Method for vitrification and laser thawing of embryos of Culter alburnus

Abstract

The present application belongs to the field of biological material / tissue cryopreservation, and particularly relates to a vitrification freezing and laser thawing method of embryos of Procypris percnurus. The embryos are sequentially transferred to low-concentration and high-concentration balancing solutions for balancing, and finally transferred to an antifreeze for standing; the embryos in the antifreeze are subjected to vitrification on an aluminum metal in liquid nitrogen for storage in liquid nitrogen; the embryos stored in liquid nitrogen are taken out, allowed to stand at room temperature, thawed under the action of near-infrared laser, and sequentially placed in high-concentration and low-concentration sucrose solutions for balancing. The Procypris percnurus cultivated by the freezing and thawing method has high survival rate and hatching rate, high storage quality, and is suitable for production application.

Description

Technical Field

[0001] This invention belongs to the field of cryopreservation of biomaterials / tissues, and specifically relates to a vitrification freezing and laser heating thawing method suitable for Culter alburnus embryos. Background Technology

[0002] The biggest hurdle in cryopreservation of fish embryos stems from the differences in size and membrane permeability among different species. The varying tightness of membrane barriers leads to significant differences in the permeability of cryoprotectants. Larger embryos dissipate heat more slowly, making them more susceptible to the formation of deadly ice crystals during freezing. Furthermore, the membrane characteristics and biochemical defense mechanisms developed in fish during evolution result in varying sensitivities and repair capabilities to cryopreservation damage such as ice crystal damage, osmotic shock, oxidative stress, and DNA damage. Their low-temperature tolerance varies drastically, and the timing of freezing differs from species to species. In short, the main challenges hindering successful cryopreservation of fish embryos can be summarized in four aspects: extremely low specific surface area, large yolk volume, low membrane permeability, and high low-temperature sensitivity. These difficulties mean that cryoprotectants and cryopreservation protocols for fish embryos are not universally applicable; specific and systematic optimization is required for different species and even different developmental stages within the same species.

[0003] To overcome these difficulties, most embryo cryopreservation research over the past few decades has focused on methods such as increasing membrane permeability, artificially removing the yolk, overexpressing aquaporins, and microinjecting CPA. However, none of these studies have successfully thawed the embryos. A possible reason is that early studies generally used slow freezing methods, and for such large, multicellular, and complex structures as embryos, the growth of ice crystals within the embryo can be fatal. Although fish embryo cryopreservation faces far greater challenges than that of mammals, there are common difficulties to overcome during vitrification. In recent years, as the importance of vitrification research on fish embryos has been increasingly recognized, some preservation methods considered feasible in mammalian studies have begun to be attempted in fish embryo cryopreservation. In 2005, Robles et al. vitrified American flounder (Pseudopleuronectes americanus) using the straw method, achieving the first successful vitrification of fish embryos after embryonic development (ROBLES V, CABRITA E, FLETCHER GL, et al. Vitrification assays with embryos from a cold tolerant sub-arctic fish species [J]. Theriogenology, 2005, 64(7): 1633-46.). Tian et al. cryopreserved Epinephelus moara embryos using PMG3T and PMG3S cryoprotectant formulations, achieving a survival rate of 5.15% and a normal development rate of 1.31%, and obtaining 44 hatched juveniles (Zhang J, Tian Y, Li Z, Wu Y, Li Z, Cheng M, Wang L, Ma W, Zhai J. Optimization of vitrification factors for embryocryopreservation of kelp grouper (Epinephelus moara). Theriogenology. 2020Jan 15;142:390-399.).

