Livestock semen cryopreservation method and application thereof

Abstract

The invention relates to a cryopreservation method for livestock semen and application of the cryopreservation method, and belongs to the technical field of semen freezing. The cryopreservation method of the livestock semen comprises the following steps: mixing the livestock semen with a semen cryopreservation solution A to obtain a livestock semen pre-freezing solution; carrying out long-distance transportation and / or low-temperature preservation on the livestock semen pre-freezing solution, centrifuging, and taking precipitate to obtain livestock sperms; mixing the livestock sperms with the semen freezing medium B, and standing at low temperature to obtain livestock semen equilibrium liquid; and pre-cooling the livestock semen equilibrium liquid and then putting into liquid nitrogen for cryopreservation to obtain the livestock frozen semen. The cryopreservation method for the livestock semen has the advantages that the method is simple and easy to implement, waste is reduced, and the quality of the livestock semen and the sperm motility rate can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of semen freezing, and in particular to a method for freezing livestock semen and application thereof. Background Art

[0002] Cryopreservation of livestock semen is not only a key link in the artificial insemination technology of modern animal husbandry, but also a core technology for the long-term preservation of genetic resources of excellent breeding bulls. In actual production, after the semen of excellent breeding bulls is collected, it needs to be processed and cryopreserved as soon as possible. However, due to the long distance between the livestock farm and the laboratory, the semen transportation time is too long or the temporary low-temperature storage operation is improper, which leads to a decrease in sperm motility and cannot meet the requirements of cryopreservation. In addition, if a large number of excellent breeding bull semen are collected in the same batch, due to limited equipment and technical personnel, the semen cannot be cryopreserved in time, which not only causes a waste of breeding bull semen, but also causes additional consumption of manpower, material and financial resources. Summary of the invention

[0003] The object of the present invention is to provide a method for freezing livestock semen and its application, so as to solve the problem in the prior art that the sperm motility of livestock semen after long-distance transportation and / or low-temperature storage cannot meet the freezing storage requirements.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for freezing livestock semen, comprising the following steps:

[0006] (1) mixing 1 mL of livestock fresh semen and 2-5 mL of semen freezing solution A to obtain a livestock semen pre-frozen solution;

[0007] (2) transporting the livestock semen pre-frozen solution over long distances and / or storing it at low temperatures, centrifuging it, and collecting the precipitate to obtain livestock sperm;

[0008] (3) mixing the livestock sperm and 5 to 8 mL of semen freezing solution B, and allowing to stand to obtain a livestock semen balancing solution;

[0009] (4) precooling the livestock semen balance solution and then freezing it in liquid nitrogen to obtain livestock frozen semen;

[0010] The sperm freezing solution A comprises a base solution and egg yolk;

[0011] The semen freezing solution B comprises semen freezing solution A and glycerol;

[0012] The base solution uses water as solvent and includes the following components in concentrations: Tris 10-50 g / L, citric acid 10-25 g / L, glucose 10-20 g / L, penicillin 0.1-1 g / L, and streptomycin 0.1-2 g / L;

[0013] In the sperm freezing solution A, the volume ratio of the base solution to the egg yolk is 70-90:10-30;

[0014] In the semen freezing solution B, the volume ratio of semen freezing solution A to glycerol is 94-96:4-6.

[0015] Preferably, the sperm motility of the livestock fresh semen in step (1) is greater than 0.8.

[0016] Preferably, the long-distance transportation distance of step (2) is 200 to 400 km, and the long-distance transportation time is 2 to 4 hours;

[0017] The temperature of the low-temperature storage in step (2) is 3 to 6° C., and the time of the low-temperature storage is 12 to 48 hours.

[0018] Preferably, the centrifugal speed in step (2) is 1000-2000 rpm, and the centrifugal time is 10-15 min.

[0019] Preferably, the standing temperature in step (3) is 3 to 6° C., and the standing time is 2 to 4 hours.

[0020] Preferably, the initial precooling temperature in step (4) is -90 to -80°C, and the precooling time is 5 to 15 minutes.

[0021] Preferably, the sperm motility of the livestock frozen semen after thawing in step (4) is >0.4.

[0022] The invention also provides application of the freezing method in freezing livestock semen.

