Ultralow-temperature cryopreservation agent for lateolabrax japonicus sperms and preservation method thereof
By using ultra-low-temperature cryopreservative composed of ethylene glycol and Hank’s solution, the complex and toxicity of the cryopreservative of the sperm in the flower bass was solved, which significantly improved the fertilization ability and reduced the freezing damage, and achieved efficient sperm preservation.
Patent Information
- Application Number
- CN202510601675.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-12
- Publication Date
- 2025-08-01
AI Technical Summary
In the existing cryopreservation scheme for the flower bass sperm, the cryopreservation agent has complex components and high cost. Some components are toxic to sperm, affecting the preservation effect and sperm mobility. During the cryopreservation of low temperature, the sperm is damaged by ultrastructure, affecting reproductive performance.
Ultra-low-temperature cryopreservative composed of ethylene glycol and Hank’s solution is used, with ethylene glycol as permeable antifreeze and Hank’s solution as diluent. It is simple to prepare, reduces toxic effects, and reduces freezing damage by inhibiting ice crystal damage and sperm movement.
The fertilization capacity of sperm in flower bass was significantly improved, from 64.1±0.7% to 83.4±3.3%, reducing freezing damage, maintaining sperm structure and function intact, and reducing costs.
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Figure CN120391420A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of fish genetic resource protection, and specifically relates to a cryopreservation agent for Japanese seabass sperm and a preservation method thereof. Background Art
[0002] Japanese seabass is an important marine economic fish in China. Its meat is delicate and delicious, it grows rapidly, has strong adaptability to temperature and salinity, and strong stress and disease resistance, and is suitable for aquaculture in seawater, brackish water and freshwater waters in the north and south of China. However, due to factors such as overfishing and environmental pollution over the years, the natural resources of Japanese seabass have declined, and the breeding varieties have also shown germplasm degradation due to inbreeding, resulting in that the reproductive performance and gamete quality cannot meet the high-yield requirements. Moreover, in production practice, there is a problem that the sexual maturity of female and male parent fish is not synchronized, which is not conducive to the stable supply of fry and seriously restricts the development of the aquaculture industry.
[0003] Cryopreservation technology is an effective means to solve the above problems; collecting the semen of sexually mature Japanese seabass for cryopreservation not only solves the problem of asynchronous sexual maturity of female and male parent fish, but also provides technical support for germplasm resource protection, distant hybridization and artificial breeding.
[0004] The cryopreservation agent is the key to determining the preservation effect; at present, mainly permeating cryoprotectants (such as methanol, glycerol, dimethyl sulfoxide, dimethylformamide, ethylene glycol, propylene glycol, etc.) or non-permeating cryoprotectants (such as trehalose, lactose, sucrose, glucose, hydroxyethyl starch, albumin, antifreeze protein, polyethylene glycol, polyvinylpyrrolidone, etc.) are mixed and prepared with salt solutions (such as NaCl, Na2CO3, NaH2PO4, Na2HPO4, MgCl2, MgSO4, KCl, CaCl2, etc.) and nutrient agents (such as amino acids, vitamins, etc.). However, the components of the cryopreservation agent used in the existing cryopreservation scheme for Japanese seabass sperm are relatively complex, which not only makes the preparation difficult and costly, but also some components may have certain toxicity to sperm, and reactions may occur between the components, which instead affects the preservation effect. The cryoprotectant used in the existing cryopreservation scheme for Japanese seabass sperm is dimethyl sulfoxide, which is the most commonly used in the field of fish sperm cryopreservation. Although the effect is good, it is easily poisoned when the concentration is too high, which affects the development of offspring. Therefore, dimethyl sulfoxide should be used at a lower concentration when the effect is good.
[0005] In addition, although cryopreservation technology has achieved certain success, sperm will inevitably be damaged during cryopreservation, affecting the normal function and reproductive performance of sperm. So far, many studies have found that cryopreservation not only causes ultrastructural damage to sperm, but also affects sperm motility, production of reactive oxygen species, mitochondrial membrane potential, enzyme activity, and DNA integrity. Currently, in the research on cryopreservation agents for sperm, those skilled in the art usually regard sperm motility as the main parameter for measuring the quality of cryopreserved sperm. As mentioned above, cryopreservation also causes ultrastructural damage, that is, high activity does not mean that the fertilization situation and development status of the offspring will also be good. It has been reported that the fertilization ability of Japanese sea bass sperm after cryopreservation is 64.1 ± 0.7% (see Ji XS et al., Cryopreservation of sea perch (Lateolabrax japonicus) spermatozoa and feasibility for production-scale fertilization. Aquaculture 2004, 241(1-4): 517-52); therefore, research on sperm cryoinjury is very necessary. Therefore, by studying sperm cryoinjury, it is beneficial to obtain higher-quality sperm, promote the preservation of Japanese sea bass germplasm resources, the development of excellent varieties, and large-scale production practice. Summary of the Invention
[0006] One of the purposes of the present invention is to provide a cryopreservation agent for Japanese sea bass sperm, which has simple composition and good effect, and significantly reduces the cryoinjury of Japanese sea bass sperm.