[0004] To achieve faster heating during thawing, more and more new heating methods are being tried in cryopreservation research, including microwave heating, magnetic induction heating, and laser heating. Laser heating is one of the more readily implemented and widely tested methods. For example, Paredes used laser heating to thaw vitrified Saccharomyces cerevisiae. The heating rate during thawing was about 107 °C / min, and the survival rate reached 80%, which is 20% higher than that of non-vitrification methods (Paredes E . Vitrification and Ultra-Rapid Laser Warming of YeastSaccharomyces cerevisiae. Cryo Letters. 2021 Jan-Feb;42(1):19-24.). Currently, the application of laser heating in cryopreservation research of aquatic organisms is still relatively limited. Smith et al. used gold nanoparticles combined with laser heating in their cryopreservation research of Pacific White Shrimp (Litopenaeus vannamei) embryos, achieving a survival rate of 54%, but none of them survived for more than 15 hours after thawing (SMITH KT, KHOSLA K, HAN G, et al. Revival of cryopreserved larvae from the important aquaculture species Pacific WhiteShrimp (Litopenaeus vannamei) using vitrification and ultra-rapid laserwarming [J]. Cryobiology, 2024, 117: 105160.). Khosla et al. were the first to introduce laser-assisted vitrification into the study of fish embryo cryopreservation. They used a combination of gold nanorods (GNRs) and laser heating, achieving a survival rate of 40%, with 3% of the juveniles surviving for more than 5 days (KHOSLA K, KANGAS J, LIU Y, et al. Cryopreservation and Laser Nanowarming of Zebrafish Embryos Followed by Hatching and Spawning [J]. Adv Biosyst, 2020, 4(11): e2000138.).

[0005] In their research, Khosla et al. developed a method for cryopreserving zebrafish embryos using microinjection combined with laser heating. This method uses a cryoprotectant formulation containing propylene glycol, methanol, and gold nanorods, injected into zebrafish embryos via microinjection, and then freezes the embryos in liquid nitrogen using a cryotope carrier. Laser heating was used during thawing, achieving, for the first time, the hatching of frozen fish embryos into adult fish. However, Khosla et al.'s cryopreserved zebrafish embryos only achieved a very low survival and hatching rate, indicating significant room for improvement. Furthermore, fish embryos vary greatly in size and physiological and biochemical properties, meaning that research conducted on the zebrafish model organism cannot be directly applied to other species. Different cryopreservation methods need to be designed for different species, lacking practical value for production applications. Moreover, microinjection technology is cumbersome and not suitable for large-scale production applications. Summary of the Invention

[0006] This invention develops a vitrification cryopreservation and thawing technology suitable for Culter alburnus embryos, optimizes the cryopreservation and thawing process, and achieves a high hatching rate during the cryopreservation and thawing of Culter alburnus embryos, which is applicable to production practice.

[0007] The technical solution of this invention is a method for vitrification freezing and laser thawing of Culter alburnus embryos, comprising the following steps: Vitrification: First, transfer the embryos to equilibration solution 1, then to equilibration solution 2, and finally to cryoprotectant for static freezing; collect the embryos from the cryoprotectant, place them on an aluminum metal plate immersed in liquid nitrogen for vitrification, and preserve them in liquid nitrogen; The antifreeze is a solvent containing melatonin and gold nanorods, and the solvent is prepared by volume percentage of the following solution: 13%-18% dimethyl sulfoxide (DMSO), 8%-12% methanol (MeOH), 13%-18% propylene glycol (PG), and the balance being Culter alburnus culture water; preferably, the solvent is prepared by volume percentage of the following solvent: 14%-16% dimethyl sulfoxide (DMSO), 9%-11% methanol (MeOH), 14%-16% propylene glycol (PG), and the balance being Culter alburnus culture water; as one embodiment, the solvent is prepared by volume percentage of the following solvent: 15% dimethyl sulfoxide (DMSO), 10% methanol (MeOH), 15% propylene glycol (PG), and the balance being Culter alburnus culture water; the concentration of melatonin is 0.1-3 mM, preferably 0.5-1 mM, more preferably 0.8-1 mM. mM, as one embodiment, 1mM is selected; the concentration of the gold nanorods is 10-50μg / mL, preferably 15-40μg / mL, more preferably 20-30μg / mL, and as one embodiment, 25μg / mL is selected; The equilibration solution 1 was prepared by diluting the antifreeze four times, and the diluent used for dilution was the culture water of Culter alburnus. The balancing solution 2 was prepared by diluting the antifreeze by two times, and the diluent used for dilution was the culture water of Culter alburnus. Laser thawing: Embryos preserved in liquid nitrogen are removed, placed at room temperature, and then thawed under near-infrared laser light. They are then sequentially placed in high-concentration and low-concentration sucrose solutions for equilibration, and finally transferred to the culture water for Culter alburnus. The sucrose concentration in the high-concentration sucrose solution is 0.2-1 mol / L, preferably 0.4-0.6 mol / L, and 0.5 mol / L is selected as one implementation method. The sucrose concentration in the low-concentration sucrose solution is 0.1-0.5 mol / L, preferably 0.2-0.3 mol / L, and 0.25 mol / L is selected as one implementation method.