[0023] The present invention has the following technical effects and advantages:

[0024] (1) The method for freezing livestock semen of the present invention has the advantages of being simple and easy to operate, reducing waste, and being able to improve the quality of livestock semen and sperm motility, thereby solving the technical problem that the sperm motility of livestock semen decreases to a level that cannot meet the requirements of cryopreservation due to long transportation time and / or improper cryopreservation treatment;

[0025] (2) The components such as Tris, citric acid, glucose, egg yolk and glycerol contained in the semen freezing solution A and the semen freezing solution B used in the present invention can protect the sperm during the long-distance transportation, low-temperature storage and thawing of the livestock semen, and penicillin and streptomycin can prevent pathogens from damaging the quality of livestock semen and the motility of sperm, so that the sperm motility of the livestock semen can still meet the requirements of semen freezing after long-distance transportation and / or low-temperature storage, and the sperm of the livestock frozen semen still has a high motility after thawing. DETAILED DESCRIPTION

[0026] The present invention provides a method for freezing livestock semen, comprising the following steps:

[0027] (1) mixing 1 mL of livestock fresh semen and 2 to 5 mL of semen freezing solution A, preferably 3 mL, to obtain a livestock semen pre-frozen solution;

[0028] (2) transporting the livestock semen pre-frozen solution over long distances and / or storing it at low temperatures, centrifuging it, and collecting the precipitate to obtain livestock sperm;

[0029] (3) mixing the livestock sperm with 5 to 8 mL of semen freezing solution B, preferably 6 mL, and allowing to stand to obtain a livestock semen balancing solution;

[0030] (4) precooling the livestock semen balance solution and then freezing it in liquid nitrogen to obtain livestock frozen semen;

[0031] The sperm freezing solution A comprises a base solution and egg yolk;

[0032] The semen freezing solution B comprises semen freezing solution A and glycerol;

[0033] The base solution uses water as solvent and includes the following components in concentrations: Tris 10-50 g / L, preferably 30.4 g / L; citric acid 10-25 g / L, preferably 17 g / L; glucose 10-20 g / L, preferably 15 g / L; penicillin 0.1-1 g / L, preferably 0.6 g / L; streptomycin 0.1-2 g / L, preferably 1 g / L;

[0034] In the sperm freezing solution A, the volume ratio of the base solution to the egg yolk is 70-90:10-30, preferably 80:20;

[0035] In the semen freezing solution B, the volume ratio of semen freezing solution A to glycerol is 94-96:4-6, preferably 95:5.

[0036] In the present invention, the sperm motility of the livestock fresh semen in step (1) is greater than 0.8.

[0037] In the present invention, the step (1) further comprises preheating the semen freezing solution A before mixing the livestock fresh semen and the semen freezing solution A, wherein the preheating method is water bath preheating;

[0038] The water bath preheating temperature is 30-35°C, preferably 32°C.

[0039] In the present invention, the transportation distance of the long-distance transportation in step (2) is 200 to 400 km, preferably 250 km; the time of long-distance transportation is 2 to 4 hours, preferably 3 hours;

[0040] The low-temperature storage temperature in step (2) is 3 to 6° C., preferably 4° C.; the low-temperature storage time is 12 to 48 hours, preferably 24 hours.

[0041] In the present invention, the centrifugal speed in step (2) is 1000-2000 rpm, preferably 1500 rpm; the centrifugal time is 10-15 min, preferably 10 min.

[0042] In the present invention, the standing temperature in step (3) is 3 to 6° C., preferably 4° C.; the standing time is 2 to 4 hours, preferably 3 hours.

[0043] In the present invention, the initial temperature of the precooling in step (4) is -90 to -80°C, preferably -85°C; the precooling time is 5 to 15 minutes, preferably 10 minutes.

[0044] In the present invention, the sperm motility of the livestock frozen semen after thawing in step (4) is greater than 0.4.

[0045] The invention also provides application of the freezing method in freezing livestock semen.

[0046] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0047] In the embodiment of the present invention, the Dorper sheep breed ram and the Suffolk sheep breed ram are provided by Ningxia Zhongmu Yilin Animal Husbandry Co., Ltd., and the Tan sheep breed ram is provided by the Yanchi Tan sheep breeding farm of Ningxia Hui Autonomous Region;

[0048] In the embodiments of the present invention, TCM-199 and fetal calf serum (FCS) were purchased from Gibco, USA, and heparin sodium was purchased from Sigma, USA;

[0049] In the embodiments of the present invention, oocyte in vitro maturation medium (BO-IVM), in vitro fertilization medium (BO-IVF), and early embryo in vitro culture medium (BO-IVC) were all purchased from IVF Bioscience.

