Method for improving in-vitro culture hatching rate of big algae and shrimp embryos and application

Abstract

The invention belongs to the technical field of aquaculture, and discloses a method for increasing the in-vitro culture hatching rate of big algae and shrimp embryos, which comprises the following steps: (1) preparing adult shrimp culture water; (2) preparing parent shrimp feed; (3) culturing big algae and shrimps; (4) preparing embryo eluent; (5) embryo in-vitro method; (6) hatching: hatching for 24 hours at the room temperature of 25 + / -1 DEG C, then disinfecting again, changing water, and removing bad eggs at regular time. According to the method disclosed by the invention, the fertilized embryos of the big algal shrimps are subjected to in-vitro incubation, so that the defects caused by direct egg-carrying incubation of the female shrimps are reduced, the limitation of egg-carrying incubation of the female shrimps is eliminated, the incubation rate of in-vitro culture of the big algal shrimps is increased, the survival rate of membrane-ruptured larvae of the big algal shrimps is improved, and a good application technology is provided for the industry; and an effective means is provided for large-scale directional breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and in particular to a method for improving the hatching rate of in vitro culture of Yamato algae shrimp embryos and its application. Background Art

[0002] Caridina japonica (De man, 1892) [Caridina]), commonly known as "Takashi Amano Shrimp", "Yamato Shrimp", pearl shrimp, Yamato algae shrimp. Among many ornamental shrimps, Yamato algae shrimp is not very eye-catching. It is an entry-level shrimp. It is very easy to raise. It is relatively common in the market and the price is relatively low. Like cherry shrimp, it is the first choice for beginners to raise shrimp. It is native to Japan and can also be called pearl shrimp because of the spots on its body.

[0003] The total length of the Yamato algae shrimp is 5cm, and it is thin and egg-shaped. The female Yamato algae shrimp has regular lines of red spots on its body, while the male shrimp has irregular spots. It is not very attractive in the aquarium, but it is still very beautiful if you look closely.

[0004] The male and female Yamato algae shrimp are relatively easy to distinguish. The red spots on the body are arranged regularly in lines, which are female shrimps, while those with irregular red spots are male shrimps. Since Yamato algae shrimps are soda shrimps, in nature, after the young shrimps shed their shells, they follow the water flow to the salt water waters near the ocean. Therefore, any attempt to breed in fresh water will only end in failure.

[0005] Yamato algae shrimp, scientific name Caridina japonica, is native to Japan and is also found in Taiwan. The maximum length of Yamato algae shrimp can reach 5 cm. The most suitable water temperature is between 22 and 26 degrees Celsius, and the water quality requires a pH value of 6.5 to 7.2.

[0006] Its name has already identified it as an algae-eating shrimp. Generally speaking, in addition to foraging for algae, Yamato algae shrimp also eat plankton, feed fragments and even aquatic plants. Yamato algae shrimp have a very strong appetite and eat continuously throughout the day. When food is insufficient, they are prone to disease and death due to malnutrition. At this time, Yamato algae shrimp may turn to eating young leaves or rotten leaves of aquatic plants. There have been records of Yamato algae shrimp eating a large amount of aquatic plants in the river in nature, harming local organisms. Therefore, while hoping that it can remove algae, it is also necessary to consider feeding it some feed to maintain its normal living state.

[0007] Their reproduction method is very interesting. The males line up one by one on the females that are about to lay eggs and collectively fertilize them. In nature, the young shrimps molt and are carried by the water flow to the salt water near the ocean. Therefore, any attempt to reproduce in fresh water will only end in failure. The young shrimps will survive in fresh water for 6 days after molting, and then die.

[0008] In most existing Yamato shrimp farming processes, the shrimp egg holding and hatching technology is not mature enough. The way the shrimp eggs are peeled, the ratio of hatching water, and the excessive bacteria and parasites in the hatching water, which easily lead to the death of shrimp eggs, are all technical bottlenecks, resulting in the current in vitro farming technology is not mature enough, the in vitro embryo hatching rate is low, and the embryo development rate after in vitro operation is almost zero. It has caused certain inconveniences to people's farming.

