Cryopreservation liquid for lateolabrax japonicus testis and ultralow-temperature cryopreservation method for lateolabrax japonicus testis
By using a specifically composed cryopreservative solution and ultra-low temperature freezing method, the problem of testis preservation in the marine fish Lateolabrax japonicus was solved, germ cell transplantation with a high survival rate was achieved, and a new way for long-term preservation and rapid reproduction of germplasm resources was provided.
Patent Information
- Application Number
- CN202510782791.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-12
- Publication Date
- 2025-10-03
AI Technical Summary
Existing technologies make it difficult to effectively preserve the reproductive cells, especially the testes, of the marine fish Lateolabrax japonicus, leading to degradation of germplasm resources and reproduction difficulties.
Ultra-low temperature cryopreservation of Lateolabrax japonicus testis was achieved by using a cryopreservation solution consisting of MEM culture medium, 35.2% buffer, 1.3M dimethyl sulfoxide, 0.1M trehalose and 10% fetal bovine serum, combined with -80℃ freezing and liquid nitrogen storage methods, as well as testis resuscitation solution and centrifugation steps.
The long-term preservation of the testes of the striped sea bass was achieved. The revived spermatogonia had a high survival rate and possessed the characteristics of reproductive stem cells. They were successfully transplanted into recipient fish, significantly broadening the application scope of germ cell transplantation.
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Figure CN120731938A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the cryopreservation of reproductive cells, in particular to a cryopreservation solution for testis of Lateolabrax japonicus and a method for ultra-low temperature cryopreservation of testis of Lateolabrax japonicus. Background Art
[0002] With the degradation of germplasm resources and the reduction of species diversity in recent years, the long-term protection of germplasm resources is particularly important. Cryopreservation provides an effective alternative for protecting endangered, precious species and high-quality germplasm resources. Cryopreservation is currently the most direct way to preserve fish resources. However, only the cryopreservation of sperm is relatively mature. Eggs or embryos have not yet been successfully preserved due to their large size and high yolk content, and maternal genetic material cannot be inherited. In recent years, primordial germ cells, spermatogonia, and oval cells have been used as alternative germplasm, and cryopreservation methods have been established in fish. Combined with fish germ cell transplantation technology, they show broad space in species protection and proxy breeding. Due to the large size, long maturity period, complex reproduction regulation, and high requirements for culture conditions of marine economic fish, most of the current research on the development and transplantation of fish germ cells is focused on model and freshwater fish, with less involvement of marine fish.
[0003] Previous research has shown that spermatogonia obtained from cryopreserved whole testes can produce functional gametes. Lateolabrax maculatus, a major commercial fish species for marine aquaculture in my country, is a highly nutritious meat and has high economic value. In the past decade, Lateolabrax has become one of my country's most important marine aquaculture fish and a preferred species for deep-sea aquaculture in modern marine ranches. However, due to its long maturation period and large size, hybridization between different Lateolabrax species has led to varietal confusion and degradation of germplasm resources. The need for new breeding models and the preservation of high-quality Lateolabrax germplasm resources is becoming increasingly prominent. The emergence of "surrogacy" technology in fish, combined with germ cell cryopreservation, has opened up new avenues for the preservation and efficient development and utilization of germplasm resources, while also providing a novel breeding model for fish surrogate aquaculture. Therefore, isolating and preserving high-quality Lateolabrax germplasm resources is of great significance, as is providing a new method for gamete preservation and rapid propagation for fish with long reproductive cycles. Summary of the Invention
[0004] One of the purposes of the present invention is to provide a cryopreservation solution for testis of Lateolabrax japonicus.
[0005] The present invention achieves the purpose through the following technical scheme: the sea bass testis cryopreservation solution comprises: MEM culture medium, 35.2% buffer (volume fraction), 1.3-1.6M dimethyl sulfoxide, 0.1-0.3M trehalose and 10% fetal bovine serum (volume fraction), and the concentrations are all the same as those in the MEM culture medium.
