036] Furthermore, the present invention provides a pure, impurity-free chemical environment through deionized water, hydrogen chloride, etc., which avoids the potential harm of heavy metal ions, microorganisms, etc. that may exist in the water to the despawning process and shrimp eggs. At the same time, sodium chloride solution, nutrient solution (DMEM) and double-antibody solution are added in a specific proportion in the in vitro culture of the culture medium to provide necessary nutrients for the development of shrimp eggs, such as amino acids, vitamins, carbohydrates, etc., to meet the energy and material requirements of the embryonic development process; the double-antibody solution can inhibit the growth of bacteria and fungi, reduce the contamination of shrimp eggs by microorganisms, and reduce the risk of death due to infection. The synergistic effect of each component optimizes the entire process of despawning and in vitro culture.

[0037] The invention has clear technical points and operating specifications for each link, from the selection and disinfection of egg-bearing shrimps, to the preparation of egg-removing solution, the egg-removing operation, and the separation, classification, and cultivation of shrimp eggs, thereby achieving standardization and high efficiency of the operating process. While ensuring a high egg-removing rate and a high survival rate of shrimp eggs, the germination rate of more than 78% can be achieved.

[0038] The present invention skillfully combines enzymatic hydrolysis technology, osmotic pressure regulation technology, environmental control technology, etc. to apply to the de-egg and in vitro culture of egg-bearing shrimps, which not only solves the problem of selecting male and female shrimps and handling fertilized eggs in the culture of in vitro egg-bearing shrimps such as Procambarus clarkii, but also provides a fast, safe and effective de-egg method and matching in vitro culture technology. This technology integration method provides a comprehensive and systematic solution for the intensive culture, strain breeding, egg collection and processing of Procambarus clarkii. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a schematic diagram of the egg removal device of the present invention.

[0040] Figure 2 It is a schematic diagram of a shrimp placement container 1 in the despawning device of the present invention.

[0041] In the figure: A, a top view of the shrimp placer; B, a front view of the shrimp placer.

[0042] Figure 3 It is a schematic diagram of the shrimp egg collector 2 in the despawning device of the present invention.

[0043] In the figure: C, a top view of the shrimp collector; D, a front view of the shrimp collector.

[0044] In the above figure: 1. shrimp placement device; 2. shrimp egg collection chamber; 3. bottom plate; 4. despawning solution; 5. shrimp with eggs; 6. fine holes; 7. shrimp eggs. DETAILED DESCRIPTION

[0045] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0046] It is understandable that the present invention is not limited to the egg removal device, and any device that can achieve the purpose of the present invention is within the protection scope of the present invention. Preferably, the schematic diagram of the egg removal device used in the present invention is as shown in Figure 1-3 The despawning device comprises a shrimp placing device 1 and an egg collector; the shrimp placing device 1 is arranged above the egg collector.

[0047] In some embodiments, the upper end area of ​​the shrimp placing device 1 is larger than the bottom area; the upper end of the shrimp placing device 1 is open, and the bottom is closed and provided with a through hole 6.

[0048] In some embodiments, the through holes 6 allow shrimp eggs to pass through but cannot allow shrimps carrying eggs and separated female shrimps to pass through.

[0049] In some embodiments, the egg collector is composed of a shrimp egg collecting chamber 2 and a bottom plate 3; the upper end of the shrimp egg collecting chamber 2 is open, and the upper end area is larger than the bottom area of ​​the shrimp placing device 1 and smaller than the upper end area of ​​the shrimp placing device 1, thereby ensuring that the shrimp placing device 1 is fixedly placed above the egg collecting chamber 2 and will not contact the bottom of the egg collecting chamber 2.

[0050] It can be understood that in the egg removal method of the present invention, the egg removal rate refers to the ratio of the fertilized eggs that fall off after the egg removal treatment to all the fertilized eggs on the egg-bearing shrimp.

[0051] Ovulation rate (%) = shed fertilized eggs / all fertilized eggs × 100%.

[0052] The survival rate refers to the proportion of fertilized eggs that can develop normally and germinate among the fertilized eggs shed after the egg shedding process.

[0053] Survival rate (%) = fertilized eggs that can germinate / fertilized eggs that fall off × 100%.