[0009] Through searching, the following journals and patent publications related to the patent application of this invention were found:

[0010] 1. Embryonic development and in vitro hatching of Caridina japonica (classification number: S917.4), including: This study used a homemade in vitro hatching device to study in vitro hatching of embryos of Caridina japonica at different developmental stages. The results showed that when the water temperature was 25.5℃, it took about 25 days for the fertilized eggs of Caridina japonica to hatch, and the developmental accumulated temperature was 637.5℃. The embryonic development went through eight stages: fertilized egg, cleavage stage, blastocyst stage, gastrula stage, pro-nauplii stage, post-nauplii stage, pro-zoa stage and intramembranous zoa stage. All in vitro embryos could hatch into larvae. The in vitro embryo hatching rate of intramembranous zoa stage was the highest, which was (80.7±2.4)%, while the non-in vitro hatching control group was (79.1±4.9)%, and the difference between the two was not significant; the in vitro embryo hatching rate of the cleavage stage was the lowest, which was (28.2±2.6)%, which was significantly different from the control group. There was no significant difference in the metamorphosis rate of stage I (ZⅠ) and stage II zoa (ZⅡ) hatched from in vitro embryos of each group. Temperature had a significant effect on the in vitro hatching of pre-zoa larvae of Japanese shrimp. In the range of 15.0℃~32.5℃, the incubation time gradually shortened with the increase of water temperature. At 15.0℃, the in vitro hatching time of pre-zoa larvae was (436.8±124.8)h, and at 32.5℃, it was shortened to (228.0±88.8)h. However, when the temperature was higher than 29.0℃, the metamorphosis rate of hatched larvae began to decrease. This study can provide a reference for the research on the reproductive biology of Japanese shrimp and the in vitro hatching technology of crustacean embryos.

[0011] 2. Research on the embryonic development of Macrobrachium rosenbergii: Ⅰ. Morphogenesis of the external structure of the embryo (classification number: Q954.4), including: The embryonic development process of Macrobrachium rosenbergii can be accurately divided into seven stages: cleavage stage, blastocyst stage, gastrula stage, pre-nauplius stage, post-nauplius stage, pre-zoa stage and zoa stage. Under the water temperature of 28℃, the whole process of embryonic development takes about 480 hours.

[0012] 3. An in vitro hatching method for 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 hatching. (2) The in vitro hatching tray is equipped with a pre-filter barrel, an ozone disinfector, a heating pipe, an aerator and an isolation net that match the water body, the fertilized eggs are placed on the isolation net, and air bubbles are set at the bottom of the in vitro hatching tray to create water flow, so that the fertilized eggs remain slightly active. The ozone machine is controlled by a time system for regular disinfection, and the fertilized eggs can hatch shrimps after about 40-50 days of hatching. By adopting the in vitro hatching method described in the present invention, the infection rate of fertilized eggs can be reduced to less than 10%, and the hatching survival rate can reach more than 90%, which greatly improves the hatching survival rate of fertilized eggs, can effectively increase the shrimp seedling yield, and has significant economic benefits.

[0013] By comparison, the patent application of the present invention is essentially different from the above-mentioned public documents. Summary of the invention

[0014] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method and application for improving the hatching rate of Yamato shrimp embryos in vitro culture.

[0015] The technical solution adopted by the present invention to solve its technical problem is:

[0016] A method for improving the hatching rate of Yamato shrimp embryos in vitro culture, comprising the following steps:

[0017] ⑴ Preparation of water for adult shrimp breeding: Yamato algae shrimp are seawater shrimp, so ordinary Yamato algae shrimp can be placed in the prepared seawater for adult shrimp breeding;

[0018] Seawater formula: distilled water and sea salt; sea salt formula: calcium (Ca) 12-24PPM, sodium (Na) 28-100PPM, potassium (K) 2-42PPM, magnesium (Mg) 10-27PPM, silicon dioxide (SiO2) 41-53PPM, iron (Fe) less than 0.02PPM and greater than 0, zinc (Zn) 0.2-4PPM, calcium carbonate (CaCO3) less than 120PPM and greater than 0, the solvent is distilled water; during preparation, each substance is dissolved one by one, stirred evenly, and oxygenated to increase the dissolved oxygen concentration of the water body, and the prepared seawater is placed for 24 hours to allow any possible sediment in the water body to completely dissolve or settle, thereby obtaining seawater;

[0019] ⑵Preparation of broodstock feed: Its components and weight proportions are as follows:

[0020] 10-15 parts of seaweed powder, 10-15 parts of chlorella, 10-15 parts of shrimp meal, 10-15 parts of Peruvian fish meal, 5-10 parts of probiotics (Lactococcus lactis), 10-15 parts of fermented corn protein, 10-15 parts of wheat flour, 10-20 parts of spinach, 1-2 parts of chitin, 1-2 parts of Ruditapes philippinarum, 1-2 parts of complex minerals, 1-2 parts of peanut vines, 1-2 parts of complex vitamins, 1-2 parts of silkworm excrement, and soybean oil. The amount of soybean oil added is to ensure that the total fat content of the feed is controlled at 6%-10%. The soybean oil should be evenly mixed in the feed to ensure that the fatty acids in the feed are evenly distributed and promote the feeding and digestion of the broodstock; during the preparation, after ultrafine grinding of various raw materials, weighing according to the formula and mixing by step-by-step expansion, finally adding soybean oil and water to stir and mix, and processing into granular feed with a diameter of 1mm in a granulator, drying at 75℃ and placing in a -20℃ refrigerator for standby use;