[0008] During vitrification freezing, the equilibration time in equilibration solution 1 is 10 ± 2 min, preferably 10 min.

[0009] During vitrification freezing, the equilibration time in equilibration solution 2 is 10 ± 2 min, preferably 10 min.

[0010] During vitrification freezing, the standing time in the antifreeze is 15±2 min, preferably 15 min.

[0011] During vitrification freezing, the vitrification time is 10 ± 2 s, preferably 10 s.

[0012] In laser thawing, the wavelength of the near-infrared laser is 780nm to 2526nm, such as 808nm, 980nm, 1064nm, or 1310nm. As one implementation method, 1064nm is selected. The power of the near-infrared laser is 5-25W, preferably 10-25W, more preferably 15-20W. As one implementation method, 15W is selected. The irradiation time of the near-infrared laser is 6-15s, preferably 8-13s, more preferably 9-12s. As one implementation method, 10s is selected. The embryo is located 10±2cm below the laser, more preferably 10cm below the laser.

[0013] During laser thawing, the incubation time at room temperature is 10 ± 2 seconds, preferably 10 seconds. The room temperature is 18℃-25℃, and more preferably 20℃-25℃. Incubation at room temperature allows the embryo to slowly warm up to -120℃ to -100℃.

[0014] In laser thawing, both high-concentration and low-concentration sucrose solutions are preheated to 30±1℃, preferably 30℃.

[0015] In laser thawing, the equilibration time in the high-concentration sucrose solution is 10 ± 2 min, preferably 10 min.

[0016] In laser thawing, the equilibration time in the low-concentration sucrose solution is 10 ± 2 min, preferably 10 min.

[0017] The culture water used in this invention is the culture water used for culturing Culter alburnus, which has been filtered through a 0.2µm filter.

[0018] This invention provides a Culter alburnus embryo, which is preserved and thawed using the vitrification freezing and laser thawing method described above.

[0019] The above-described Culter alburnus embryo provided by the present invention can be used to cultivate Culter alburnus.

[0020] The present invention also provides a Culter alburnus, which is cultivated from Culter alburnus embryos preserved and thawed by the vitrification freezing and laser thawing method described above.

[0021] This invention describes vitrification cryopreservation of Culter alburnus embryos in the tail bud stage, and compares the hatching rate and malformation rate of embryos thawed using the laser heating method employed in this invention and the water bath heating method commonly used in traditional studies. The results show that the hatching rate of Culter alburnus embryos obtained using the method described in this invention reaches (18.83±0.57)%.

[0022] This invention marks the first successful vitrification cryopreservation of Culter alburnus embryos.

[0023] The operation process of this invention can be carried out in aquaculture sites, which is convenient for production practice.

[0024] The thawed Culter alburnus embryos obtained by this invention can successfully hatch into juvenile fish, and the preservation quality is high. Attached Figure Description

[0025] Figure 1 Screening of cryoprotectants for Culter alburnus embryos. (a) The toxicity of six commonly used organic solvents to Culter alburnus embryos was tested; (b) The heating rate of four selected organic solvents in GNRs-assisted laser heating was tested.

[0026] Figure 2 The temperature rise curves of V1-V12 antifreeze under laser heating.

[0027] Figure 3 The laser heating conditions were screened. (a) Laser intensity screening: laser intensities of 5W, 10W, 15W, 20W, and 25W were used for heating, and the heating curves were detected. (b) GNRs concentration screening: GNRs concentrations of 10μg / mL, 25μg / mL, and 50μg / mL were used for laser heating, and the heating curves were detected.

[0028] Figure 4 A schematic diagram showing the screening results of thawing methods for vitrification cryopreservation of Culter alburnus embryos. Among them, (a) detection of hatching rate of Culter alburnus embryos after water bath heating and laser heating; (b) morphological observation of Culter alburnus embryos after water bath heating and laser heating. Detailed Implementation

[0029] The room temperature mentioned in the following experiments of this invention is controlled at 20℃-25℃.