[0054] In in vitro culture, the germination rate refers to the ratio of the number of germinations that finally emerge after in vitro culture to the number of fertilized eggs that can germinate normally.

[0055] Germination rate (%) = number of germination / fertilized eggs that can germinate × 100%.

[0056] It is understandable that the fertilized eggs that can germinate refer to the fertilized eggs that survive the egg-laying process and can hatch and germinate normally.

[0057] It is understandable that in the present invention, when preparing brooding shrimps, the selected brooding lobsters need to be soaked in a 4 mg / L potassium permanganate solution for about half an hour to achieve the effect of preliminary disinfection. If the soaking time is too long, the shrimps or eggs will die.

[0058] It is understood that the enzymes used in the present invention include pepsin; they may also include trypsin and alkaline protease, and when the three proteases are included, the mass ratio of trypsin: pepsin: alkaline protease is 3:1:1. In the present invention, it is preferred to use pepsin alone, and its egg removal rate, survival rate and emergence rate are all higher than those of the three mixed enzymes.

[0059] It is understandable that in S5, after the fertilized eggs have fallen off, the de-egged female shrimp is fished out using a net. For the unseparated egg-bearing shrimp, the shrimp eggs can be safely and carefully separated. For example, the remaining shrimp eggs in the abdomen of the parent shrimp can be carefully and slowly peeled off by using sterile and disinfected tweezers. During use, the temperature of the tweezers should be kept below 22°C.

[0060] It is understood that the culture dishes used must be sterilized by ultraviolet light before use, maintained at room temperature, normal humidity, and kept in a shaking state at all times.

[0061] In the present invention, when the in vitro culture duration is about 10 days, shrimp eggs with a higher initial development degree will emerge in large quantities, and eggs with a lower initial development degree will emerge later, and their emergence rate will also be slightly lower. However, when it is generally cultivated to about 15 days, the period of large-scale emergence of shrimp eggs of different development degrees has basically been completed. During the period of large-scale emergence, the mortality rate of shrimp eggs will also increase, which may be related to the difficulty of embryo emergence. After the period of large-scale emergence has passed, the shrimp eggs that cannot emerge become dead eggs, and then the emergence rate will hardly increase again.

[0062] The following are specific embodiments.

[0063] Example 1

[0064] The method for removing eggs from lobsters comprises the following steps:

[0065] S1: Prepare the shrimp with eggs and pre-treat them

[0066] Select 3 kg of female lobsters with normal body shape, no disease or harm, healthy limbs, abundant vitality, and mature fertilized eggs in the abdomen. The size of female lobsters is about 70 grams each. Before the spawning operation, place them under the same water temperature and light conditions as the spawning conditions for future use.

[0067] Soak the selected lobsters with eggs in a 4mg / L potassium permanganate solution for about half an hour to achieve preliminary disinfection and avoid the death of shrimp or eggs.

[0068] S2: After the egg removal device is processed, add an oxygenating stone connected to an oxygenating pump.

[0069] Before the egg removal operation, Figure 1 An oxygenating stone connected to an oxygenating pump is added to the egg-removing device shown, and the egg-removing device is sterilized and disinfected using ultraviolet rays, and then washed several times with deionized water and dried for standby use.

[0070] S3: Pour 4 kg of deionized water at 24°C into the egg removal device, add 36 g of analytical grade sodium chloride, stir thoroughly, add 40 g of pepsin (3000 U / mg) after it dissolves, stir thoroughly until it is completely dissolved, add hydrochloric acid to adjust the pH value of the egg removal solution to 7, and obtain the egg removal solution.

[0071] S4: Turn on the oxygen pump, and after ten minutes of oxygenation, add 3 kg of prepared shrimp with eggs. During the whole process, keep the water temperature at about 24℃, and continue to illuminate, oxygenate, and control the temperature. Gently stir the lobsters in the despawning tank every ten minutes or so. The despawning operation lasts for 120 minutes, and the despawning rate of fertilized eggs is 91.7%.

[0072] S5: After the fertilized eggs have fallen off, the female shrimps are fished out with a net and transferred to a net cage for recovery. This process needs to be quick to reduce the casualties of the female shrimps. The fertilized eggs that have fallen off in the shrimp egg collector of the shrimp egg separation device are collected with a 100-mesh filter. After the dead shrimp eggs are eliminated, the de-egg solution on the surface of the surviving fertilized eggs is cleaned with physiological saline. At this point, the survival rate of all shrimp eggs is maintained at 97%.

