Anti-freezing agent and anti-freezing diluent for cryopreservation of semen of Wujin pigs

Abstract

The invention provides a Wujin pig seminal plasma protein crude extract and application of the Wujin pig seminal plasma protein crude extract in an anti-freezing agent and an anti-freezing diluent for freeze preservation of Wujin pig seminal fluid. The Wujin pig seminal plasma protein crude extract is derived from Wujin pig seminal plasma, does not contain other exogenous bioactive substances, has a better freezing protection effect on sperms of Wujin pigs and is safer, and the acrosome integrity rate of frozen sperms of the Wujin pigs can be remarkably increased. According to the present invention, with the anti-freezing diluent, the vitality and the movement performance of the cryopreserved Wujin pig sperm can be significantly improved, the active oxygen level is reduced, the mitochondrial membrane potential and the energy metabolism are improved, and the oxidation resistance is enhanced, such that the vitality of the Wujin pig sperm can be easily improved, and the mitochondrial function can be easily maintained; the research provides key technical support for establishment of a Wujin pig semen cryopreservation technology, and has great practical significance for Wujin pig breeding and population propagation.

Description

Technical Field

[0001] The invention relates to the biological field, and in particular to a crude extract of black pig seminal plasma protein and application of the crude extract in an antifreeze agent and an antifreeze diluent for cryopreservation of black pig semen. Background Art

[0002] Wujin pig is a local pig breed listed in the "Chinese Pig Breeds", named after its distribution in the Wumeng Mountains and the Jinsha River Basin. Its main characteristics are strong physique, strong resistance to adversity, strong adaptability, tolerance to roughage and cold, carcass lean meat rate of 37% to 42%, and high intramuscular fat, tender and delicious meat. Ham cured with Wujin pig or pigs with Wujin pig blood as ham raw materials is rich in nutrition, tender meat, oily but not greasy, salty and sweet, and is the most distinctive and potential local specialty product in Yunnan Province's livestock products. In order to save, protect and develop and utilize Wujin pig resources, it is necessary to carry out research on the protection and development and utilization of Wujin pigs.

[0003] In this context, cryopreservation is a safe and effective method for preserving genetic resources. Common cryopreservation methods mainly include frozen semen, frozen somatic cells and frozen embryos. Frozen semen can be preserved for a long time. In theory, it is not restricted by time, region and the life span of the boar, which can maximize the utilization efficiency of the boar. Although semen cryopreservation technology is becoming more and more popular, due to the large difference in lipid content and component characteristics of pig semen from other mammals (cattle, sheep, etc.), it is particularly sensitive to low temperatures and has poor freezing resistance. The survival rate of semen after freezing is low, the conception rate of using frozen semen for breeding is low, and the relevant technology is still immature. Moreover, semen cryopreservation includes two closely related and independent processes: freezing and thawing. The freezing and thawing process of pig semen also undergoes the transformation of two forms, "solid" and "liquid", so the thawing process of pig frozen semen is as important as the freezing process of pig semen. The antifreeze and antifreeze diluent in the prior art are not suitable for the cryopreservation of semen of the specific local pig breed of Wujin pig, and the effect of improving sperm motility and fertilization rate is not ideal.

[0004] Seminal plasma is a fluid mixture secreted in the testicles, epididymis, and accessory glands, containing a variety of factors that regulate sperm function. Existing research results have confirmed that the protective effect of adding seminal plasma to frozen semen is not consistent, and the efficiency of seminal plasma preservation shows great differences between studies. This variability in the efficiency of seminal plasma preservation may be related to the variability of the composition of seminal plasma in ions, small molecules, or proteins between semen, but it is still unknown which specific components have side effects on the cryopreservation of semen. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of existing semen freezing diluents, and to provide a crude extract of black pig seminal plasma protein, and its application in antifreeze agents and antifreeze diluents for cryopreservation of black pig semen.

