New Use of MA-5 and Diluent for Preservation of Porcine Semen at Room Temperature

Abstract

The present invention discloses a new use of MA-5 and a dilution solution for preserving pig semen at room temperature, belonging to the technical field of biological reproduction. The present invention uses MA-5 for preserving pig semen at room temperature and provides a dilution solution for preserving semen at room temperature, with the following composition: 25.0-30.0 g / L of D-glucose, 2.0-2.7 g / L of disodium EDTA, 6.5-7.5 g / L of sodium citrate, 2.5-3.5 g / L of citric acid, 0.5-1.5 g / L of sodium bicarbonate, 5.0-6.0 g / L of Tris, 50-1.5 million IU / L of penicillin, 50-1.5 million IU / L of streptomycin, 20-600,000 IU / L of polymyxin, and 0.01-10 nmol / L of MA-5. This dilution solution can enable the motility of pig semen to still reach over 80% after being stored at room temperature for one week, effectively solving the timeliness problem of fresh semen artificial insemination.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological reproduction, and particularly relates to a new use of MA-5 and a diluent for preserving pig semen at room temperature. Background Art

[0002] In China, the technology of artificial insemination of pigs started relatively late. Artificial insemination experiments began in the 1950s and were promoted and applied in the 1960s. However, with the reform and opening up, the number of imported breeds increased. The estrus of imported breeds was not obvious, and it was impossible to accurately identify estrus, making it difficult to master the insemination time, resulting in infertility after multiple inseminations of sows, a decrease in conception rate, and a decrease in litter size. These problems that occurred during the artificial insemination process hindered the popularization of artificial insemination technology. In the 1990s, with the further development of artificial insemination technology, the artificial insemination technology was gradually recognized and promoted by large-scale pig farms. And with the development of world trade, a large number of foreign pork flooded into China, causing a huge impact on the development of China's pig industry. Facing the increasingly fierce market competition, it is necessary to reduce costs, raise excellent breeds, and improve the competitiveness of products. The artificial insemination technology has gradually been popularized throughout the country with its unique advantages. The artificial insemination technology has relatively high requirements for the quality of boar semen, and non-standard semen preservation will also affect the effect of artificial insemination.

[0003] The preservation of semen is divided into preservation at room temperature (17°C), preservation at low temperature (4°C), and cryopreservation (-196°C). Due to the characteristics of the structure of pig sperm, that is, the ratio of cholesterol / phospholipid in pig sperm is low and the ratio of unsaturated fatty acids is high, pig sperm is very sensitive to cold shock at low temperature and reactive oxygen free radicals. Therefore, during the preservation of pig semen, the method of preservation at room temperature is usually adopted. Although "pig semen at room temperature" (GB23238-2009) can be preserved for more than 72 hours when the motility is 60%, for the intensive, large-scale, and specialized production of pigs, this motility level still cannot meet the actual production needs at present. At the same time, extending the preservation time of pig semen also helps to further promote the artificial insemination technology of pigs. However, as the preservation time prolongs, the reactive oxygen free radicals generated by pig sperm will gradually increase, and excessive reactive oxygen free radicals will damage sperm, resulting in a decrease in sperm motility and other problems, affecting the preservation quality.

[0004] During the room-temperature preservation of boar semen, it is affected by various factors such as temperature, osmotic pressure, pH, dilution ratio, antioxidant, buffer, bacteria, etc. The two major factors that cause the continuous decline of semen quality with the extension of preservation time are reactive oxygen species and microorganisms. Excessive reactive oxygen species will damage the structure and function of sperm, while the growth of microorganisms will consume a large amount of nutrients of sperm and increase the risk of reducing porcine reproductive function. At present, the main formulations of diluents developed at home and abroad are as follows: sodium citrate - saccharide, Tris - citric acid - saccharide, and disodium EDTA - sodium citrate - saccharide. Diluents with different formulations have differences in specific component compositions and different protective effects on sperm. The commercial Modena diluent extends the preservation time of semen to a certain extent compared with other diluents, but the Modena diluent lacks antioxidant substances and cannot effectively scavenge the reactive oxygen free radicals generated by sperm during semen preservation, which is not conducive to semen preservation. In addition, during the preservation process, sperm continuously consumes energy, and when the energy supply is insufficient, sperm will die rapidly.