[0073] An in vitro culture method for lobster eggs comprises the following steps:

[0074] (1) grading the shrimp eggs separated above, generally selecting brown-black or black-white shrimp eggs according to their color and transparency, and selecting shrimp eggs with a certain degree of transparency, which indicates that they are more mature and have a higher survival rate in in vitro culture;

[0075] (2) Prepare the shaker, culture medium and culture dishes

[0076] Before using the shaker, turn on the ultraviolet light in the shaker one hour in advance to fully disinfect the shaker, then adjust the shaker temperature to 26°C and the speed to 120r / min;

[0077] To prepare the culture medium, first weigh 5.844 g of NaCl and add it to 1000 ml of deionized water, shake it evenly to obtain a sodium chloride solution, then select six sterile culture dishes, add 2 mL of NaCl solution and 1 mL of double antibody respectively, then add 1 mL of DMEM, and adjust the pH to 7.0 to obtain the culture medium.

[0078] (3) Select shrimp eggs in the black and white periods and add them to the culture medium, and try to keep the number of shrimp eggs in each culture medium small and moderate. Finally, seal the culture dish with a sealing film and place it in a shaker that has been treated in advance, culture at a constant temperature, observe the culture status every three days, and add culture medium so that the amount of culture medium is always maintained at the initial level. A large number of shrimp eggs can be observed to emerge in about 12 days, and the emergence rate can reach 82.2%.

[0079] Example 2

[0080] The method for removing eggs from lobsters comprises the following steps:

[0081] S1: Prepare the shrimp with eggs and pre-treat them

[0082] Select 3 kg of female lobsters with normal body shape, no disease or harm, healthy limbs, abundant vitality, and mature fertilized eggs in the abdomen. The size of female lobsters is about 70 grams each. Before the spawning operation, place them under the same water temperature and light conditions as the spawning conditions for future use.

[0083] Soak the selected lobsters with eggs in a 4mg / L potassium permanganate solution for about half an hour to achieve preliminary disinfection and avoid the death of shrimp or eggs.

[0084] S2: After the egg removal device is processed, add an oxygenating stone connected to an oxygenating pump.

[0085] Before the egg removal operation, Figure 1 An oxygenating stone connected to an oxygenating pump is added to the egg-removing device shown, and the egg-removing device is sterilized and disinfected using ultraviolet rays, and then washed several times with deionized water and dried for standby use.

[0086] S3: Pour 4 kg of deionized water at 24°C into the egg removal device, add 24 g of analytical grade sodium chloride, stir thoroughly, add 24 g of pepsin (3000 U / mg) after it dissolves, stir thoroughly until it is completely dissolved, add hydrochloric acid to adjust the pH value of the egg removal solution to 7, and obtain the egg removal solution.

[0087] S4: Turn on the oxygen pump. After ten minutes of oxygenation, add 3 kg of prepared shrimp with eggs. Keep the water temperature at about 22°C during the whole process, and continue to illuminate, oxygenate and control the temperature. Gently stir the lobsters in the despawning tank every ten minutes. The despawning operation lasts for 100 minutes. At this time, the despawning rate of fertilized eggs is 87.7%.

[0088] S5: After the fertilized eggs have fallen off, the female shrimps are fished out with a net and transferred to a net cage for recovery. This process needs to be quick to reduce the casualties of the female shrimps. The fertilized eggs that have fallen off in the shrimp egg collector of the shrimp egg separation device are collected with a 100-mesh filter. After the dead shrimp eggs are eliminated, the de-egg solution on the surface of the surviving fertilized eggs is cleaned with physiological saline. At this point, the survival rate of all shrimp eggs is maintained at more than 97%.