[0006] Preferably, the Lateolabrax japonicus testis cryopreservation solution comprises: MEM culture medium, 35.2% buffer (volume fraction), 1.3M dimethyl sulfoxide, 0.1M trehalose and 10% fetal bovine serum (volume fraction), and the concentrations are all the same as those in MEM culture medium.
[0007] The 100% formulation of the buffer solution is: 55.27 mM Hepes, 375.48 mM NaCl, 7.28 mM KCl, 23.10 mM KH2PO4, 3.82 mM Na2HPO4, 3.64 mM sodium pyruvate, 2.6 mM CaCl2·2H2O, and 1.4 mM MgCl2·6H2O. Adjust the pH to 7.8, dissolve in ultrapure water, sterilize with a 0.22 μm filter membrane, and store at 4°C.
[0008] Another object of the present invention is to provide a method for ultra-low temperature freezing and preservation of Lateolabrax japonicus testes.
[0009] The present invention achieves the purpose through the following technical scheme: a method for ultra-low temperature cryopreservation of Lateolabrax japonicus testis, wherein the Lateolabrax japonicus testis is added into a cryopreservation solution, then frozen at -80 DEG C, and then transferred to liquid nitrogen for cryopreservation.
[0010] The ratio of testis to cryopreservation solution is 1g:10-20mL.
[0011] Freeze at -80°C for no more than 90 minutes.
[0012] The method of the present invention further comprises a resuscitation step: after the frozen testis is thawed, it is placed in a testis resuscitation solution for rehydration, centrifuged, further washed with the testis resuscitation solution, and finally placed on ice for resuscitation.
[0013] The spermatozoa resuscitation solution uses MEM medium as the base solution, supplemented with 5% fetal bovine serum (FBS), 25 mM Hepes and 2 mM L-glutamine, and sterilized by filtration through a 0.22 μm filter membrane.
[0014] The recovery time on ice should not exceed 15 minutes.
[0015] Beneficial effects
[0016] 1. The present invention provides a cryopreservation solution for the testes of Lateolabrax japonicus, which can achieve long-term preservation of the testes of Lateolabrax japonicus. The resuscitated cells have the characteristics of reproductive stem cells, thereby achieving long-term preservation of germplasm resources.
[0017] 2. Using the testis cryopreservation solution provided by the present invention, the testis of the sea bass is ultra-low temperature frozen and preserved. The spermatogonia prepared after revival are observed under a microscope. It can be seen that the spermatogonia after cryopreservation are still alive, with good cell viability. The spermatogonia survival rate is above 50%, and there is no difference in cell activity and integrity compared with the whole tissue preparation.
[0018] The inventors transplanted germ cells frozen for one year into recipient fish (medaka and zebrafish) five days after hatching. Microscopic observation 60 days later revealed that the Lateolabrax japonicus germ cells had successfully migrated and integrated into the recipient fish's gonads, demonstrating that germ cells extracted from frozen testes can be used for cell transplantation. This study further demonstrated that germ cells prepared from Lateolabrax japonicus testes after long-term cryopreservation retained the properties of germline stem cells, exhibited no decrease in transplantability during the one-year cryopreservation period, and demonstrated significant colonization.
[0019] 3. The present invention establishes a simple and easy-to-operate method for long-term cryopreservation and on-demand thawing of testes, providing a reliable donor guarantee for germ cell transplantation, thereby significantly broadening the application scope of fish germ cell transplantation and being suitable for breeding test bases.