[0006] Specifically, the present invention first provides a crude extract of black pig seminal plasma protein, and the extraction method of the crude extract of black pig seminal plasma protein is:

[0007] Semen of healthy Wujin pig boars was collected by hand-gripping method, and the middle part of semen was taken and filtered through 3 layers of gauze. Semen with sperm motility above 80% was isothermally diluted with sperm washing solution at a ratio of 1:1; wherein, the sperm washing solution was: 30g Tris, 10g citric acid, 12g glucose, and 6mM cysteine ​​in 1L system;

[0008] After standing, the semen was divided into 15 mL centrifuge tubes, centrifuged at 2000-2500 rpm for 3-5 minutes, and supernatant I was taken. At the same time, the lower concentrated part was isothermally diluted again with sperm washing solution at a ratio of 1:1, and centrifuged at 2000-2500 rpm for 3-5 minutes. Supernatant II was taken and combined with supernatant I for crude extraction of seminal plasma protein;

[0009] The above supernatant pool is dialyzed for 12-36 hours using a dialysis bag with a molecular weight cutoff of 12000-16000Da, then concentrated to 10-30% volume, ethanol is added to make its ethanol concentration 60-85%, alcohol precipitation is performed, and then centrifuged at 3000-5000 rpm for 5-15 minutes, the supernatant is discarded, and the precipitate is the black gold pig seminal plasma protein extract, which is resuspended in PBS and quantified to 1 mg / ml.

[0010] Furthermore, the present invention provides an antifreeze diluent for freezing and preserving black pig semen containing the black pig semen protein extract.

[0011] Furthermore, the antifreeze diluent is specifically:

[0012] Basic freezing solution (100 ml): 5 g trehalose, 5 g glucose, 1 g lactose, 0.2 g vitamin C, 1.6 g sodium citrate, 100 IU / L (final concentration) antibacterial agent, 0.05 g adenosine triphosphate, 0.02 g glutathione, 0.01 g oleic acid (18:1, n-9), 0.01 g docosapentaenoic acid (22:5, n-6), 0.01 g docosahexaenoic acid (DHA, 22:6, n-3);

[0013] Solution I: Add 5 ml of antifreeze polypeptide (HGEEGASGVRGAF, 1 mg / ml, dissolved in 0.01 M PBS) and 10 ml of Wujin pig seminal plasma protein extract (1 mg / ml) to the basic freezing solution;

[0014] Solution II: Add 6% glycerol to solution I.

[0015] Wherein, the antibacterial agent is selected from one or more of penicillin, gentamicin, amphotericin B, and penicillin-streptomycin.

[0016] Furthermore, the present invention provides a method for cryopreservation of Wujin pig semen using the antifreeze diluent, the method comprising the following steps:

[0017] Fresh semen of Wujin pigs was collected by hand-gripping method. The middle part of semen was taken and filtered through 3 layers of gauze. Semen with sperm motility above 80% was isothermally diluted with sperm washing solution (based on 1L system, Tris 30g, citric acid 10g, glucose 12g, cysteine ​​6mM) and the sperm density after dilution was 8×10 8 -10×10 8 Calculate the total amount of freezing solution required based on the dilution concentration of 100 ml / mL. Add half the required total volume of liquid I to dilute the fresh semen, mix well and transfer to a refrigerator or 4℃ low temperature environment, cool to 0-4℃ in a water bath for 1.5-2h to complete the first step of dilution; add liquid II with the same volume as liquid I at the same temperature to complete the second step of dilution, and balance at 4℃ for 2-6h. The diluted and balanced semen is aspirated into a 0.25mL capillary at 0-4℃, sealed with polyvinyl alcohol powder, and the first step of cooling is to quickly place the capillary on a freezing rack 4-5cm away from the surface of liquid nitrogen, fumigate for 4-5min, and the second step of cooling is to put the capillary into liquid nitrogen for long-term storage.