[0021] ⑶Cultivation of Yamato shrimp: The adult shrimps are placed at a rate of 3 / 20 shrimps are placed in a breeding tank, cultured with prepared breeding water, and fed with prepared broodstock feed until the female shrimps are pregnant with eggs;

[0022] (4) Preparation of embryo eluate: Under a sterile environment, mix 20 PPM collagenase, glutaraldehyde, EDTA2 sodium, Tween 20, and 1X PBS buffer, and then add water to make a 1% aqueous solution to obtain embryo eluate. The embryo eluate is stored in a refrigerator at 4°C.

[0023] ⑸ Method for embryo separation: Wet clean cotton wool with embryo washing solution, place the wet cotton wool on the part of the existing broodstock that has just been incubated with eggs, that is, has been incubated with eggs for no more than 10 hours, and leave it for 1 minute. Wash the eggs off the broodstock with hatching water, disinfect with low-concentration potassium permanganate solution for 10 seconds, and store at 4℃ for future use. All equipment used in the whole process is disinfected and sterilized; the prepared egg washing solution can minimize the damage to the broodstock and the required fertilized eggs, and completely separate the fertilized eggs without harming the broodstock. It can reduce and prevent the infection of aquatic organisms and microorganisms.

[0024] (6) Incubation method: Take out the preserved fertilized eggs, observe the stage of the cells under a stereoscope, and take photos 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 it stand for 10 to 15 seconds, then use a sterilized plastic straw to suck out all the eggs, put them into a sterilized container, pour in the prepared hatching water, and slowly add 5-10 ml / egg of hatching water. After incubating at room temperature of 25±1℃ for 24 hours, disinfect and change the water again and remove bad eggs regularly.

[0025] Furthermore, the steps for preparing the hatching water are as follows:

[0026] Accurately measure filtered seawater with a salinity of 13‰, accurately weigh EDTA, PBS buffer, and DMEM respectively, pour them into the sterilized seawater, add 5g EDTA, 3ml PBS buffer, and 2g DMEM to every 1L of filtered seawater, filter and sterilize with a 0.22μm filter membrane to obtain hatching water; seal and store at room temperature for later use.

[0027] Further, each 1L PBS buffer was prepared as follows:

[0028] Weigh 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4, dissolve in 800 ml filtered seawater with a salinity of 13‰, adjust the pH of the solution to 7.4 with HCl, and finally add filtered seawater to make up to 1 L.

[0029] Furthermore, the configured hatching solution has a shelf life of no more than one month.

[0030] Furthermore, the preparation steps of the low concentration potassium permanganate solution are as follows:

[0031] Weigh potassium permanganate reagent, then add distilled water. Add 0.0001 g of potassium permanganate reagent to every 1 L of distilled water, mix well, and seal for storage. This is the prepared low-concentration potassium permanganate solution.

[0032] Furthermore, in step (4), the eggs in the abdomen of the parent shrimp are eluted with embryo elution solution and hatching water.

[0033] The method for improving the hatching rate of in vitro culture embryos of Yamato algae shrimp as described above is used to improve the survival rate of membrane-breaking larvae of Yamato algae shrimp.

[0034] The advantages and positive effects achieved by the present invention are:

[0035] 1. The method of the present invention incubates the fertilized embryos of Yamato algae shrimp in vitro, thereby reducing the disadvantages of direct hatching of eggs by mother shrimps, getting rid of the limitation of hatching eggs by mother shrimps, and increasing the hatching rate of in vitro culture of Yamato algae shrimp. The hatching rate of the present invention is about 75%, which is about 8% higher than the hatching rate of fertilized eggs of ordinary Yamato algae shrimps. The in vitro culture technology of the present invention can achieve a hatching rate of up to 100% under some breeding environments, and does not produce some adverse conditions of in vitro culture, thereby improving the survival rate of membrane-breaking larvae of Yamato algae shrimp, providing a good application technology for the industry and an effective means for large-scale directional breeding.

[0036] 2. The method of the present invention provides a technical platform for in vitro fertilization and embryo manipulation of Yamato shrimp, and provides necessary auxiliary technology for gene editing of economic crustaceans.