[0030] Example 1: Investigation of heating rate under organic solvents and laser heating for cryopreservation (1) Select healthy male and female Culter alburnus, inject oxytocin, wait 8 hours to collect the embryos, and culture the embryos in the incubator until the tail bud stage; (2) Take DMSO, MeOH, EG, PG, DMF and Gly solutions with a volume ratio of 5%, 10% and 15% respectively and mix them with the remaining volume ratio of the culture water of Culter alburnus (after filtration) to prepare toxicity test solutions; use a three-step method to transfer the embryos to the toxicity test solutions: first, transfer the embryos to the 5% concentration of each toxicity test solution and equilibrate for 10 min; then transfer them to the 10% concentration of each toxicity test solution and equilibrate for 10 min; finally transfer them to the 15% concentration of each toxicity test solution and let them stand for 15 min; use the three-step method again to transfer the embryos from the toxicity test solutions to the culture water: first, transfer the embryos to a 0.5M sucrose solution preheated to 30℃ and equilibrate for 10 min; then transfer them to a 0.25M sucrose solution preheated to 30℃ and equilibrate for 10 min; finally transfer them to the culture water to continue culturing; continue culturing until hatching and count the hatching rate.

[0031] The results showed that DMSO (dimethyl sulfoxide), EG (ethylene glycol), and PG (propylene glycol) had very low toxicity to Culter alburnus embryos, with no significant difference in survival rate compared to the control group. The survival rate in the MeOH group was slightly lower, but still at a high level, and its toxicity was relatively low. In contrast, DMF (N,N-dimethylformamide) and Gly (glycerol) exhibited extremely high toxicity, with almost all embryos dying. Figure 1 (a)

[0032] (3) We selected DMSO, MeOH, EG and PG for subsequent research and tested their heating rates under GNRs-assisted laser heating. We took 50% by volume of DMSO, MeOH, EG and PG respectively, mixed them with the remaining volume of water, and added GNRs (25 μg / mL) as the vitrification detection solution; we took 30 μL of the above vitrification detection solution, added it to a wheat tube, and inserted the temperature sensor probe into the center of the liquid and fixed it with tape.

[0033] The results showed that the addition of organic solvents significantly increased the heating rate under laser heating, and the difference in heating rate was not significant. Therefore, both can be used for the formulation of subsequent composite antifreeze agents. Figure 1 (b)

[0034] Example 2: Screening of Composite Antifreeze (1) Select healthy male and female Culter alburnus, inject oxytocin, wait 8 hours to collect the embryos, and culture the embryos in the incubator until the tail bud stage; (2) Prepare V1-12 antifreeze according to the formula in Table 1. Load each of the 12 antifreezes into a 250μL straw, fill it completely, and then seal it with a straw stopper. Quickly insert the straw containing the antifreeze into liquid nitrogen and let it stand for 2 minutes to freeze it completely. (3) Take out the straw, let it stand at room temperature for 10 seconds, and then quickly insert it into a 30°C water bath to thaw; observe whether ice forms in the straw during the whole process to determine its vitrification properties.

[0035] The results showed that although most antifreeze formulations could reach a glassy state in liquid nitrogen, recrystallization occurred during thawing. Among them, V11 exhibited relatively excellent glassy properties, successfully achieving a crystallization-free freeze-thaw process in all three replicates (Table 2).

[0036] Subsequently, we tested the heating rate of these 12 antifreeze agents under GNRs-assisted laser heating. We took 30 μL of the above vitrification test solution, added it to a straw, and inserted the temperature sensor probe into the center of the liquid, securing it with tape. We then inserted the straw into liquid nitrogen. After the temperature sensor showed a temperature of -190°C, we removed it and slowly heated it to -120°C at room temperature. Finally, we quickly placed it 10 cm below a 15W laser with a wavelength of 1064 nm and recorded the heating rate. The results showed that V11 also exhibited the highest heating rate, consistent with the vitrification test results. Figure 2 ).

[0037] Table 1. Formulation of embryo vitrification antifreeze (the remaining volume of solvent is filtered water used for Culter alburnus culture). The volume percentages of DMSO, MeOH, EG, and PG in Table 1 represent the volume proportions of each organic solvent in all solvents added to the preparation of the embryo vitrification antifreeze.