[0020] Due to the long maturity period and large size of the striped seabass, it is not convenient to transport it to the laboratory for breeding and research. At the same time, it is usually necessary to go to the breeding base for sampling, which results in a large consumption of time and labor costs. In order to effectively save time and reduce costs, the present invention adopts the technology of freezing and preserving the testes of the striped seabass. This method is simple to operate and has high practicality and operability. By freezing the testes, the spermatogonial stem cells of the striped seabass can be preserved for a long time without the need for frequent fishing or transportation, and can be revived on demand when needed to carry out subsequent experiments. Compared with traditional live transportation or regular sampling, the cryopreservation of testes not only reduces dependence on the environment, but also maximizes research efficiency and the sustainability of experiments. In addition, the present invention can better maintain the cell activity of the testes during the preservation process, providing reliable guarantees for subsequent cell transplantation, gene editing and other biotechnology experiments, and significantly enhancing the research and application potential of spermatogonial cells of the striped seabass. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 Comparison results of different osmotic agents in cryopreservation solution.
[0022] Figure 2 Comparison results of different concentrations of dimethyl sulfoxide in the cryopreservation solution.
[0023] Figure 3 Comparison of the results of adding different non-permeabilizing agents to the cryopreservative solution containing 1.3 M dimethyl sulfoxide.
[0024] Figure 4 Comparison results of adding fetal bovine serum, fresh egg yolk, bovine serum albumin, and skim milk powder to the cryopreservation solution containing 1.3 M dimethyl sulfoxide and 0.1 M trehalose.
[0025] Figure 5Chimeric images of PKH-26-labeled Lateolabrax japonicus germ cells were observed in the recipient 60 days after transplantation. DETAILED DESCRIPTION
[0026] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be fully described below in conjunction with the drawings in the embodiments of the present application, so as to fully understand the purpose, effect and application prospects of the present invention. The following examples are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention.
[0027] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.
[0028] 1. Reagents and Methods
[0029] 1. Reagents
[0030] MEM medium (51200038, Gbico), fetal bovine serum (10099141C, Gbico), Hepes buffer (15630080, Gbico), L-glutamine (25030081, Gbico), collagenase IV (C8160, Solebro), DNase I (11284932001, Roche), L-15 medium (11415064, Gbico), trypsin-EDTA (0.5%) (15400054, Gbico), Percoll (P1644, Sigma-Aldrich), 10xPBS (P1022, Solebol) 1xPBS (10010023, Gbico), PKH-26 (154214-55-8, MCE), 0.4% trypan blue solution (T8154, Sigma-Aldrich), MS-222 anesthetic (E10521, Sigma-Aldrich), penicillin-streptomycin (double antibody) (15140122, Gbico), dimethyl sulfoxide (0219605580, MP Biomedicals), trehalose (T100012, Aladdin), bovine serum albumin (A8010, Solebol), and skim milk powder (D8340, Solebol).
[0031] In this experiment, Lateolabrax japonicus was purchased from China Resources Supermarket in Guangzhou, Guangdong Province.
[0032] Sperm washing solution: Use MEM medium as the base solution, add 5% FBS, 25 mM Hepes and 2 mM L-glutamine, filter through a 0.22 μm filter membrane for sterilization, and store at 4°C.
[0033] Sperm cryopreservation solution: MEM medium as the base solution, supplemented with 35.2% buffer, 10% FBS, 1.3 M dimethyl sulfoxide, and 0.1 M trehalose. The 100% buffer solution consists of 55.27 mM Hepes, 375.48 mM NaCl, 7.28 mM KCl, 23.10 mM KH2PO4, 3.82 mM Na2HPO4, 3.64 mM sodium pyruvate, 2.6 mM CaCl2·2H2O, and 1.4 mM MgCl2·6H2O. Adjust the pH to 7.8, dissolve in ultrapure water, sterilize with a 0.22 μm filter, and store at 4°C.
[0034] Sperm recovery solution: MEM medium was used as the base solution, supplemented with 5% FBS, 25 mM Hepes, and 2 mM L-glutamine, and sterilized by filtration through a 0.22 μm filter membrane.
[0035] Sperm digestion solution: Use L-15 culture medium as the base, add 2 mg / mL collagenase IV, 0.1% DNase I and 5% FBS, filter and sterilize with a 0.22 μm filter membrane, and add 0.5% trypsin-EDTA to a final concentration of 0.25% to continue digestion.