[0037] 3. In modern biotechnology, hybrid breeding has become very mature, and molecular breeding technology can better improve the yield and quality of animals and plants. Varieties of aquatic animals that can carry out in vitro embryo culture can become alternative varieties for molecular breeding of crustaceans. The method of the present invention can provide the necessary preliminary technology for obtaining embryonic stem cells of crustaceans at different stages.

[0038] 4. The method of the present invention can provide the necessary preliminary technology for some omics technologies such as single-cell transcriptome sequencing, proteome sequencing, metabolome sequencing, etc. in crustacean development research.

[0039] 5. The in vitro embryo culture method provided by the present invention can hatch the fertilized embryos of Yamato algae shrimp in vitro, getting rid of the various limitations of the mother shrimp holding eggs for hatching, thereby improving the in vitro hatching rate of Yamato algae shrimp embryos, shortening the number of days required for the hatching of fertilized eggs, and also to a certain extent improving the survival rate of the larvae of Yamato algae shrimp after the fertilized eggs break the membrane.

[0040] 6. The broodstock feed used in the method of the present invention can increase the number and quality of eggs carried by female shrimps to a certain extent, and improve the physical fitness of broodstock to a certain extent.

[0041] 7. The embryo eluting solution used in the method of the present invention can remove the fertilized eggs of the broodstock one by one without being affected by artificial external forces and without damaging the fertilized eggs.

[0042] 8. The hatching water used in the method of the present invention can protect the fertilized eggs from interference from aquatic microorganisms during hatching, thereby improving the in vitro hatching rate of the Yamato algae shrimp of the present patent.

[0043] 9. The specific embryo in vitro culture technique of the method of the present invention is simple and easy to operate without too many restrictions. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 These are pictures continuously recorded during the in vitro culture of the Yamato algae shrimp of the present invention; wherein, 1-2. blastocyst stage; 3-4. gastrula stage; 5-6. pre-nauplius stage; 7-8. post-nauplius stage; 9-10. pre-Daphnia stage; 11-15. intramembranous daphnia stage; 16-20. ruptured larvae;

[0045] Figure 2These are continuous recording pictures during the specific implementation of the parallel experiment in the present invention; wherein, 1-2. blastocyst stage; 3-4. gastrula stage; 5-6. pre-nauplius stage; 7-8. post-nauplius stage; 9-11. pre-Daphnia stage; 12-15. intramembranous Daphnia stage; 16-20. ruptured larvae. DETAILED DESCRIPTION

[0046] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0047] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0048] A method for improving the hatching rate of Yamato shrimp embryos in vitro culture, comprising the following steps:

[0049] ⑴ Preparation of water for adult shrimp breeding: Yamato algae shrimp are seawater shrimp, so ordinary Yamato algae shrimp can be placed in the prepared seawater for adult shrimp breeding;

[0050] Seawater formula: distilled water and sea salt; sea salt formula: calcium (Ca) 12-24PPM, sodium (Na) 28-100PPM, potassium (K) 2-42PPM, magnesium (Mg) 10-27PPM, silicon dioxide (SiO2) 41-53PPM, iron (Fe) less than 0.02PPM and greater than 0, zinc (Zn) 0.2-4PPM, calcium carbonate (CaCO3) less than 120PPM and greater than 0, the solvent is distilled water; during preparation, each substance is dissolved one by one, stirred evenly, and oxygenated to increase the dissolved oxygen concentration of the water body, and the prepared seawater is placed for 24 hours to allow any possible sediment in the water body to completely dissolve or settle, thereby obtaining seawater;

[0051] ⑵Preparation of broodstock feed: Its components and weight proportions are as follows:

[0052] 10-15 parts of seaweed powder, 10-15 parts of chlorella, 10-15 parts of shrimp meal, 10-15 parts of Peruvian fish meal, 5-10 parts of probiotics (Lactococcus lactis), 10-15 parts of fermented corn protein, 10-15 parts of wheat flour, 10-20 parts of spinach, 1-2 parts of chitin, 1-2 parts of Ruditapes philippinarum, 1-2 parts of complex minerals, 1-2 parts of peanut vines, 1-2 parts of complex vitamins, 1-2 parts of silkworm excrement, and soybean oil. The amount of soybean oil added is to ensure that the total fat content of the feed is controlled at 6%-10%. The soybean oil should be evenly mixed in the feed to ensure that the fatty acids in the feed are evenly distributed and promote the feeding and digestion of the broodstock; during the preparation, after ultrafine grinding of various raw materials, weighing according to the formula and mixing by step-by-step expansion, finally adding soybean oil and water to stir and mix, and processing into granular feed with a diameter of 1mm in a granulator, drying at 75℃ and placing in a -20℃ refrigerator for standby use;

[0053] ⑶Cultivation of Yamato shrimp: The adult shrimps are placed at a rate of 3 / 20 shrimps are placed in a breeding tank, cultured with prepared breeding water, and fed with prepared broodstock feed until the female shrimps are pregnant with eggs;

[0054] (4) Preparation of embryo eluate: Under a sterile environment, mix 20 PPM collagenase, glutaraldehyde, EDTA2 sodium, Tween 20, and 1X PBS buffer, and then add water to make a 1% aqueous solution to obtain embryo eluate. The embryo eluate is stored in a refrigerator at 4°C.