[0018] Beneficial Effects

[0019] The crude extract of seminal plasma protein of black pig used in the present invention is derived from the seminal plasma of black pig itself and does not contain other exogenous bioactive substances. Moreover, it has been verified through experiments that the crude extract of seminal plasma protein has better and safer cryoprotective effect on the sperm of black pig itself because it contains macromolecular bioactive substances such as proteins, RNAs, lipids and DNA. Through repeated verification and multiple experiments, it has been proved that the substance can significantly improve the acrosome integrity rate of black pig frozen semen. Moreover, the antifreeze diluent obtained on this basis can significantly improve the vitality and motility of frozen black pig semen, reduce the level of active oxygen, improve mitochondrial membrane potential and energy metabolism, and enhance antioxidant capacity, all of which are helpful to improve the vitality of black pig sperm and maintain mitochondrial function. This study provides key technical support for the establishment of black pig semen cryopreservation technology, which has great practical significance for the reproduction and population expansion of black pigs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0021] Figure 1 The morphology of Wujin pig semen before and after cryopreservation is compared. A is normal Wujin pig sperm, B is Wujin pig sperm with a broken tail, C is Wujin pig sperm with a folded tail, and D is Wujin pig sperm agglutination. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0023] The Wujin pigs were selected from the Wujin pig Huomao line of the Wujin pig breeding professional cooperative in Yiliang County, Zhaotong City, Yunnan Province, including 20 boars and 30 sows.

[0024] Example 1 Extraction of crude protein extract from black pig seminal plasma

[0025] Semen from healthy black gold pig boars was collected by hand-gripping method. The mid-section semen was taken and filtered through 3 layers of gauze. Semen with sperm motility above 80% was isothermally diluted 1:1 with sperm washing solution (based on 1L system, Tris 30g, citric acid 10g, glucose 12g, cysteine ​​6mM) and placed in a thermos bottle and immediately transported back to the laboratory.

[0026] After standing, the semen was divided into 15 mL centrifuge tubes, centrifuged at 2000-2500 rpm for 3-5 minutes, and supernatant I was taken. At the same time, the lower concentrated part was isothermally diluted again with sperm washing solution at a ratio of 1:1, and centrifuged at 2000-2500 rpm for 3-5 minutes. Supernatant II was taken and combined with supernatant I for crude extraction of seminal plasma protein;

[0027] The above supernatant pool was dialyzed for 12-36 hours using a dialysis bag with a molecular weight cutoff of 12000-16000Da, then concentrated to 10-30% volume, ethanol was added to make the ethanol concentration 60-85%, and then precipitated by alcohol, and then centrifuged at 3000-5000 rpm for 5-15 minutes, the supernatant was discarded, and the precipitate was the black pig seminal plasma protein extract, which was resuspended in PBS and quantified to 1 mg / ml. The dialysate was used as a small molecule dialysis control after alcohol precipitation, centrifugation, etc. as the above sample.

[0028] Example 2 Configuration of diluent for cryopreservation of black pig semen

[0029] According to the applicant's previous research results, a black pig frozen semen diluent is prepared according to the following formula, including the following components:

[0030] Basic freezing solution (100ml): 5g trehalose, 5g glucose, 1g lactose, 0.2g vitamin C, 1.6g sodium citrate, 100IU / L (final concentration) antibacterial agent, 0.05g adenosine triphosphate, 0.02g glutathione, 0.01g oleic acid (18:1, n-9), 0.01g docosapentaenoic acid (22:5, n-6), 0.01g docosahexaenoic acid (DHA, 22:6, n-3).

[0031] Solution I: Add 5 ml of antifreeze peptide (HGEEGASGVRGAF, 1 mg / ml, dissolved in 0.01 M PBS) to the basic freezing solution. At the same time, add 10 ml of Wujin pig seminal plasma protein extract and its small molecule dialysis control to verify the distribution of active ingredients. In addition, set up a blank control to verify the effect of adding seminal plasma.

[0032] Solution II: Add 6% glycerol to solution I.

[0033] Wherein, the antibacterial agent is selected from one or more of penicillin, gentamicin, amphotericin B, and penicillin-streptomycin.