[0007] Another purpose of the present invention is to provide a cryopreservation method for Japanese sea bass sperm.
[0008] To achieve the above purposes, the present invention adopts the following technical solutions.
[0009] A cryopreservation agent for Japanese sea bass sperm, characterized in that it is composed of ethylene glycol and Hank's solution; the volume ratio of ethylene glycol to Hank's solution is 1:10.
[0010] The pH value of the cryopreservation agent is 7.8, and the osmotic pressure is 334 mOsmol / Kg.
[0011] A cryopreservation method for Japanese sea bass sperm, characterized in that it includes the following steps:
[0012] Mix the semen with Hank's solution at a volume ratio of 1:1 - 3, then add ethylene glycol at 10% of the volume of Hank's solution, and mix well to obtain the cryopreservation sample; place the sample at 4°C for 10 minutes of equilibration, then load it into a straw, horizontally place it at a height of 5 - 7 cm from the liquid nitrogen surface and stay for 10 minutes for cooling; then put the sample into liquid nitrogen for storage.
[0013] When thawing the said sample, take the straw out of the liquid nitrogen, immediately thaw it in a water bath at 40°C for 6 seconds, then dry the straw and cut it open with scissors to let the sample flow into a centrifuge tube; store the thawed sample at 4°C.
[0014] Compared with the prior art, the present invention has the following beneficial effects:
[0015] The present invention simplifies the formula of the cryopreservation agent, only uses one kind of permeating cryoprotectant ethylene glycol and one kind of mixed salt solution, without using any nutrient agent. Therefore, not only is the preparation simple and the cost reduced to 20% of the commonly used commercially available cryopreservation agent, but also the adverse effects of the toxicity of the cryopreservation agent itself on sperm are reduced, and a better preservation effect is obtained. Moreover, the ultra-low temperature cryopreservation agent of the present invention uses ethylene glycol as the permeating cryoprotectant, which can enter the sperm to lower the freezing point of the solution and reduce ice crystal damage; using Hank's solution as the diluent, on the one hand, it is close to the osmotic pressure of seminal plasma to inhibit sperm movement and conserve energy; on the other hand, Hank's solution contains glucose as a substrate, which can interact with the phospholipid bilayer to reduce ice crystal damage. The combination of the two can significantly reduce cryodamage. It has been experimentally confirmed that using the ultra-low temperature cryopreservation agent of the present invention can greatly improve the fertilization ability of frozen sperm, which is increased from 64.1 ± 0.7% reported previously to 83.4 ± 3.3%. Description of the Drawings
[0016] Figure 1 It is a comparison chart of the preservation effects of different diluents, permeating cryoprotectants and concentrations in Examples 1 - 2;
[0017] Figure 2 It is a comparison chart of the preservation effects of different cryogenic conditions in Examples 3 - 6;
[0018] Figure 3 It is a scanning electron micrograph of fresh sperm and sperm after cryopreservation and thawing;
[0019] Figure 4 It is a transmission electron micrograph of fresh sperm and sperm after cryopreservation and thawing;
[0020] Figure 5 It is a comparison chart of the ROS content, the percentage of high mitochondrial membrane potential and the SOD content of fresh sperm and frozen sperm after cryopreservation;
[0021] Figure 6 γ-H2AX immunostaining fluorescence images of fresh sperm and thawed cryopreserved sperm.
[0022] Figure 7 Comparison chart of fertilization rates of Japanese seabass sperm cryopreserved with the cryopreservative of the present invention and fresh sperm. Detailed implementation manners
[0023] The present invention will be further described in detail through specific implementation cases below, so that those skilled in the art can better understand and implement the technical solutions of the present invention. All reagents used in the experimental examples of the present invention below are commercially available products.