[0036] Percoll gradient solution: 100% Percoll was prepared by 10xPBS:Percoll=1:9, and diluted with 1xPBS to form 10% Percoll solution, 30% Percoll solution and 40% Percoll solution.
[0037] 2. Sperm cryopreservation method:
[0038] 1) The testes of 6-month-old Lateolabrax japonicus (500-600 g) (gonadal index: 0.042 ± 0.013%) were removed. Excess fat and blood were removed from the testes using scissors and sharp forceps in pre-chilled PBS. The cleaned testes were disinfected by soaking in 75% alcohol for 10 seconds and then washed five times with pre-chilled PBS containing 1% penicillin-streptomycin (antibody).
[0039] 2) In a clean bench, transfer the washed testis to pre-cooled testis washing solution and cut the testis into 1mm pieces with scissors. 3 Wash the tissue blocks with 3 volumes of testis washing solution and centrifuge at 1500 rpm for 5 minutes at 4°C. Repeat twice. Take 3 fresh gonads from each experimental group as control.
[0040] 3) Add cryopreservation solution to the testis, with the ratio of testis to cryopreservation solution being 1 g:10-20 mL.
[0041] 4) Mix well and place in a 2 mL cryovial. Place the cryovial and programmed cooling box on ice for 1 hour. Transfer the cryovial to a programmed cooling box and freeze at -80°C for 90 minutes. Transfer the cryovial to liquid nitrogen and freeze for 2-3 days before thawing. Wash the fresh testis and cut into 1 mm pieces. 3 Wash the tissue blocks with 3 volumes of spermatozoa washing solution, centrifuge at 1500 rpm for 5 minutes at 4 °C, and repeat twice.
[0042] 3. Resuscitation methods:
[0043] 1) Remove the cryovial from liquid nitrogen and place on ice. Thaw the cryovial in a 10°C water bath for 1 minute. Gently remove the freezing medium adhering to the testis sample using a tissue. Transfer the testis to a new centrifuge tube in a clean hood and rehydrate in sperm resuscitation solution. Centrifuge at 1500 rpm for 5 minutes at 4°C. Wash three more times with sperm resuscitation solution and place on ice (recovery time should not exceed 15 minutes).
[0044] 2) The washed, centrifuged tissue pellet and fresh testis pellet were added to the testis digestion solution and digested in a shaker at 37°C (80 rpm) for 1 hour. To prevent cell clumping, trypsin-EDTA was added to the original testis digestion solution to a final concentration of 0.25%. Digestion was continued in a shaker for 1.5 hours. Finally, L-15 medium containing 5% FBS was added to terminate the digestion. After completion, the cells were sieved through 200-mesh and 70-mesh sieves, respectively. The washed fresh testes were directly digested in the testis digestion solution and sieved.
[0045] 3) After harvesting the cells, centrifuge at 1500 rpm for 5 minutes at room temperature, then resuspend and wash twice in L-15 medium. After centrifugation, resuspend the cells in L-15 medium to obtain a single-cell suspension of testes. Place on ice until ready to use.
[0046] 4. Purification of spermatogonial stem cells:
[0047] Percoll gradient purification of spermatogonial stem cells:
[0048] 1) Prepare Percoll stock solution in 10xPBS at a ratio of 10xPBS:Percoll = 1:9 to make 100% Percoll. Dilute the desired gradient Percoll solution in 1xPBS.
[0049] 2) Prepare 10%, 30%, and 40% Percoll solutions, adding a small amount of trypan blue solution (final concentration 0.01%) to the 10% and 40% Percoll solutions as an indicator;
[0050] 3) Layer the different Percoll solutions in descending order of density. Add 2 mL of 40%, 30%, and 10% Percoll solutions to a 15 mL centrifuge tube to prepare the Percoll gradient centrifugation working solution.