[0034] Fresh semen of Wujin pigs was collected by hand-gripping method. The middle part of semen was taken and filtered through 3 layers of gauze. Semen with sperm motility above 80% was isothermally diluted with sperm washing solution (based on 1L system, Tris 30g, citric acid 10g, glucose 12g, cysteine ​​6mM) and the sperm density after dilution was 8×10 8 -10×10 8 Calculate the total amount of freezing solution required based on the dilution concentration of 100 ml / mL. Add half the required total volume of liquid I to dilute the fresh semen, mix well and transfer to a refrigerator or 4℃ low temperature environment, cool to 0-4℃ in a water bath for 1.5-2h to complete the first step of dilution; add liquid II with the same volume as liquid I at the same temperature to complete the second step of dilution, and balance at 4℃ for 2-6h. The diluted and balanced semen is aspirated into a 0.25mL capillary at 0-4℃, sealed with polyvinyl alcohol powder, and the first step of cooling is to quickly place the capillary on a freezing rack 4-5cm away from the surface of liquid nitrogen, fumigate for 4-5min, and the second step of cooling is to put the capillary into liquid nitrogen for long-term storage.

[0035] Liquid nitrogen freezing effect test: After taking out the frozen sperm tube from liquid nitrogen with tweezers, expose it to air for 1-2 seconds, put it into a constant temperature water bath at 38℃ for 30 seconds, incubate it at 37℃ for 5 minutes, and test the semen motility, plasma membrane integrity rate, and acrosome integrity rate.

[0036] Semen motility: Sperm motility is measured and evaluated by the percentage of sperm forward movement and motility. According to the standards and requirements of the Manual, the Beijing Weili Color Computer-Assisted Semen Analysis System is used to process and analyze the semen after thawing, and the forward movement and sperm motility before and after freezing are measured. Forward movement (PR) is the linear or large circular movement actively presented by sperm, and non-forward movement (NP) refers to all other non-forward sperm movements, such as small circular swimming and tail swinging. Sperm motility is the total activity rate of sperm, expressed as NP+PR.

[0037] Plasma membrane integrity: The hypotonic sperm swelling (HOST) method is used to detect the plasma membrane integrity. Sperm is mixed with a hypotonic fructose sodium citrate solution (13.5 g / L fructose, 7.35 g / L sodium citrate), incubated at 37°C for 60 min, and observed under a phase contrast microscope at 1000 times magnification. If the sperm plasma membrane is intact, the sperm head will absorb water and swell, and the sperm tail will swell and curl.

[0038] Acrosome integrity: isothiocyanate labeled peanut agglutinin (FITC-PNA) was used to detect acrosome integrity. The FITC-PNA kit was purchased from Bio-Technology. Semen was mixed with ethD-1 and incubated at 37°C for 15 minutes. 5 μl of the mixture was pipetted onto a glass slide, dehydrated with 95% ethanol for 30 seconds, and 15 μl of FITC-PNA solution was added. The reaction was carried out at 4°C for 30 minutes, and then rinsed with PBS. Observed with a fluorescence microscope at 1000 times. The number of sperm observed was not less than 200, and at least 5 fields of view.

[0039] Table 1 Effect of Wujin pig antifreeze protein on the quality of semen cryopreservation

[0040] index Small molecule control Crude seminal plasma extract No Additive Survival rate (%) 57.23±0.14 74.19±0.48 68.38±0.37 vitality(%) 35.24±0.29 54.84±0.93 48.11±0.25 VSL(μm / s) 24.34±0.58 39.41±0.59 31.05±0.32 VCL(μm / s) 52.44±0.20 68.81±0.70 60.21±0.63 VAP(μm / s) 27.68±0.38 43.27±0.39 40.20±0.23 Plasma membrane integrity rate (%) 65.28±0.25 66.37±0.49 64.26±0.16 Acrosome integrity rate (%) 71.79±0.31 75.81±0.24 70.23±0.71