[0050] Example 1: Preparation of sperm cryopreservation solution

[0051] (1) Preparation of basic solution: 30.4 g Tris, 17 g citric acid, 15 g glucose, 0.6 g penicillin, and 1 g streptomycin were added to 1 L ultrapure water and mixed well to obtain a basic solution;

[0052] (2) Preparation of sperm cryopreservation solution A: 80 mL of the base solution and 20 mL of egg yolk were mixed evenly to obtain sperm cryopreservation solution A;

[0053] (3) Preparation of semen cryopreservation solution B: Mix 95 mL of semen cryopreservation solution A and 5 mL of glycerol to obtain semen cryopreservation solution B.

[0054] Example 2: Preparation of sperm cryopreservation solution

[0055] (1) Preparation of basic solution: 10 g Tris, 25 g citric acid, 20 g glucose, 0.1 g penicillin, and 0.1 g streptomycin were added to 1 L ultrapure water and mixed well to obtain basic solution;

[0056] (2) Preparation of sperm cryopreservation solution A: 90 mL of the base solution and 10 mL of egg yolk were mixed evenly to obtain sperm cryopreservation solution A;

[0057] (3) Preparation of semen cryopreservation solution B: Mix 94 mL of semen cryopreservation solution A and 6 mL of glycerol to obtain semen cryopreservation solution B.

[0058] Example 3: Preparation of sperm cryopreservation solution

[0059] (1) Preparation of basic solution: 50 g Tris, 10 g citric acid, 10 g glucose, 1 g penicillin, and 2 g streptomycin were added to 1 L ultrapure water and mixed well to obtain a basic solution;

[0060] (2) Preparation of sperm cryopreservation solution A: 70 mL of the base solution and 30 mL of egg yolk were mixed evenly to obtain sperm cryopreservation solution A;

[0061] (3) Preparation of semen cryopreservation solution B: Mix 96 mL of semen cryopreservation solution A and 4 mL of glycerol to obtain semen cryopreservation solution B.

[0062] Example 4: Cryopreservation of Dorper ram semen

[0063] (1) Fresh semen collection: The pseudo-vaginal method disclosed in the article published by Liang Guorong et al. (Liang Guorong, Liu Bohe, Zhang Mingliang, et al. Application and promotion effect of normal temperature artificial insemination technology of sheep [J]. Gansu Animal Husbandry and Veterinary Medicine, 2022, 52(10): 38-40) was used to collect fresh semen of Dorper sheep rams;

[0064] (2) Prefreezing and long-distance transportation of semen: The semen freezing solution A prepared in Example 1 was preheated to 32° C. in a water bath to obtain preheated semen freezing solution A; 1 mL of fresh semen of Dorper ram with a sperm motility of 0.88 was taken and mixed evenly with 3 mL of preheated semen freezing solution A in a centrifuge tube to obtain a Dorper ram semen pre-frozen solution; then, the centrifuge tube containing the Dorper ram semen pre-frozen solution was wrapped with 8 layers of gauze and placed upright in an ice box for long-distance transportation, the transportation distance was 250 km, the transportation time was 3 h, and the Dorper ram semen pre-frozen solution was gradually cooled by the ice box during transportation;

[0065] (3) Cryopreservation of semen: The Dorper ram semen pre-frozen solution that has been transported over long distances is transferred to a 4°C refrigerator for cryopreservation for 24 hours, and then the Dorper ram semen pre-frozen solution is centrifuged at 1500 rpm for 10 minutes to obtain Dorper ram sperm; the Dorper ram sperm and 6 mL of the semen freezing solution B prepared in Example 1 are mixed evenly in a centrifuge tube, and then allowed to stand at 4°C for 3 hours to obtain Dorper ram semen equilibrium solution;

[0066] (4) Cryopreservation of semen: Place an aluminum box on a liquid nitrogen surface for precooling. Use an ultra-low temperature thermometer to measure when the temperature of the aluminum box drops to -85°C. Place a straw containing Dorper ram semen balance solution in the aluminum box for continuous precooling for 10 minutes. Then, place the straw in liquid nitrogen for freezing for 5 minutes to obtain Dorper ram frozen semen.