[0051] 4) Carefully add 2 mL of the prepared testis single-cell suspension to the top layer of 6 mL of Percoll gradient centrifugation working solution. Centrifuge at 2000 rpm (acceleration 2, deceleration 4) in a horizontal centrifuge (3-18KS, Sigma) for 30 minutes at room temperature. After centrifugation, remove the suspension slowly and steadily.
[0052] 5) Slowly aspirate and collect cells at the junction of 30% and 10% Percoll solutions, dilute the collected cells with 10 volumes of PBS solution, centrifuge at 1500 rpm for 5 minutes at room temperature, discard the supernatant, and resuspend the spermatogonial stem cells with PBS.
[0053] 5. Spermatogonial stem cell transplantation
[0054] 1) The spermatogonial stem cells collected at the junction of 10% and 30% Percoll solutions were precipitated to obtain a cell concentration of 10 7 Spermatogonial stem cells at a concentration of 5 cells / mL were washed twice with PBS, centrifuged at 1500 rpm at room temperature for 5 minutes, and stained with PKH-26 at a final concentration of 0.5 μM. After incubation at room temperature for 15 minutes, the cells were centrifuged and washed twice with PBS. Finally, the cells were resuspended in L-15 medium and placed on ice for cell transplantation experiments.
[0055] 2) Pre-transplantation preparation. Prepare a microinjection glass needle with a tip diameter of 40-50 μm. Connect the microinjection system to the dissecting microscope. Use a pipette tip to transfer the prepared cell suspension into the capillary needle, then mount it on the microinjector. Before injection, recipient fish (medaka and zebrafish) 5 days post-embryonic were anesthetized with 0.001% MS-222 in seawater and freshwater, respectively, and then transferred to a 2% agarose-coated culture dish for microinjection. A blank control group of juveniles was not transplanted and was maintained under the same conditions.
[0056] 3) Under a dissecting microscope, microinject the cells into the abdominal cavity of recipient larvae near the genital ridge using a microinjector, injecting approximately 100 spermatogonial stem cells per larvae. Once completed, return the larvae to the experimental tank for continued culture.
[0057] 4) 60 days after transplantation, observation under a Leica DMi8 fluorescence inverted microscope (Leica) revealed that the spermatogonial stem cells of the donor sea bass had successfully integrated into the recipient gonads, demonstrating that the spermatogonial stem cells possessed the characteristics of germline stem cells.
[0058] 2. Study on the composition of cryopreservation solution
[0059] (1) Screening of osmotic protective agents in the cryopreservation solution of Lateolabrax japonicus testes
[0060] 1. Prepare 5 mL of dimethyl sulfoxide, glycerol, and methanol at the same concentration (1.3 M) using MEM culture medium and 35.2% buffer to obtain a freezing solution.
[0061] 2. Dissect 6-month-old Lateolabrax japonicus, remove excess fat and blood from the testes using scissors and sharp forceps in pre-cooled PBS, weigh the testes, and take 3 g of testes; wash them 5 times with pre-cooled PBS.
[0062] 3. After washing, transfer the testis to pre-cooled testis washing solution and cut the testis into 1mm pieces with scissors. 3 Wash the tissue blocks with about 3 times the volume of testis washing solution, centrifuge at 1500 rpm and 4℃ for 5 minutes, repeat twice, and take 3 fresh gonads from each experimental group as control.
[0063] 4. Divide the testes into 10 equal portions. Cryopreservation of 9 portions in the same concentration of osmotic fluid is performed, and the remaining portion is used for cell preparation. Add 10 ml of cryopreservation fluid to each portion, mix thoroughly, and transfer to a cryovial.
[0064] 5. Place the cryovials and programmed cooling box on ice for 1 hour, transfer the cryovials to the programmed cooling box and freeze at -80°C for 90 minutes, transfer the cryovials to liquid nitrogen and store frozen for 3 days.