[0038] Table 212 Vitrification Performance Tests for Antifreeze Formulations Example 3: Investigation of Laser Power and GNRs Concentration We first screened the laser power using a vitrification solution containing 50% DMSO and 25 μg / mL GNRs. We prepared a 25 μg / mL GNRs detection solution using a 50% (volume) DMSO solution (with the remaining 50% water) as the base antifreeze. 30 μL of the detection solution was added to a straw, and the temperature sensor probe was inserted into the center of the liquid and secured with tape. The straw was then immersed in liquid nitrogen. After the temperature sensor reached -190°C, it was removed and slowly heated to -120°C at room temperature. The straw was then rapidly placed 10 cm below lasers of 5W, 10W, 15W, 20W, and 25W power at a wavelength of 1064 nm, and the heating rate was recorded to screen for the optimal laser power.

[0039] The results showed that the heating rate increased with increasing laser intensity. However, when the laser power reached 15W, further increasing the power did not significantly increase the heating rate of the solution. Therefore, 15W was chosen as the power for subsequent studies. Figure 3 (a)

[0040] Next, we conducted laser heating and temperature rise rate tests on antifreeze containing different GNR concentrations at a power of 15W. First, using a 50% (v / v) DMSO solution as the base antifreeze, we prepared laser detection solutions with GNR concentrations of 10 μg / mL, 25 μg / mL, and 50 μg / mL. We then pipetted 30 μL of each laser detection solution containing different GNR concentrations into a straw, inserted a temperature sensor probe into the center of the liquid, and secured it with tape. The straw was then immersed in liquid nitrogen. After the temperature sensor showed a temperature of -190°C, it was removed and slowly heated to -120°C at room temperature. It was then quickly placed 10 cm below a 15W laser with a wavelength of 1064 nm, and the temperature rise rate was recorded to screen for the optimal GNR concentration.

[0041] The results showed that adding GNRs significantly improved the laser heating effect. The heating rate reached its maximum after the GNRs concentration was increased to 25 μg / mL. Therefore, 25 μg / mL was selected as the GNRs concentration for subsequent experiments. Figure 3 (b)

[0042] Example 4: Comparative Study of the Freezing and Thawing Scheme of the Present Invention with Traditional Schemes The process of using this invention for cryopreservation and thawing of Culter alburnus embryos is as follows: (1) Select healthy male and female Culter alburnus, inject oxytocin, wait 8 hours to collect the embryos, and culture the embryos in the incubator until the tail bud stage; (2) Prepare a basic antifreeze according to the volume ratio of 15% dimethyl sulfoxide (DMSO), 10% methanol (MeOH), 15% propylene glycol (PG), the remainder being filtered Culter alburnus culture water and 1 mM melatonin. Add gold nanorods (GNRs) at a final concentration of 25 μg / mL as the antifreeze (CPA) for this experiment. Dilute to one-quarter concentration as equilibration solution 1 (ES1) and half concentration as equilibration solution 2 (ES2). Use filtered Culter alburnus culture water as the diluent. (3) The embryos were transferred to the cryoprotectant using a three-step method: first, the embryos were transferred to ES1 and equilibrated for 10 minutes; then transferred to ES2 and equilibrated for 10 minutes; finally, they were transferred to the cryoprotectant, left to stand for 15 minutes, and divided into two groups. (4) Use a freezing loop to pick up a single Culter alburnus embryo, place it on a metal block immersed in liquid nitrogen to cool it down for 10 seconds to achieve vitrification, and then insert it into liquid nitrogen for short-term preservation (1 min). (5) One group used the three-step method to transfer the embryos from liquid nitrogen to culture water again: first, the embryos were taken out of liquid nitrogen, left to stand at room temperature for 10 seconds, and then transferred to a 0.2 g / mL sucrose solution preheated to 30°C and equilibrated for 10 min; then transferred to a 0.1 g / mL sucrose solution preheated to 30°C and equilibrated for 10 min; finally, transferred to culture water for continued culture, which was the water bath thawing group; Another group used laser heating to thaw the embryos: the embryos were first removed from liquid nitrogen, left to stand at room temperature for 10 seconds, and then placed 10 cm below a 15W laser with a wavelength of 1064nm. After 10 seconds of laser irradiation, the embryos were observed to thaw. Then, the embryos were transferred to culture water using the same three-step method as in Example 1 and continued to be cultured. This group was the laser thawing group.