[0089] An in vitro culture method for lobster eggs comprises the following steps:

[0090] (1) grading the shrimp eggs separated above, generally selecting brown-black or black-white shrimp eggs according to their color and transparency, and selecting shrimp eggs with a certain degree of transparency, which indicates that they are more mature and have a higher survival rate in in vitro culture;

[0091] (2) Prepare the shaker, culture medium and culture dishes

[0092] Before using the shaker, turn on the ultraviolet light in the shaker one hour in advance to fully disinfect the shaker, then adjust the shaker temperature to 25°C and the speed to 100r / min;

[0093] To prepare the culture medium, first weigh 5.844 g of NaCl and add it to 1000 ml of deionized water, shake it evenly to obtain a sodium chloride solution, then select six sterile culture dishes, add 2 mL of NaCl solution and 1 mL of double antibody respectively, then add 1 mL of DMEM, and adjust the pH to 7.0 to obtain the culture medium.

[0094] Select shrimp eggs from the black and white periods and add them to the culture medium, and try to keep the number of shrimp eggs in each culture medium as small as possible and in a moderate amount. Finally, seal the culture dish with a sealing film and place it in a pre-treated shaker for constant temperature culture. Observe the culture status every three days, and add culture medium to keep the amount of culture medium at the initial level. A large number of shrimp eggs can be observed to emerge in about 12 days, and the emergence rate can reach 80.7%.

[0095] Example 3

[0096] The method for removing eggs from lobsters comprises the following steps:

[0097] S1: Prepare the shrimp with eggs and pre-treat them

[0098] Select 3 kg of female lobsters with normal body shape, no disease or harm, healthy limbs, abundant vitality, and mature fertilized eggs in the abdomen. The size of female lobsters is about 60 grams each. Before the spawning operation, place them under the same water temperature and light conditions as the spawning conditions for future use.

[0099] Soak the selected lobsters with eggs in a 4mg / L potassium permanganate solution for about half an hour to achieve preliminary disinfection and avoid the death of shrimp or eggs.

[0100] S2: After the egg removal device is processed, add an oxygenating stone connected to an oxygenating pump.

[0101] Before the egg removal operation, Figure 1 An oxygenating stone connected to an oxygenating pump is added to the egg-removing device shown, and the egg-removing device is sterilized and disinfected using ultraviolet rays, and then washed several times with deionized water and dried for standby use.

[0102] S3: Pour 4 kg of deionized water at 26°C into the egg removal device, add 30 g of analytical grade sodium chloride, stir thoroughly, add 30 g of pepsin (3000 U / mg) after it dissolves, stir thoroughly until it is completely dissolved, add hydrochloric acid to adjust the pH value of the egg removal solution to 7, and obtain the egg removal solution.

[0103] S4: Turn on the oxygen pump, and after ten minutes of oxygenation, add 3 kg of prepared shrimp with eggs. During the whole process, keep the water temperature at about 26℃, and continue to illuminate, oxygenate, and control the temperature. Gently stir the lobsters in the despawning tank every ten minutes or so. The despawning operation lasts for 150 minutes, and the despawning rate of fertilized eggs is 87.8%.

[0104] S5: After the fertilized eggs have fallen off, the female shrimps are fished out with a net and transferred to a net cage for recovery. This process needs to be quick to reduce the casualties of the female shrimps. The fertilized eggs that have fallen off in the shrimp egg collector of the shrimp egg separation device are collected with a 100-mesh filter. After the dead shrimp eggs are eliminated, the de-egg solution on the surface of the surviving fertilized eggs is cleaned with physiological saline. At this point, the survival rate of all shrimp eggs is maintained at more than 90%.

[0105] An in vitro culture method for lobster eggs comprises the following steps:

[0106] (1) grading the shrimp eggs separated above, generally selecting brown-black or black-white shrimp eggs according to their color and transparency, and selecting shrimp eggs with a certain degree of transparency, which indicates that they are more mature and have a higher survival rate in in vitro culture;

[0107] (2) Prepare the shaker, culture medium and culture dishes

[0108] Before using the shaker, turn on the ultraviolet light in the shaker one hour in advance to fully disinfect the shaker, then adjust the shaker temperature to 28°C and the speed to 150r / min;

[0109] To prepare the culture medium, first weigh 5.844 g of NaCl and add it to 1000 ml of deionized water, shake it evenly to obtain a sodium chloride solution, then select six sterile culture dishes, add 2 mL of NaCl solution and 1 mL of double antibody respectively, then add 1 mL of DMEM, and adjust the pH to 7.0 to obtain the culture medium.