[0041] From the results, we can see that ( Figure 1): Since the plasma membrane of boar sperm is rich in unsaturated phospholipids and the cholesterol molecules are weak, this constitutes the high sensitivity of boar sperm to cold shock, and the differences between breeds are huge. The non-added freezing buffer used in this application has a good cryoprotective effect in other pig breeds, but it is not satisfactory in Wujin pigs. Analysis may be due to differences in semen composition and protein expression between different pig breeds. In addition, previous studies have also confirmed that individual differences between boars have a significant impact on sperm motility and quality after recovery. When collecting and freezing the semen of Landrace pigs, it was observed that 30% of the semen of boars showed very low motility after recovery. In addition, even for the same pig, significant differences in semen quality were observed after cryopreservation of its semen at different times, ages, and seasons. However, the above plasma membrane integrity rate (%) and acrosome integrity rate (%) results suggest that both large and small components in Wujin pig seminal plasma have a significant protective effect on the integrity of the plasma membrane and the integrity of the acrosome, confirming that adding seminal plasma components to the buffer can improve the integrity of the membrane and the acrosome. Moreover, oleic acid, docosapentaenoic acid and docosahexaenoic acid added in the above buffer are also the most abundant fatty acids in the plasma membrane of boar sperm. Adding the above ingredients to the freezing buffer can achieve a good effect of protecting the plasma membrane and acrosome. However, from the perspective of indicators such as vitality, viability, and trajectory speed, the macromolecular crude extract components in seminal plasma may achieve a better promotion effect, while the small molecules therein inhibit the above indicators. In the previous semen storage process, especially in cryopreservation, seminal plasma is usually removed before cryopreservation because researchers believe that the presence of seminal plasma will reduce the survival rate of sperm after recovery. However, studies in recent years have found that not all proteins from seminal plasma are harmful to sperm, and the amount of seminal plasma added does not show a dose-related relationship. There is still no clear statement on whether the above indicators are positively induced or reversely inhibited. The above results of the present application can confirm that removing low molecular weight proteins (<12000-16000Da) can improve the cryopreservation effect and increase the vitality and viability of frozen sperm, but the specific components still need further detailed research.

[0042] Sperm fertilization ability test: Use the method of culturing in vitro embryos to select mature oocytes and incubate them with thawed sperm. Use a stereomicroscope to observe the ratio of sperm heads entering the oocyte cytoplasm, or observe the cleavage rate after 48 hours of embryo culture. The specific operation is: before in vitro culture, add 600μl of culture solution to the four-well plate, cover it with 300μl of embryo-grade mineral oil, and put it in the incubator for at least 2 hours in advance. Wash the oocytes fertilized in vitro for 24 hours with the balanced culture solution 3 times, then move them into the four-well plate, about 30 per well, put them into the air bag, culture them with mixed gas (5% O2+5% CO2+90% N2), and place them in a saturated humidity incubator at 38.5℃ and 5% CO2. The cleavage rate is counted at 48 hours, and the blastocyst rate is counted at 7 days.

[0043] Table 2 Effect of Wujin pig antifreeze protein on sperm fertilization ability

[0044] index Small molecule control Crude seminal plasma extract No Additive Cleavage rate (%) 51.23±0.40 72.91±0.79 69.42±0.23 Blastocyst rate (%) 33.96±0.25 48.11±0.62 44.15±0.84

[0045] The above results show that the macromolecular crude extract of seminal plasma can effectively improve the motility of sperm after freezing. While ensuring the fertilization ability of sperm, it can effectively prolong the survival time of sperm in vitro, and improve the breeding efficiency of excellent male Wujin pigs. The cleavage rate and blastocyst rate also increase, which is helpful to improve the fertilization ability of sperm. It can be used as a good preservation additive in the cryopreservation of Wujin pig sperm. In contrast, the effect of the small molecule control is significantly inferior to the negative control without addition. The analysis may be due to the damage of cell membrane, cholesterol loss, capacitation-like changes and DNA fragmentation caused by freezing and thawing, and the destruction of disulfide bonds may also lead to reduced stability of nucleoprotein structure. The small molecule components in seminal plasma may cause intracellular ROS imbalance during the freezing and thawing process, induce lipid peroxidation, and cause apoptosis-like changes and DNA oxidative damage to cells. In addition, in connection with the analysis of the effects of the above small molecule components on sperm motility, it is possible to damage organelles such as mitochondria during the freezing and thawing process, reduce the activity of these organelles, thereby affecting sperm motility. Mitochondrial damage also increases the production of reactive oxygen species, which leads to the final result, but the specific reasons need further analysis.

[0046] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples, unless they are contradictory.