[0067] Example 5: Cryopreservation of Suffolk ram semen

[0068] (1) Fresh semen collection: The pseudo-vaginal method disclosed in the article published by Liang Guorong et al. (Liang Guorong, Liu Bohe, Zhang Mingliang, et al. Application and promotion effect of normal temperature artificial insemination technology of sheep [J]. Gansu Animal Husbandry and Veterinary Medicine, 2022, 52(10): 38-40) was used to collect fresh semen of Suffolk sheep rams;

[0069] (2) Prefreezing and long-distance transportation of semen: The semen freezing solution A prepared in Example 1 was preheated to 32° C. in a water bath to obtain preheated semen freezing solution A; 1 mL of fresh semen of Suffolk ram with a sperm motility of 0.86 was taken and mixed evenly with 3 mL of preheated semen freezing solution A in a centrifuge tube to obtain a Suffolk ram semen pre-frozen solution; then, the centrifuge tube containing the Suffolk ram semen pre-frozen solution was wrapped with 8 layers of gauze and placed upright in an ice box for long-distance transportation, the transportation distance was 250 km, the transportation time was 3 h, and the Suffolk ram semen pre-frozen solution was gradually cooled by the ice box during transportation;

[0070] (3) Cryopreservation of semen: The Suffolk sheep ram semen pre-frozen solution that has been transported over long distances is transferred to a 4°C refrigerator for cryopreservation for 48 hours, and then the Suffolk sheep ram semen pre-frozen solution is centrifuged at 1500 rpm for 10 minutes to obtain the Suffolk sheep ram sperm; the Suffolk sheep ram sperm and 6 mL of the semen freezing solution B prepared in Example 1 are mixed evenly in a centrifuge tube, and then allowed to stand at 4°C for 3 hours to obtain the Suffolk sheep ram semen equilibrium solution;

[0071] (4) Cryopreservation of semen: Place an aluminum box on a liquid nitrogen surface for precooling. Use an ultra-low temperature thermometer to measure when the temperature of the aluminum box drops to -85°C. Place a straw containing Suffolk ram semen balance solution in the aluminum box for continuous precooling for 10 minutes. Then, place the straw in liquid nitrogen for cryopreservation for 5 minutes to obtain Suffolk ram frozen semen.

[0072] Example 6: Cryopreservation of Tan Sheep Ram Semen

[0073] (1) Fresh semen collection: The pseudo-vaginal method disclosed in the article published by Liang Guorong et al. (Liang Guorong, Liu Bohe, Zhang Mingliang, et al. Application and promotion effect of normal temperature artificial insemination technology of sheep [J]. Gansu Animal Husbandry and Veterinary Medicine, 2022, 52(10): 38-40) was used to collect fresh semen of Tan sheep rams;

[0074] (2) Prefreezing and long-distance transportation of semen: The semen freezing solution A prepared in Example 1 was preheated to 32° C. in a water bath to obtain preheated semen freezing solution A; 1 mL of fresh semen of a Tan sheep breed ram with a sperm motility of 0.89 was taken and mixed evenly with 3 mL of preheated semen freezing solution A in a centrifuge tube to obtain a Tan sheep breed ram semen pre-frozen solution; then, 8 layers of gauze were wrapped around the centrifuge tube containing the Tan sheep breed ram semen pre-frozen solution and the tube was placed upright in an ice box for long-distance transportation, with a transportation distance of 400 km and a transportation time of 4 h. During the transportation process, the Tan sheep breed ram semen pre-frozen solution was gradually cooled by the ice box;

[0075] (3) Low temperature storage of semen: The pre-frozen semen solution of Tan sheep ram that has been transported over long distances is transferred to a refrigerator at 4°C for low temperature storage for 24 hours, and then the pre-frozen semen solution of Tan sheep ram is centrifuged at 1500 rpm for 10 minutes to obtain the precipitate to obtain Tan sheep ram sperm; the Tan sheep ram sperm and 6 mL of the semen freezing solution B prepared in Example 1 are mixed evenly in a centrifuge tube, and then allowed to stand at 4°C for 3 hours to obtain Tan sheep ram semen equilibrium solution;

[0076] (4) Cryopreservation of semen: Place an aluminum box on a liquid nitrogen surface for precooling. Use an ultra-low temperature thermometer to measure when the temperature of the aluminum box drops to -85°C. Place a straw containing Tan sheep ram semen balance solution in the aluminum box for continuous precooling for 10 minutes. Then, place the straw in liquid nitrogen for freezing for 5 minutes to obtain Tan sheep ram frozen semen.