[0055] ⑸ Method for embryo separation: Wet clean cotton wool with embryo washing solution, place the wet cotton wool on the part of the existing broodstock that has just been incubated with eggs, that is, has been incubated with eggs for no more than 10 hours, and leave it for 1 minute. Wash the eggs off the broodstock with hatching water, disinfect with low-concentration potassium permanganate solution for 10 seconds, and store at 4℃ for future use. All equipment used in the whole process is disinfected and sterilized; the prepared egg washing solution can minimize the damage to the broodstock and the required fertilized eggs, and completely separate the fertilized eggs without harming the broodstock. It can reduce and prevent the infection of aquatic organisms and microorganisms.

[0056] (6) Incubation method: Take out the preserved fertilized eggs, observe the stage of the cells under a stereoscope, and take photos 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 it stand for 10 to 15 seconds, then use a sterilized plastic straw to suck out all the eggs, put them into a sterilized container, pour in the prepared hatching water, and slowly add 5-10 ml / egg of hatching water. After incubating at room temperature of 25±1℃ for 24 hours, disinfect and change the water again and remove bad eggs regularly.

[0057] Preferably, the steps for preparing the hatching water are as follows:

[0058] Accurately measure filtered seawater with a salinity of 13‰, accurately weigh EDTA, PBS buffer, and DMEM respectively, pour them into the sterilized seawater, add 5g EDTA, 3ml PBS buffer, and 2g DMEM to every 1L of filtered seawater, filter and sterilize with a 0.22μm filter membrane to obtain hatching water; seal and store at room temperature for later use.

[0059] Preferably, each 1L PBS buffer is prepared as follows:

[0060] Weigh 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4, dissolve in 800 ml filtered seawater with a salinity of 13‰, adjust the pH of the solution to 7.4 with HCl, and finally add filtered seawater to make up to 1 L.

[0061] Preferably, the configured hatching solution has a shelf life of no more than one month.

[0062] Preferably, the preparation steps of the low concentration potassium permanganate solution are as follows:

[0063] Weigh potassium permanganate reagent, then add distilled water. Add 0.0001 g of potassium permanganate reagent to every 1 L of distilled water, mix well, and seal for storage. This is the prepared low-concentration potassium permanganate solution.

[0064] Preferably, in step (4), the eggs in the abdomen of the broodstock are eluted with embryo elution solution and hatching water.

[0065] The method for improving the hatching rate of in vitro culture embryos of Yamato algae shrimp as described above is used to improve the survival rate of membrane-breaking larvae of Yamato algae shrimp.

[0066] Specifically, the relevant preparation and detection are as follows:

[0067] A method for improving the hatching rate of Yamato shrimp embryos in vitro culture, comprising the following steps:

[0068] ⑴ Preparation of water for adult shrimp breeding: Yamato algae shrimp are seawater shrimp, so ordinary Yamato algae shrimp can be placed in the prepared seawater for adult shrimp breeding;

[0069] Seawater formula: distilled water and sea salt; sea salt formula: calcium (Ca) 12-24PPM, sodium (Na) 28-100PPM, potassium (K) 2-42PPM, magnesium (Mg) 10-27PPM, silicon dioxide (SiO2) 41-53PPM, iron (Fe) less than 0.02PPM and greater than 0, zinc (Zn) 0.2-4PPM, calcium carbonate (CaCO3) less than 120PPM and greater than 0, the solvent is distilled water; dissolve them one by one, stir them evenly, and perform oxygenation treatment to increase the dissolved oxygen concentration of the water body. The prepared seawater is placed for 24 hours to allow any possible sediment in the water body to completely dissolve or settle, and obtain seawater;

[0070] ⑵Preparation of broodstock feed: Its components and weight proportions are as follows:

[0071] 10-15 parts of seaweed powder, 10-15 parts of chlorella, 10-15 parts of shrimp meal, 10-15 parts of Peruvian fish meal, 5-10 parts of probiotics (Lactococcus lactis), 10-15 parts of fermented corn protein, 10-15 parts of wheat flour, 10-20 parts of spinach, 1-2 parts of chitin, 1-2 parts of Ruditapes philippinarum, 1-2 parts of complex minerals, 1-2 parts of peanut vines, 1-2 parts of complex vitamins, 1-2 parts of silkworm feces, and soybean oil. The amount of addition is to ensure that the total fat content of the feed is controlled at 6%-10%. The soybean oil should be evenly mixed in the feed to ensure that the fatty acids in the feed are evenly distributed and promote the feeding and digestion of the broodstock; during the preparation, after ultrafine grinding of various raw materials, weighing according to the formula and mixing by step-by-step expansion, finally adding soybean oil and water to stir and mix, and processing into granular feed with a diameter of 1mm in a granulator, drying at 75℃ and placing in a -20℃ refrigerator for standby use;

[0072] ⑶ Cultivation of Yamato algae shrimp: Place adult shrimps in a breeding tank at a rate of 20 per m3, cultivate them with the breeding water prepared in step ⑴, feed the adult shrimps with the broodstock feed prepared in step ⑵, and wait for the female shrimps to carry eggs;

[0073] (4) Preparation of embryo eluate: Under a sterile environment, mix 20 PPM collagenase, glutaraldehyde, EDTA2 sodium (small amount), Tween 20, and 1X PBS buffer in a certain proportion, and then add water to make a 1% aqueous solution to obtain embryo eluate. The embryo eluate is stored in a refrigerator at 4°C.

[0074] ⑸ Method for embryo separation: Wet clean cotton wool with embryo washing solution, place the wet cotton wool on the part of the existing shrimp that has just been holding eggs (holding eggs for no more than 10 hours) where the eggs need to be removed, leave it for 1 minute, wash off the eggs on the shrimp with hatching water, disinfect with low-concentration potassium permanganate solution for 10 seconds, store at 4℃ for standby use, and sterilize the equipment used in the whole process; the prepared egg washing solution can minimize the damage to the shrimp and the required fertilized eggs, and completely separate the fertilized eggs without harming the shrimp. It can reduce and prevent the infection of aquatic organisms and microorganisms.

[0075] (6) Incubation method: Take out the preserved fertilized eggs, observe the stage of the cells under a stereoscope, and take photos 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 it stand for 10 to 15 seconds, then use a sterilized plastic straw to suck out all the eggs, put them into a sterilized container, pour in the prepared hatching water, and slowly add 5-10 ml / egg of hatching water. After incubating at room temperature of 25±1℃ for 24 hours, disinfect and change the water again and remove bad eggs regularly.

[0076] The steps for preparing the hatching water in step (5) are as follows:

[0077] Accurately measure filtered seawater with a salinity of 13‰, sterilize, take out, and cool to room temperature; accurately weigh EDTA, PBS buffer, and DMEM respectively, pour them into the sterilized seawater, add 5g EDTA, 3ml PBS buffer, and 2g DMEM to every 1L of sterilized seawater, filter and sterilize with a 0.22μm filter membrane to obtain hatching water; seal and store, and place at room temperature for later use.

[0078] The sterilization is carried out by placing the sample in a high temperature and high pressure sterilization pot for 2 hours.

[0079] Preparation of 1L PBS buffer:

[0080] Weigh 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4, dissolve in 800 ml filtered seawater with a salinity of 13‰, adjust the pH of the solution to 7.4 with HCl, and finally add filtered seawater to make up to 1 L.

[0081] The hatching water prepared in step (5) shall not be used for more than one month.

[0082] The preparation steps of the low concentration potassium permanganate solution are as follows:

[0083] Weigh potassium permanganate reagent, then add distilled water. Add 0.1 mg of potassium permanganate reagent to every 1 L of distilled water, mix well, and seal for storage. This is the prepared low-concentration potassium permanganate solution.

[0084] More specifically, the relevant preparation and detection are as follows:

[0085] A method for improving the hatching rate of Yamato shrimp embryos in vitro culture, comprising the following steps:

[0086] a. Method for embryo separation: Wet clean absorbent cotton with embryo washing solution, place the wet absorbent cotton on the part of the broodstock where the eggs need to be removed, leave it for 1 minute, wash off the eggs on the broodstock with hatching water, disinfect with low concentration potassium permanganate for 10 seconds, and store at 4°C for later use. The contents not described in detail are the same as the above contents (i.e. the contents recorded in "Specifically, the relevant preparation and detection are as follows:").