[0110] (3) Select shrimp eggs in the black and white periods and add them to the culture medium, and try to keep the number of shrimp eggs in each culture medium small and moderate. Finally, seal the culture dish with a sealing film and place it in a pre-treated shaker for constant temperature culture. Observe the culture status every three days, and add culture medium so that the amount of culture medium is always maintained at the initial level. A large number of shrimp eggs can be observed to emerge in about 12 days, and the emergence rate can reach 78.6%.

[0111] Example 4

[0112] A lobster despawning method is basically the same as Example 1, except that the enzyme used in the despawning solution is pepsin. In this example, pepsin is replaced with a mixture of trypsin: pepsin: alkaline protease in a mass ratio of 3:1:1, totaling 40 grams, and the amount of sodium chloride added remains unchanged.

[0113] The specific preparation method of the egg removal solution is as follows: 4 kg of deionized water at 24°C, 36 g of analytical pure sodium chloride is added, and the mixture is stirred thoroughly. After the mixture is dissolved, 40 g of a mixture of trypsin: pepsin: alkaline protease in a mass ratio of 3:1:1 is added, and the mixture is stirred thoroughly until all the mixture is dissolved. Hydrochloric acid is added to adjust the pH value of the egg removal solution to 7. The egg removal rate of the comparative example is only 88.8%, and the survival rate is 94.3%.

[0114] An in vitro culture method for lobster eggs is basically the same as that of Example 1, except that the shrimp eggs of this comparative example are used. The emergence rate after in vitro culture is 78.3%.

[0115] Comparative Example 1

[0116] A lobster despawning method is basically the same as Example 1, except that the pH of the despawning solution is different. In this comparative example, the pH is adjusted to 3.0.

[0117] The specific preparation method of the egg removal solution is as follows: add 36 grams of analytical grade sodium chloride to 4 kg of deionized water at 24°C, stir thoroughly, add 40 grams of pepsin (>3000U / mg) after it dissolves, stir thoroughly until it is completely dissolved, and add hydrochloric acid to adjust the pH value of the egg removal solution to 3.

[0118] The oviposition rate of the control group was 93.6% and the survival rate was 67%.

[0119] An in vitro culture method for lobster eggs is basically the same as that of Example 1, except that the shrimp eggs of this comparative example are used, and the emergence rate after in vitro culture is 70.4%.

[0120] Comparative Example 2

[0121] A lobster despawning method is basically the same as Example 1, except that the water temperature used to prepare the despawning solution is 28°C.

[0122] The specific preparation method of the egg-removing solution is as follows: add 36 grams of analytical grade sodium chloride to 4 kg of deionized water at 26°C, stir thoroughly, add 40 grams of pepsin (3000U / mg) after it dissolves, stir thoroughly until it is completely dissolved, and add hydrochloric acid to adjust the pH value of the egg-removing solution to 7.

[0123] The egg removal rate of this comparison group was only 87.3% and the survival rate was 91%.

[0124] An in vitro culture method for lobster eggs is basically the same as that of Example 1, except that the shrimp eggs of this comparative example are used. The emergence rate after in vitro culture is 72.4%.

[0125] Comparative Example 3

[0126] A lobster de-egg method is basically the same as that of Example 1, except that the specification of the egg-bearing shrimp is 40g per tail.

[0127] The oviposition rate of this comparison group was only 85.7% and the survival rate was 92.3%.

[0128] An in vitro culture method for lobster eggs is basically the same as that of Example 1, except that the shrimp eggs of this comparative example are used. The emergence rate after in vitro culture is 77.4%.

[0129] Comparative Example 4

[0130] A lobster detaching method, the same as that in Example 1.

[0131] In vitro culture method: The shrimp eggs obtained by the above-mentioned despawning method were cultured in vitro. The in vitro culture method was basically the same as that in Example 1, except that the ratio of the culture medium was changed. In this comparative example, the culture medium was prepared by first weighing 5.844 g of NaCl and adding 1000 ml of deionized water, shaking evenly to obtain a sodium chloride solution, then selecting a sterile culture dish, adding 2 mL of NaCl solution and 1 mL of double antibody in sequence, then adding 2 mL of DMEM, and adjusting the pH to 7.0 to obtain a culture medium. The emergence rate of this comparative example was 69.2%.