[0077] Experimental Example 1: Detection of sperm motility in livestock semen

[0078] The sperm motility of the Dorper ram semen, Suffolk ram semen and Tan ram semen described in Examples 4 to 6 at different processing stages was tested, specifically:

[0079] (1) Sperm motility of fresh semen: The sperm motility of fresh semen of various rams was examined and counted using an ordinary optical microscope to obtain the sperm motility of fresh semen of various rams;

[0080] (2) Sperm motility of pre-frozen semen after transportation: The sperm motility of various ram semen pre-frozen semen after long-distance transportation was examined and counted using an ordinary optical microscope to obtain the sperm motility of various ram semen pre-frozen semen after transportation;

[0081] (3) Sperm motility of the semen pre-frozen solution after cryopreservation: The sperm motility of various ram semen pre-frozen solutions after cryopreservation was examined and counted using an ordinary optical microscope to obtain the sperm motility of various ram semen pre-frozen solutions after cryopreservation;

[0082] (4) Sperm motility after thawing: The frozen semen of various rams that had been frozen in liquid nitrogen for 5 min were thawed in a water bath at 38.8°C for 17 s. The sperm motility of the frozen semen of various rams after thawing was examined and counted using an ordinary optical microscope to obtain the sperm motility of the semen of various rams after thawing;

[0083] The sperm motility test results of various ram semens at different processing stages described in Examples 4 to 6 are shown in Table 1.

[0084] Table 1 Sperm motility of ram semen

[0085]

[0086] The results show that, when the semen cryopreservation solution A, the semen cryopreservation solution B and the livestock semen cryopreservation method prepared in Example 1 were used to treat Dorper ram semen, Suffolk ram semen and Tan sheep ram semen, the sperm motility of various ram semen after long-distance transportation and low-temperature storage was greater than 0.7, all of which met the requirements for semen cryopreservation, and the sperm of various ram frozen semen still had a high motility after thawing.

[0087] Experimental Example 2: Effect of frozen livestock sperm on in vitro fertilization

[0088] The frozen semen of Dorper ram, Suffolk ram and Tan ram described in Examples 4 to 6 were thawed in a water bath at 38.8°C and then subjected to in vitro fertilization experiments, specifically:

[0089] (1) Ovarian collection and transportation: Dorper sheep ovaries, Suffolk sheep ovaries, and Tan sheep ovaries were collected from the slaughterhouse, immediately placed in 33°C saline with double antibiotics (i.e., 1% penicillin and 1% streptomycin), and transported to the laboratory within 4 hours;

[0090] (2) Ovarian processing: Excess fat tissue and connective tissue around each sheep ovary were cut off, and the ovaries were washed once with 75% alcohol, and then rinsed three times with physiological saline with double antibody. Each sheep ovary was placed in a 10 mm culture dish and added with egg collection fluid. Then, the follicles within 6 mm in diameter of each sheep ovary were cut by cutting method, and the cumulus oocyte complexes (COCs) with normal morphology, uniform cytoplasm, and no less than 3 layers of granulosa cells were collected under SMZ745 stereo microscope (Nikon, Japan) using a mouth pipette; the formula of the egg collection fluid was: TCM-199 + 2% FCS + 10 μg / mL sodium heparin + 1% penicillin + 1% streptomycin;

[0091] (3) In vitro maturation of oocytes: The collected sheep COCs were transferred into oocyte in vitro maturation medium (BO-IVM) that had been incubated for 4 hours in advance, and then cultured for 24 hours at 38.5°C, 5% CO2, and saturated humidity to obtain mature oocytes;

[0092] (4) In vitro fertilization: 45 mature oocytes from each sheep were added to 400 μL of in vitro fertilization solution (BO-IVF), and the final concentration of each was 3.0×10 6 The capacitated sperm of Dorper ram frozen semen, Suffolk ram frozen semen and Tan ram frozen semen with a concentration of 100 / mL were thawed and incubated at 38.5°C, 5% CO2 and saturated humidity for 24 hours to obtain presumptive fertilized eggs.