[0087] b. Incubation method: Take out the preserved fertilized eggs, observe the period of the cells under a stereoscope, and take photos 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 it stand for 10-15 seconds, then use a sterilized plastic pipette to suck out all the eggs, put them into a sterilized 100ml beaker, and pour in the prepared hatching water. The amount of hatching water is slowly added at a rate of 10ml / (per egg). After 24 hours, suck out the fertilized eggs with a plastic pipette, put them into a certain amount of low-concentration potassium permanganate solution, let it stand for 10-15 seconds, and then suck them out. Rinse the beaker with a low-concentration potassium permanganate solution, and put the sucked out sterilized fertilized eggs into the rinsed beaker. Pour in hatching water in proportion and continue to culture. Observe once a day and record photos under a stereomicroscope.

[0088] Record the results ( Figure 1 ):

[0089] a. Blastocyst stage: Day 1-2.

[0090] b. Enterostasis stage: Day 3-4.

[0091] c. Pre-larval stage: 5-6 days.

[0092] d. Post-nauplius stage: Day 7-8.

[0093] e. Pre-zoa stage: 9-10 days.

[0094] f. Intramembranous zoa larvae stage: Day 11-15.

[0095] g. Broken membrane larvae: 16-20 days.

[0096] Parallel test ( Figure 2 ):

[0097] Record the results:

[0098] a. Blastocyst stage: Day 1-2.

[0099] b. Enterostasis stage: Day 3-4.

[0100] c. Pre-larval stage: 5-6 days.

[0101] d. Post-nauplius stage: Day 7-8.

[0102] e. Pre-zoa stage: Day 9-11.

[0103] f. Intramembranous zoa larvae stage: 12-15 days.

[0104] g. Broken membrane larvae: 16-20 days.

[0105] pass Figure 1 and Figure 2 It can be seen that:

[0106] 1. The hatching rate of the fertilized eggs of the Yamato algae shrimp in vitro of the present invention is about 75%, which is about 8% higher than the hatching rate of the fertilized eggs of the ordinary Yamato algae shrimp. The in vitro culture technology of the present invention can achieve a hatching rate of up to 100% in some breeding environments, and does not produce some adverse conditions of in vitro culture.

[0107] 2. The number of days for the fertilized eggs of Yamato algae shrimp to break the membrane after the in vitro culture of the present invention is similar to that of the fertilized eggs of Yamato algae shrimp under normal breeding conditions, which is about 15 to 20 days. Currently, there is no other method for in vitro culture of fertilized eggs of Yamato algae shrimp.

[0108] 3. The embryo washing solution used for separating eggs before in vitro operation of the present invention can ensure that the fertilized eggs are completely separated to the greatest extent and the fertilized eggs are not affected by the chemical components in the egg washing solution and their development is not affected. There is no deformity, cessation of development or death after rupture of membranes.

[0109] 4. The feed formula for feeding the Yamato algae shrimp broodstock operated by the present invention can increase the number of Yamato algae shrimp broodstock incubation eggs to a certain extent, and the egg-holding period of the broodstock of the Yamato algae shrimp broodstock is shorter than that of the broodstock of the ordinary compound feed. The normal egg-holding period is usually 20-30 days, and the specific time is also greatly affected by the ambient temperature. Under the condition of a certain ambient temperature, the broodstock feed of the present invention can control the time to within 20 days, and can also enhance the physical fitness of the Yamato algae shrimp broodstock, and the shrimp has a good state and vitality.

[0110] 5. The in vitro culture technology of the Yamato algae shrimp of the present invention can prevent the fertilized eggs of the Yamato algae shrimp from being infected by aquatic microorganisms during the hatching process, thereby preventing the fertilized eggs from becoming bad eggs. Generally, in vitro culture is easily infected by bacteria. Since each step of the method of the present invention is sterile and the formula is good, this situation is avoided to a great extent.

[0111] 6. The in vitro culture technology of the present invention is simple to operate and does not have high requirements on the conditions of the surrounding environment. Generally, in vitro culture requires the use of various homemade in vitro incubation devices, which have complex conditions and many requirements. The method of the present invention is relatively simple to operate and easy to implement.