[0132] Comparative Example 5

[0133] A lobster despawning method is basically the same as Example 1, except that the despawning time is extended to 180 minutes. The despawning rate of the comparative example is 92% and the survival rate is 86%.

[0134] An in vitro culture method for lobster eggs is basically the same as that of Example 1, except that the shrimp eggs of this comparative example are used. The emergence rate after in vitro culture is 72.3%.

[0135] The above is only a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and changes directly derived or associated by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included in the protection scope of the present invention.

Claims

1. A lobster egg removal solution, characterized in that: The egg-removing liquid for lobsters with eggs is obtained by dissolving protease and sodium chloride in water and adding acid to adjust the pH value; The mass ratio of the protease, sodium chloride and water is 0.006-0.010:0.006-0.009:

1.

2. The egg-removing liquid for lobsters with eggs according to claim 1, characterized in that: The enzyme includes pepsin; And / or, the acid includes at least one of hydrochloric acid, acetic acid, and citric acid; And / or, the pH adjustment refers to adjusting the pH to 7; And / or, the temperature of the water is 22-26°C.

3. A method for removing eggs, characterized in that: The oocyte removal method comprises the following steps: S1: prepare the shrimps with eggs, pre-process them and set them aside; S2: After the egg removal device is treated, an aeration stone connected to an aeration pump is added; S3: adding water, sodium chloride, and protease to the egg removal device in sequence, stirring to dissolve, and adding acid to adjust the pH to obtain the egg removal solution according to any one of claims 1 to 2; S4: Turn on the oxygen pump, add oxygen for 10 minutes, put the shrimp with eggs in step S1 into the de-oviposition solution, keep the temperature constant, continue to add oxygen, and de-oviposition under continuous light for 100-150 minutes; S5: Collect female shrimp and shrimp eggs separately.

4. The method for removing eggs according to claim 3, characterized in that: In step S1, the size of the egg-bearing shrimp is 50-70g per tail.

5. The method for removing eggs according to claim 3, characterized in that: In step S2, the despawning device comprises a shrimp placing device (1) and an egg collector; the shrimp placing device (1) is arranged above the egg collector.

6. The method for removing eggs according to claim 5, characterized in that: The upper end area of ​​the shrimp placing device (1) is larger than the bottom area; the upper end of the shrimp placing device (1) is open, and the bottom is closed and provided with a through hole (6); Preferably, the through hole (6) allows shrimp eggs to pass through but cannot pass through the shrimps carrying eggs and the separated female shrimps.

7. The method for removing eggs according to claim 5, characterized in that: The egg collector is composed of a shrimp egg collection chamber (2) and a bottom plate (3); the upper end of the shrimp egg collection chamber (2) is open, and the upper end area is larger than the bottom area of ​​the shrimp placement device (1) and smaller than the upper end area of ​​the shrimp placement device (1), thereby ensuring that the shrimp placement device (1) is fixedly placed above the egg collection chamber (2) and does not contact the bottom of the egg collection chamber (2).

8. The method for removing eggs according to claim 3, characterized in that: In step S1, the mass volume ratio of the egg-bearing shrimp to the egg-removing liquid is 3kg:4-5L.

9. A method for culturing lobster in vitro, characterized in that: The steps include: (1) obtaining shrimp eggs according to the despawning method according to any one of claims 3 to 8, and grading and treating the eggs for later use; (2) Prepare the shaker, culture medium and culture dishes; (3) Add the shrimp eggs to the culture medium, seal it and place it in a shaker, add the culture medium every 3-4 days, and keep the amount of the culture medium at the initial state; after 12-15 days of culture, the shrimp eggs will become shrimp fry.

10. The in vitro culture method according to claim 9, characterized in that: In step (2), the preparation of the shaking table includes sterilizing the shaking table, setting the temperature of the shaking table to 25-28° C. and the speed to 100-150 r / min; In step S3, the culture solution includes sodium chloride solution, nutrient solution, and double antibody solution, and the pH value is 6-7; The concentration of the sodium chloride solution is 25-45 mmol / L, the nutrient solution is DMEM nutrient solution; the double antibody solution is HyClone penicillin-streptomycin 100 times solution; The volume ratio of the sodium chloride solution, the nutrient solution and the double antibody solution is 2:1:1.

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