[0047] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A crude extract of black pig seminal plasma protein, characterized in that The extraction method of crude protein extract of Wujin pig seminal plasma is as follows: Use the hand-holding method to collect semen from healthy black pig boars, take the middle part of the semen, filter it through 3 layers of gauze, and dilute the semen with sperm motility above 80% with sperm washing solution at a ratio of 1:1 isothermally; After standing, the semen was divided into 15 mL centrifuge tubes, centrifuged at 2000-2500 rpm for 3-5 minutes, and supernatant I was taken. At the same time, the lower concentrated part was isothermally diluted again with sperm washing solution at a ratio of 1:1, and centrifuged at 2000-2500 rpm for 3-5 minutes. Supernatant II was taken and combined with supernatant I for crude extraction of seminal plasma protein; The supernatant pool is dialyzed for 12-36 hours using a dialysis bag with a molecular weight cutoff of 12000-16000Da, then concentrated to 10-30% volume, ethanol is added to make its ethanol concentration 60-85%, alcohol precipitation is performed, and then centrifuged at 3000-5000 rpm for 5-15 minutes, the supernatant is discarded, and the precipitate is the black gold pig seminal plasma protein extract.

2. The crude extract of black pig seminal plasma protein as described in claim 1, wherein the sperm washing solution is specifically: based on 1L system, it is 30g Tris, 10g citric acid, 12g glucose, and 6mM cysteine.

3. The crude extract of black pig seminal plasma protein according to claim 1, wherein the method further comprises resuspending the black pig seminal plasma protein extract in PBS buffer to a quantitative concentration of 1 mg / ml and then preserving it.

4. An antifreeze diluent for freezing and preserving black pig semen containing the black pig semen protein extract as claimed in any one of claims 1 to 3.

5. The antifreeze diluent for cryopreservation of black pig semen according to claim 4, characterized in that: The antifreeze diluent is specifically: Basic freezing solution (100 ml): 5 g trehalose, 5 g glucose, 1 g lactose, 0.2 g vitamin C, 1.6 g sodium citrate, 100 IU / L (final concentration) antibacterial agent, 0.05 g adenosine triphosphate, 0.02 g glutathione, 0.01 g oleic acid (18:1, n-9), 0.01 g docosapentaenoic acid (22:5, n-6), 0.01 g docosahexaenoic acid (DHA, 22:6, n-3); Solution I: Add 5 ml of antifreeze polypeptide (HGEEGASGVRGAF, 1 mg / ml, dissolved in 0.01 M PBS) and 10 ml of Wujin pig seminal plasma protein extract (1 mg / ml) to the basic freezing solution; Solution II: Add 6% glycerol to solution I.

6. The antifreeze diluent for cryopreservation of black pig semen as claimed in claim 5, wherein the antibacterial agent is selected from one or more of penicillin, gentamicin, amphotericin B, and penicillin-streptomycin.

7. A method for cryopreservation of black pig semen using the antifreeze diluent for cryopreservation of black pig semen as claimed in any one of claims 4 to 7.

8. The method for cryopreservation of black pig semen as claimed in claim 7, comprising the following steps: Collect fresh Wujin pig semen. Collect healthy Wujin pig semen by hand-holding method. Take the middle semen, filter it through 3 layers of gauze, and dilute the semen with sperm motility above 80% with sperm washing solution isothermally. The sperm density after dilution is 8×10 8 -10×10 8 Calculate the total amount of freezing solution required based on the dilution concentration of 100 g / mL; add half the volume of solution I to dilute the fresh semen, mix well and transfer to a refrigerator or a 4°C low-temperature environment, and cool to 0-4°C in a water bath for 1.5-2 hours to complete the first step of dilution; add a volume of solution II equal to that of solution I at the same temperature to complete the second step of dilution, and equilibrate at 4°C for 2-6 hours; aspirate the diluted and equilibrated semen into a 0.25 mL capillary at 0-4°C, seal it with polyvinyl alcohol powder, and cool it down in the first step, i.e. quickly place the capillary on a freezing rack 4-5 cm away from the surface of liquid nitrogen, and fumigate it for 4-5 minutes. In the second step of cooling, the capillary is placed in liquid nitrogen for long-term storage.

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