[0065] 6. Thawing: Remove the cryovial from liquid nitrogen and place on ice. Thaw the cryovial in a 10°C water bath for 1 minute. Gently remove the freezing medium adhering to the testis sample with a paper towel. Transfer the testis to a new centrifuge tube in a clean hood and rehydrate in testis thawing solution. Centrifuge at 1500 rpm for 5 minutes at 4°C. Rinse three more times with testis thawing solution and place on ice.
[0066] 7. Preparation of Sperm Cells: Add 10 mL of Sperm Digestion Solution to each testis sample and digest in a 15 mL centrifuge tube. Digest at 80 rpm at 37°C for 1 hour. To prevent cell clumping, add trypsin-EDTA to the original Sperm Digestion Solution to a final concentration of 0.25%. Continue digesting on a shaker for 1.5 hours. Finally, add L-15 medium supplemented with 5% FBS to terminate digestion. After completion, sieve the cells through 200-mesh and 70-mesh sieves, respectively.
[0067] 8. After harvesting the cells, centrifuge at 1500 rpm for 5 minutes at room temperature, then resuspend and wash twice in L-15 medium. After centrifugation, resuspend the cells in L-15 medium to obtain a testicular cell suspension. Count the total number of cells and determine the number of viable cells using a cell counter. Cell viability (%) = frozen tissue-isolated cells / freshly isolated cells.
[0068] The experimental results are shown in Figure 1 By comparing the cell number and activity, it was found that dimethyl sulfoxide was the best permeability protective solution at the same concentration.
[0069] (II) Screening of the concentration of dimethyl sulfoxide as the permeability protectant in the cryopreservation solution of Lateolabrax testis
[0070] 5 mL of dimethyl sulfoxide of different concentrations (1 M, 1.3 M and 1.6 M) were prepared based on MEM culture medium and 35.2% buffer to obtain freezing preservation solutions.
[0071] The subsequent operations are the same as (1).
[0072] The experimental results are shown in Figure 2 By comparing the cell number and activity, it was found that 1.3M dimethyl sulfoxide was the best component of the cryopreservative solution.
[0073] (III) Screening of non-permeable protective agents in the cryopreservation solution of Lateolabrax japonicus testes
[0074] Based on 1.3M dimethyl sulfoxide as a protective agent, we screened non-permeable protective agents (trehalose, glucose, and sucrose) and set up two concentrations (0.1M and 0.3M) for a total of 6 groups of experiments. 10mL of cryopreservative solution was prepared for each group.
[0075] The subsequent operations are the same as (1).
[0076] The experimental results are shown in Figure 3 By comparing the cell number and activity, it was found that 1.3M dimethyl sulfoxide and 0.1M trehalose were the best components of the cryopreservation solution.
[0077] (IV) Screening of fetal bovine serum, fresh egg yolk, bovine serum albumin and skimmed milk powder in the cryopreservation solution of Lateolabrax japonicus testis
[0078] Based on the 1.3M dimethyl sulfoxide and 0.1M trehalose components, four groups of experiments were further screened, including fetal bovine serum (10% by volume), fresh egg yolk (10% by volume), bovine serum albumin (1.5% by mass), and skim milk powder (1.5% by mass). 10 ml of freezing solution was prepared for each group.
[0079] The subsequent operations are the same as (1).
[0080] The experimental results are shown in Figure 4 By comparing the cell number and activity, it was found that MEM medium, 35.2% buffer, 1.3M dimethyl sulfoxide, 0.1M trehalose and 10% fetal bovine serum were the best components of the cryopreservation solution.
[0081] 3. Ultra-low temperature cryopreservation and recovery of Lateolabrax japonicus testes
[0082] Based on the results of the first point, a cryopreservation solution was prepared, including MEM culture medium, 35.2% buffer, 1.3M dimethyl sulfoxide, 0.1M trehalose and 10% fetal bovine serum. Six-month-old striped seabass were dissected, and germ cells prepared from the testes of the striped seabass that had been cryopreserved for one year were transplanted into recipient fish (medaka and zebrafish) 5 days after hatching. Germ cell transplantation prepared from fresh testicular tissue served as a positive control, and juvenile fish that were not transplanted served as a negative control. The methods for testicular cryopreservation, recovery, testicular stem cell purification and spermatogonial stem cell transplantation were the same as above. In the spermatogonial stem cell transplantation, the juvenile fish in the transplanted and non-transplanted groups were raised under the same conditions.