[0024] Experimental Example 1: Comparative experiment of different diluents
[0025] Ethylene glycol was selected as the permeating cryoprotectant and 9 diluents with different compositions were used to prepare 9 cryopreservatives for ultra-low temperature storage. The compositions of the diluents are shown in Table 1.
[0026] The steps for ultra-low temperature cryopreservation were as follows: Japanese seabass semen was mixed with the diluent at a volume ratio of 1:3, and then ethylene glycol was added at 10% of the volume of the diluent and mixed evenly; after equilibration at 4°C for 10 min, the sample was loaded into a 0.25 ml straw, horizontally placed on a floating board 5 cm above the liquid nitrogen surface and left to cool for 10 min; then, it was stored in liquid nitrogen for 48 hours. During thawing, the straw was taken out of liquid nitrogen and immediately thawed in a water bath at 40°C for 6 s.
[0027] Based on the sperm motility after thawing, the results showed that when 10% EG was used as the cryoprotectant and Hank's solution or Ringer solution was used as the diluent, the sperm motility after thawing was 71.1±7.6% and 68.7±2.8% respectively, and the difference between the two was not significant (see Figure 1 .A).
[0028] Table 1
[0029]
[0030] Experimental Example 2: Comparative experiment of different permeating cryoprotectants and optimal concentrations
[0031] (1) Comparative experiment of different permeating cryoprotectants
[0032] In this experiment, 7 permeating cryoprotectants - dimethyl sulfoxide, glycerol, ethylene glycol, dimethylacetamide, dimethylformamide, glycerol, and a combination of 5% dimethyl sulfoxide and 5% ethylene glycol were selected. Then, Hank's or Ringer solution was used as the diluent, and the permeating cryoprotectant was added at 10% of the volume of the diluent to prepare different cryopreservatives for ultra-low temperature storage. The steps for ultra-low temperature cryopreservation were the same as those in Example 1.
[0033] The results with Hank's solution as the diluent are as follows Figure 1 .B shows that when ethylene glycol is selected as the cryoprotectant, the sperm motility is the highest after thawing, and the effects of other permeating cryoprotectants are far less than that of ethylene glycol. When using Ringer as the diluent, the results are shown in Figure 1 .D. When ethylene glycol is used as the cryoprotectant, the sperm motility after thawing is significantly lower than the above results, indicating that Ringer as the diluent is not stable.
[0034] (2) Conduct concentration gradient screening on the optimal cryoprotectant
[0035] Using Hank's solution as the diluent, ethylene glycol is added to it at 5%, 7.5%, 10%, 12.5%, 15%, and 20% of the diluent volume respectively to prepare different cryopreservation agents for ultra-low temperature. The ultra-low temperature cryopreservation steps are the same as those in Example 1.
[0036] The results are as follows Figure 1 .C shows that when the addition amount of ethylene glycol is 10%, the sperm motility after thawing is the highest, reaching 67.5±7.4%. However, when the addition amount of ethylene glycol is 7.5%, the difference in sperm motility after thawing is not significant.
[0037] Therefore, Hank’s is selected as the diluent and 10% EG as the cryoprotectant will be used in the subsequent experiments.
[0038] Experimental Example 3: Comparative experiment on the cooling height
[0039] The cryopreservation agent used in this example is composed of EG and Hank's solution with a volume ratio of 1:10.
[0040] The ultra-low temperature cryopreservation steps are the same as those in Example 1, except that: the samples stay on the floating plates with the freezing heights from the liquid nitrogen surface set at 1, 3, 5, 7, and 9 cm respectively for 10 minutes to cool down, and the preservation effects at different cooling heights are compared.
[0041] The results are as follows Figure 2 .A shows that the sperm motilities after thawing at the cooling heights of 5 cm and 7 cm are 72.0±8.6% and 69.0±7.3% respectively, and the difference between them is not significant, which is significantly higher than other treatment groups.
[0042] Experimental Example 4: Comparative experiment on the cooling time
[0043] The cryopreservation agent used in this example is composed of EG and Hank's solution with a volume ratio of 1:10.
[0044] The ultra-low temperature cryopreservation steps were the same as those in Example 1, except that: on the floating plate at a height of 5 cm from the liquid nitrogen surface, the temperature was reduced for 5, 10, and 20 minutes respectively, and the preservation effects at different temperature reduction times were compared.