[0093] (5) Early embryo in vitro culture: The presumed fertilized eggs of each sheep were transferred into early embryo in vitro culture medium (BO-IVC) and cultured at 38.5°C, 5% CO2, and saturated humidity for 24 h. The cleavage was observed under a SMZ745 stereo microscope and the cleavage rate was calculated. Then, on the 7th day of culture, the blastocyst development was examined and the blastocyst development rate was calculated. The results are shown in Table 2.

[0094] The calculation formula of cleavage rate is: cleavage rate (%) = number of cleavages / number of oocytes × 100%;

[0095] The calculation formula of blastocyst development rate is: blastocyst rate (%) = number of blastocysts / number of cleavages × 100%.

[0096] Table 2 Results of in vitro fertilization of capacitated sperm of rams

[0097]

[0098] The results showed that, when the semen freezing solution A, the semen freezing solution B and the freezing method for livestock semen prepared in Example 1 were used to treat Dorper ram semen, Suffolk ram semen and Tan sheep ram semen, the capacitated sperm thawed after freezing of various ram semen still had normal fertilization ability, and after in vitro fertilization, the cleavage rate and blastocyst development rate of the fertilized eggs were within the normal range.

[0099] As can be seen from the above embodiments, the present invention provides a method for freezing livestock semen and its application. After the livestock semen is transported over long distances and cryopreserved using the method for freezing livestock semen of the present invention, the sperm motility of the livestock semen can still meet the requirements for semen freezing, and the sperm of the frozen semen still has a very high motility after thawing, and the cleavage rate and blastocyst development rate after in vitro fertilization are both within the normal range, indicating that the fertilization ability of the frozen semen is normal. It shows that the method for freezing livestock semen of the present invention has the advantages of being simple and easy to operate, reducing waste, and being able to improve the quality of livestock semen and sperm motility.

[0100] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for freezing livestock semen, characterized in that: The steps include: (1) mixing 1 mL of livestock fresh semen and 2-5 mL of semen freezing solution A to obtain a livestock semen pre-frozen solution; (2) transporting the livestock semen pre-frozen solution over long distances and / or storing it at low temperatures, centrifuging it, and collecting the precipitate to obtain livestock sperm; (3) mixing the livestock sperm and 5 to 8 mL of semen freezing solution B, and allowing to stand to obtain a livestock semen balancing solution; (4) precooling the livestock semen balance solution and then freezing it in liquid nitrogen to obtain livestock frozen semen; The sperm freezing solution A comprises a base solution and egg yolk; The semen freezing solution B comprises semen freezing solution A and glycerol; The base solution uses water as solvent and includes the following components in concentrations: Tris 10-50 g / L, citric acid 10-25 g / L, glucose 10-20 g / L, penicillin 0.1-1 g / L, and streptomycin 0.1-2 g / L; In the sperm freezing solution A, the volume ratio of the base solution to the egg yolk is 70-90:10-30; In the semen freezing solution B, the volume ratio of semen freezing solution A to glycerol is 94-96:4-6.

2. The cryopreservation method according to claim 1, characterized in that: The sperm motility of the livestock fresh semen in step (1) is greater than 0.

8.

3. The cryopreservation method according to claim 2, characterized in that: The long-distance transportation distance of step (2) is 200 to 400 km, and the long-distance transportation time is 2 to 4 hours; The temperature of the low-temperature storage in step (2) is 3 to 6° C., and the time of the low-temperature storage is 12 to 48 hours.

4. The cryopreservation method according to claim 3, characterized in that: The centrifugal speed in step (2) is 1000-2000 rpm, and the centrifugal time is 10-15 min.

5. The cryopreservation method according to claim 4, characterized in that: The standing temperature in step (3) is 3 to 6° C., and the standing time is 2 to 4 hours.

6. The cryopreservation method according to claim 5, characterized in that: The initial precooling temperature in step (4) is -90 to -80°C, and the precooling time is 5 to 15 minutes.

7. The cryopreservation method according to claim 6, characterized in that: The sperm motility of the livestock frozen semen after thawing in step (4) is greater than 0.

4.

8. Use of the cryopreservation method according to any one of claims 1 to 7 in cryopreservation of livestock semen.

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