[0083] The experimental results are shown in Figure 5 PKH-26-stained spermatogonia were transplanted into recipient fish (medaka and zebrafish). PKH-26-labeled germ cells of Lateolabrax japonicus were observed in the recipients 60 days after transplantation. Embedded fluorescently stained germ cells were observed in the gonads of the fresh testes of the positive control group. Figure 5 BC), zebrafish ( Figure 5 F); in the testis group after cryopreservation, donor cells were also observed in the recipient fish, such as Lateolabrax japonicus, and Oystercatcher ( Figure 5 D), zebrafish ( Figure 5 G); while the negative control group showed no fluorescence at the same position, medaka ( Figure 5 A), zebrafish ( Figure 5 E).
[0084] The above embodiment merely represents one embodiment of the present invention. While the description is relatively specific and detailed, it should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make various modifications and improvements without departing from the scope of the present invention, and all such modifications and improvements fall within the scope of protection of the present invention.
Claims
1. A freezing solution for testis of Lateolabrax japonicus, characterized by: The components include MEM culture medium, 35.2% buffer, 1.3-1.6M dimethyl sulfoxide, 0.1-0.3M trehalose and 10% fetal bovine serum, and the concentrations are all the same as those in MEM culture medium.
2. The Lateolabrax testis cryopreservation solution according to claim 1, wherein The components include MEM medium, 35.2% buffer, 1.3 M dimethyl sulfoxide, 0.1 M trehalose and 10% fetal bovine serum, and the concentrations are all the same as those in MEM medium.
3. A method for ultra-low temperature cryopreservation of Lateolabrax testis, characterized in that: The testis of Lateolabrax japonicus is added into a freezing preservation solution, then frozen at -80°C, and then transferred to liquid nitrogen for freezing preservation; the components of the freezing preservation solution include MEM culture medium, 35.2% buffer, 1.3-1.6M dimethyl sulfoxide, 0.1-0.3M trehalose and 10% fetal bovine serum, and the concentrations are all the same as those in the MEM culture medium.
4. The method for ultra-low temperature freezing and preservation of Lateolabrax testis according to claim 3, wherein: The components of the freezing preservation solution include MEM culture medium, 35.2% buffer, 1.3M dimethyl sulfoxide, 0.1M trehalose and 10% fetal bovine serum, and the concentrations are all the same as those in the MEM culture medium.
5. The method for ultra-low temperature cryopreservation of Lateolabrax japonicus testis according to claim 3, wherein: The ratio of the testis to the freezing preservation solution is 1g:10-20mL.
6. The method for ultra-low temperature cryopreservation of Lateolabrax japonicus testis according to claim 3, wherein: The -80°C freezing time does not exceed 90 minutes.
7. The method for ultra-low temperature cryopreservation of Lateolabrax japonicus testis according to claim 3, wherein: The method also includes a recovery step in which the frozen testes are thawed, placed in a testes recovery solution for rehydration, centrifuged, further washed with the testes recovery solution, and finally placed on ice for recovery.
8. The method for ultra-low temperature cryopreservation of Lateolabrax japonicus testis according to claim 7, wherein: The testis resuscitation solution uses MEM culture medium as a base solution, supplemented with 5% FBS, 25 mM Hepes and 2 mM L-glutamine, and sterilized by filtration with a 0.22 μm filter membrane.
9. The method for ultra-low temperature cryopreservation of Lateolabrax japonicus testis according to claim 7, characterized in that: The time for resuscitation on ice does not exceed 15 minutes.