[0043] (6) Continue culturing until hatching, and count the hatching rate.

[0044] The results showed that the hatching rate of frozen Culter alburnus embryos obtained by this method reached (18.83±0.57)%, which was significantly higher than that obtained by the traditional method. Figure 4 (a) Traditional methods generally only yield temporarily surviving embryos after thawing, with few cases of hatching into juvenile fish and an extremely low hatching rate. Morphological observation of thawed embryos revealed that only 27.8% of embryos thawed by laser showed deformities, while 57.9% of embryos thawed by water bath showed deformities. The hatching rate of the present invention is significantly higher than that of traditional methods, and the thawed embryos of the present invention show intact body segments without obvious deformities.

[0045] Note: The above examples are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention; and all technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for vitrification freezing and laser thawing of Culter alburnus embryos, characterized in that the steps include: include: Vitrification: First, transfer the embryos to equilibration solution 1, then to equilibration solution 2, and finally to cryoprotectant for static freezing; collect the embryos from the cryoprotectant, place them on an aluminum metal plate immersed in liquid nitrogen for vitrification, and preserve them in liquid nitrogen; The antifreeze is a solvent containing melatonin and gold nanorods, and the solvent is prepared by the following solution in volume percentage: 13%-18% dimethyl sulfoxide, 8%-12% methanol, 13%-18% propylene glycol, and the balance being the water used for raising Culter alburnus; the concentration of melatonin is 0.1-3 mM, and the concentration of gold nanorods is 10-50 μg / mL; The balancing solution 1 was prepared by diluting the antifreeze four times, and the diluent used for dilution was the culture water of Culter alburnus. The balancing solution 2 was prepared by diluting the antifreeze by two times, and the diluent used for dilution was the culture water of Culter alburnus. Laser thawing: The embryos stored in liquid nitrogen are taken out, left to stand at room temperature, and then thawed under the action of near-infrared laser. They are then placed in high-concentration and low-concentration sucrose solutions for equilibration, and finally transferred to the culture water for Culter alburnus. The sucrose concentration in a high-concentration sucrose solution is 0.2-1 mol / L, while the sucrose concentration in a low-concentration sucrose solution is 0.1-0.5 mol / L.

2. The vitrification freezing and laser thawing method according to claim 1, characterized in that, The antifreeze is a solvent containing melatonin and gold nanorods, and the solvent is prepared by the following solutions in volume percentage: 14%-16% dimethyl sulfoxide (DMSO), 9%-11% methanol (MeOH), 14%-16% propylene glycol (PG), and the balance being the water used for raising Culter alburnus; the concentration of melatonin is 0.5-1 mM, and the concentration of gold nanorods is 15-40 μg / mL.

3. The vitrification freezing and laser thawing method according to claim 1, characterized in that, During vitrification freezing, the equilibration time in equilibration solution 1 is 10±2 min, the equilibration time in equilibration solution 2 is 10±2 min, and the settling time in the antifreeze is 15±2 min.

4. The vitrification freezing and laser thawing method according to claim 1, characterized in that, During laser thawing, the wavelength of the near-infrared laser is 780nm~2526nm, the power of the near-infrared laser is 5-25W, and the irradiation time of the near-infrared laser is 6-15s.

5. The vitrification freezing and laser thawing method according to claim 1, characterized in that, During laser thawing, the settling time at room temperature was 10±2 s, the equilibration time in high-concentration sucrose solution was 10±2 min, and the equilibration time in low-concentration sucrose solution was 10±2 min.

6. The vitrification freezing and laser thawing method according to claim 1, characterized in that, In laser thawing, the sucrose concentration in the high-concentration sucrose solution is 0.4-0.6 mol / L, and the sucrose concentration in the low-concentration sucrose solution is 0.2-0.3 mol / L.

7. The vitrification freezing and laser thawing method according to claim 1, characterized in that, In laser thawing, both high-concentration and low-concentration sucrose solutions are preheated.

8. A Culter alburnus embryo, characterized in that, Preservation and revival by the vitrification freezing and laser thawing method according to any one of claims 1-7.

9. The application of the Culter alburnus embryo as described in claim 8 in the cultivation of Culter alburnus.

10. A type of Culter alburnus, characterized in that, It is cultivated from the Culter alburnus embryo described in claim 8.

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