[0045] The results are as Figure 2 .B shows that the post-thaw motility of sperm with a temperature reduction time of 10 minutes was 72.0 ± 8.6%, which was higher than that of other treatment groups.
[0046] Experimental Example 5: Comparative experiment on the dilution ratio of semen to diluent
[0047] The ultra-low temperature cryopreservation agent used in this example was composed of EG and Hank's solution with a volume ratio of 1:10.
[0048] The ultra-low temperature cryopreservation steps were the same as those in Example 1, except that: sperm and Hank's solution were mixed and diluted at volume ratios of 2:1, 1:1, 1:3, 1:5, 1:7, and 1:9 respectively.
[0049] The results are as Figure 2 .C shows that when the ratio of sperm to Hank's solution was 1:1, the sperm motility was the highest (74.5 ± 10.3%), but there was no significant difference compared with the situation of 1:3 (68.9 ± 7.3%), and both were significantly higher than the post-thaw sperm motility at other dilution ratios. Therefore, the dilution ratio of semen to diluent used in the present invention was 1:1 to 3; however, for the purpose of facilitating the full utilization of semen, the dilution ratio of semen to diluent was preferably 1:3.
[0050] Experimental Example 6: Comparative experiment on equilibration time
[0051] The ultra-low temperature cryopreservation agent used in this example was composed of EG and Hank's solution with a volume ratio of 1:10.
[0052] The ultra-low temperature cryopreservation steps were the same as those in Example 1, except that: the equilibration times were set to 5, 10, 20, and 30 minutes respectively.
[0053] The experimental results showed that the post-thaw sperm motility was the highest (70.6 ± 6.3%) after equilibration at 4°C for 10 minutes. Although there was no significant difference compared with equilibration for 5 minutes (64.4 ± 6.7%), it was significantly higher than the post-thaw sperm motility after equilibration for 30 minutes and 60 minutes (see Figure 2 .D).
[0054] Example 7: Research experiment on the freezing loss and damage of sperm by the ultra-low temperature cryopreservation agent of the present invention
[0055] Experimental group: The cryopreservative used was a solution composed of EG and Hank's solution at a volume ratio of 1:10. The cryopreservation steps were as follows: The semen was mixed with the diluent at a volume ratio of 1:3, and then ethylene glycol was added at 10% of the volume of the diluent and mixed evenly; after equilibration at 4°C for 10 min, the samples were loaded into 0.25 ml straws, horizontally placed on a floating board 5 cm above the liquid nitrogen surface and left to cool for 10 min; then, they were stored in liquid nitrogen for 48 hours. During thawing, the straws were taken out of the liquid nitrogen and immediately thawed in a water bath at 40°C for 6 s.
[0056] Control group: The difference from the experimental group was that no cryopreservative was used and cryopreservation was carried out directly.
[0057] The sperm of the experimental group and the control group after thawing, as well as fresh sperm, were observed using a scanning electron microscope and a transmission electron microscope. The results are shown in Figure 3 and 4 .
[0058] The ROS content, the percentage of high mitochondrial membrane potential, and the SOD content of the sperm of the experimental group and the control group after thawing, as well as fresh sperm, were measured. The results are shown in Figure 5 ; and γ-H2AX immunofluorescence staining analysis was performed to observe the DNA damage. The results are shown in Figure 6 .
[0059] Figure 3 In, A - C are scanning electron micrographs of fresh sperm at different magnifications, showing that the surface of fresh sperm is smooth and the head (H) and flagellum (F) are intact; D - F are scanning electron micrographs of sperm of the experimental group after thawing at different magnifications, showing that the sperm have normal morphology and the head (H) and flagellum (F) are complete; G - I are scanning electron micrographs of sperm of the control group after thawing at different magnifications, showing that the surface of the sperm is dissolved and the head (H) and flagellum (F) are completely broken.