[0112] Although the embodiments of the present invention are 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 present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for improving the hatching rate of Yamato shrimp embryos in vitro culture, characterized in that: Here are the steps: ⑴ Preparation of water for adult shrimp breeding: Yamato algae shrimp are seawater shrimp, so ordinary Yamato algae shrimp can be placed in the prepared seawater for adult shrimp breeding; Seawater formula: distilled water and sea salt; sea salt formula: calcium 12-24PPM, sodium 28-100PPM, potassium 2-42PPM, magnesium 10-27PPM, silicon dioxide 41-53PPM, iron less than 0.02PPM, zinc 0.2-4PPM, calcium carbonate less than 120PPM, and the solvent is distilled water; during preparation, each substance is dissolved one by one, stirred evenly, and oxygenated to increase the dissolved oxygen concentration of the water body, and the prepared seawater is placed for 24 hours to obtain seawater; ⑵Preparation of broodstock feed: Its components and weight proportions are as follows: 10-15 parts of seaweed powder, 10-15 parts of chlorella, 10-15 parts of shrimp meal, 10-15 parts of Peruvian fish meal, 5-10 parts of probiotics, 10-15 parts of fermented corn protein, 10-15 parts of wheat flour, 10-20 parts of spinach, 1-2 parts of chitosan, 1-2 parts of Manila clams, 1-2 parts of composite minerals, 1-2 parts of peanut vines, 1-2 parts of composite vitamins, 1-2 parts of silkworm excrement, and soybean oil. The amount of soybean oil added is to ensure that the total fat content of the feed is controlled at 6%-10%, and the soybean oil should be evenly mixed in the feed; during preparation, various raw materials are ultra-finely crushed, weighed according to the formula, and mixed by step-by-step expansion, and finally soybean oil and water are added and stirred, and processed into granular feed with a diameter of 1mm in a granulator, and then dried at 75℃ and placed in a -20℃ refrigerator for standby use; ⑶Cultivation of Yamato shrimp: The adult shrimps are placed at a rate of 3 / 20 shrimps are placed in a breeding tank, cultured with prepared breeding water, and fed with prepared broodstock feed until the female shrimps are pregnant with eggs; (4) Preparation of embryo eluate: Under a sterile environment, mix 20 PPM collagenase, glutaraldehyde, EDTA2 sodium, Tween 20, and 1X PBS buffer, and then add water to make a 1% aqueous solution to obtain embryo eluate. The embryo eluate is stored in a refrigerator at 4°C. ⑸ Method for embryo separation: Wet clean cotton wool with embryo washing solution, place the wet cotton wool on the part of the existing broodstock that has just been holding eggs, that is, holding eggs for no more than 10 hours, where the eggs need to be removed, leave it for 1 minute, wash off the eggs on the broodstock with hatching water, disinfect with low-concentration potassium permanganate solution for 10 seconds, and store at 4°C for future use. All equipment used in the whole process are disinfected and sterilized; (6) Incubation method: Take out the preserved fertilized eggs, observe the stage of the cells under a stereoscope, and take photos 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 it stand for 10 to 15 seconds, then use a sterilized plastic straw to suck out all the eggs, put them into a sterilized container, pour in the prepared hatching water, and slowly add 5-10 ml / egg of hatching water. After incubating at room temperature of 25±1℃ for 24 hours, disinfect and change the water again and remove bad eggs regularly.

2. The method for improving the hatchability of Yamato shrimp embryos in vitro culture according to claim 1, characterized in that: The preparation steps of the hatching water are as follows: Accurately measure filtered seawater with a salinity of 13‰, accurately weigh EDTA, PBS buffer, and DMEM respectively, pour them into the sterilized seawater, add 5g EDTA, 3ml PBS buffer, and 2g DMEM to every 1L of filtered seawater, filter and sterilize with a 0.22μm filter membrane to obtain hatching water; seal and store at room temperature for later use.

3. The method for improving the hatchability of Yamato shrimp embryos in vitro culture according to claim 2, characterized in that: Preparation of 1L PBS buffer: Weigh 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4, dissolve in 800 ml filtered seawater with a salinity of 13‰, adjust the pH of the solution to 7.4 with HCl, and finally add filtered seawater to make up to 1 L.

4. The method for improving the hatchability of Yamato shrimp embryos in vitro culture according to claim 2 or 3, characterized in that: The prepared hatching solution should not be used for more than one month.

5. The method for improving the hatchability of Yamato shrimp embryos in vitro culture according to claim 1, characterized in that: The preparation steps of the low concentration potassium permanganate solution are as follows: Weigh potassium permanganate reagent, then add distilled water. Add 0.0001 g of potassium permanganate reagent to every 1 L of distilled water, mix well, and seal for storage. This is the prepared low-concentration potassium permanganate solution.

6. The method for improving the hatchability of Yamato shrimp embryos in vitro culture according to claim 1, characterized in that: In the step (4), the eggs in the abdomen of the parent shrimp are washed out with embryo eluting solution and hatching water.

7. Use of the method for improving the hatching rate of in vitro culture embryos of Yamato algae shrimp as claimed in any one of claims 1 to 6 in improving the survival rate of membrane-broken larvae of Yamato algae shrimp.

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