[0060] Figure 4 A - C in are transmission electron micrographs of fresh sperm in oblique section, oblique section, and cross section respectively, showing that they have a complete inverted U-shaped nucleus (N) and oval mitochondria (M), and showing that the plasma membrane (*), nuclear membrane (∨), and flagellum (T) are slightly swollen. Figure 4 D - G in are transmission electron micrographs of sperm of the experimental group after thawing in oblique section, oblique section, and cross section respectively, specifically showing that the plasma membrane and nuclear membrane of the thawed sperm are swollen, but most of the sperm retain intact organelles and membrane structures. Figure 4In it, H-I is the transmission electron micrograph of the sperm in the control group after thawing, specifically showing that the plasma membrane (*) and nuclear membrane (∨) of the sperm after thawing are dissolved, the nucleus is severely deformed, and it is completely apoptotic and fragmented. Therefore, through the observation of scanning and transmission electron microscopy, it can be found that compared with the control group, the experimental group's solution can significantly reduce the ultrastructural damage of sperm, and even achieve an effect similar to that of fresh sperm. When sperm is frozen, it will inevitably be damaged structurally. In this experiment, by using Hank’s and ethylene glycol as cryopreservation agents and combining this preservation method, the vast majority of sperm after thawing can maintain a complete structure, with only a slight swelling of the membrane structure, and very good results have been achieved, which will not affect the normal function of sperm and the normal development of offspring.
[0061] Figure 5 The results show that the ROS content of the sperm in the experimental group after thawing is significantly lower than that of the sperm in the control group after thawing, while the percentage of high mitochondrial membrane potential and SOD content are significantly higher than those of the sperm in the control group after thawing; in other words, the cryopreservation agent of the present invention reduces the oxidative damage of Japanese seabass sperm.
[0062] Figure 6 In it, fresh sperm, sperm in the control group after thawing, and sperm in the experimental group after thawing are compared horizontally. Specifically, A-C are the DAPI nuclear staining maps of fresh sperm, sperm in the control group after thawing, and sperm in the experimental group after thawing respectively, D-F are the γ-H2AX immunofluorescence staining maps of fresh sperm, sperm in the control group after thawing, and sperm in the experimental group after thawing respectively, and G-I are the dual-channel confocal synthesis maps of fresh sperm, sperm in the control group after thawing, and sperm in the experimental group after thawing respectively; blue is sperm, and green fluorescence represents DNA damage. The results show that the sperm in the experimental group after thawing is not much different from fresh sperm in terms of DNA loss, and is significantly better than the sperm in the control group after thawing.
[0063] Experimental Example 8: Comparison experiment on the fertilization rate of fresh sperm and frozen sperm
[0064] The cryopreservation agent used in this example is composed of EG and Hank’s solution with a volume ratio of 1:10.
[0065] The cryopreservation steps are as follows: Mix semen and diluent at a volume ratio of 1:3, then add ethylene glycol at 10% of the volume of the diluent and mix well; after equilibrating at 4°C for 10 min, load the sample into a 0.25 ml straw, place it horizontally on a floating plate 5 cm above the liquid nitrogen surface and stay for 10 min to cool down; then, store it in liquid nitrogen for 48 hours. When thawing, take out the straw from liquid nitrogen and immediately thaw it in a water bath at 40°C for 6 s.
[0066] Take the thawed semen for artificial insemination experiments (using fresh semen as the control group). The specific operation is as follows: The sperm-egg ratio of fresh semen and frozen semen is controlled at about 100,000:1; after mixing sperm and eggs gently (for 3 s), then slowly add 5 ml of clean seawater to activate sperm; 15 minutes after sperm-egg contact, adjust the seawater volume in the container to 100 ml and inflate, and incubate in the dark at 21 °C; after incubation for 2.5 h when the fertilized eggs enter the 16-cell stage, conduct the statistics of fertilization rate; the fertilization rate is the proportion of fertilized eggs among the observed eggs.
[0067] The results of the artificial insemination experiments are as Figure 7 shown, indicating that there is no significant difference in the fertilization rate between frozen semen and fresh semen, and it reaches 83.4 ± 3.3%.
Claims
1. A cryopreservative for Japanese seabass spermatozoa, characterized in that, It consists of ethylene glycol and Hank's solution; the volume ratio of ethylene glycol to Hank's solution is 1:
10.
2. A method for cryopreservation of Japanese seabass spermatozoa, characterized in that, It includes the following steps: Mix semen with Hank's solution at a volume ratio of 1:1 - 3, then add ethylene glycol with a volume of 10% of the Hank's solution volume, and mix well to obtain a cryopreservation sample; place the sample at 4°C for 10 minutes for equilibration, then load it into a straw, horizontally place it at a height of 5 - 7 cm from the liquid nitrogen surface and stay for 10 minutes for temperature reduction; then put the sample into liquid nitrogen for preservation.
3. The cryopreservation method of Japanese seabass sperm according to claim 2, characterized in that, Semen is mixed with Hank's solution at a volume ratio of 1:3.