[0005] Mitochonic acid-5(MA-5) is a newly synthesized plant hormone indole-3-acetic acid derivative, and its full English name is (4-(2,4-difluorophenyl)-2-(1H-indole-3-yl)-4-oxobutanoic acid. At present, MA-5 is mainly applied in apoptosis and kidney diseases, and there is no prior art to disclose its application in the room-temperature preservation of boar semen. Summary of the Invention

[0006] Based on the requirement of semen preservation in the expansion of the artificial insemination radiation range of improved male livestock stations to surrounding enterprises, farmers (farms), through the screening and ratio optimization of energy supply and antioxidant components in the diluent, a diluent formulation that can efficiently preserve boar semen (preserved for more than one week at room temperature) is developed. Compared with commercial diluents (such as Modena diluent), it can better overcome the adverse effects of actual situations such as the semen collection time limit and the distance of insemination sites on giving full play to the utilization rate of improved boars and realizing the sharing of improved breed resources.

[0007] The technical solution of the present invention is as follows:

[0008] The application of MA-5 in the room-temperature preservation of boar semen. Preferably, the application includes the application in improving semen preservation duration, sperm motility rate, sperm motility, plasma membrane integrity rate, acrosome integrity rate, mitochondrial membrane potential, ATP content, and reducing the ROS level after sperm preservation.

[0009] A room-temperature preservation diluent for porcine semen, which is composed of the following components: 25.0 - 30.0 g / L of D-glucose, 2.0 - 2.7 g / L of disodium EDTA, 6.5 - 7.5 g / L of sodium citrate, 2.5 - 3.5 g / L of citric acid, 0.5 - 1.5 g / L of sodium bicarbonate, 5.0 - 6.0 g / L of Tris, 500,000 - 1,500,000 IU / L of penicillin, 500,000 - 1,500,000 IU / L of streptomycin, 200,000 - 600,000 IU / L of polymyxin, and 0.01 - 10 nmol / L of MA-5.

[0010] In a specific embodiment, the room-temperature preservation diluent for porcine semen is composed of the following components: 27.5 g / L of D-glucose, 2.35 g / L of disodium EDTA, 6.9 g / L of sodium citrate, 2.9 g / L of citric acid, 1.0 g / L of sodium bicarbonate, 5.65 g / L of Tris, 1,000,000 IU / L of penicillin, 1,000,000 IU / L of streptomycin, 400,000 IU / L of polymyxin, and 0.01 nmol / L of MA-5.

[0011] In a specific embodiment, the room-temperature preservation diluent for porcine semen is composed of the following components: 27.5 g / L of D-glucose, 2.35 g / L of disodium EDTA, 6.9 g / L of sodium citrate, 2.9 g / L of citric acid, 1.0 g / L of sodium bicarbonate, 5.65 g / L of Tris, 1,000,000 IU / L of penicillin, 1,000,000 IU / L of streptomycin, 400,000 IU / L of polymyxin, and 0.1 nmol / L of MA-5.

[0012] In a specific embodiment, the room-temperature preservation diluent for porcine semen is composed of the following components: 27.5 g / L of D-glucose, 2.35 g / L of disodium EDTA, 6.9 g / L of sodium citrate, 2.9 g / L of citric acid, 1.0 g / L of sodium bicarbonate, 5.65 g / L of Tris, 1,000,000 IU / L of penicillin, 1,000,000 IU / L of streptomycin, 400,000 IU / L of polymyxin, and 1 nmol / L of MA-5.

[0013] In a specific embodiment, the room-temperature preservation diluent for porcine semen is composed of the following components: 27.5 g / L of D-glucose, 2.35 g / L of disodium EDTA, 6.9 g / L of sodium citrate, 2.9 g / L of citric acid, 1.0 g / L of sodium bicarbonate, 5.65 g / L of Tris, 1,000,000 IU / L of penicillin, 1,000,000 IU / L of streptomycin, 400,000 IU / L of polymyxin, and 10 nmol / L of MA-5.

[0014] A method for room-temperature preservation of porcine semen, which comprises the following steps:

[0015] Mix the above-mentioned diluent stored at room temperature with porcine semen and store it at 15-20°C.

[0016] In the above method for storing porcine semen at room temperature, the mixing specifically includes the following steps: Dilute the freshly collected porcine semen with the above-mentioned diluent stored at room temperature, and dilute the density of porcine sperm to 1.5-2.0×10 8 per mL.

[0017] In the above method for storing porcine semen at room temperature, before mixing, preheat the diluent stored at room temperature to 37-38.5°C to keep it isothermal with the fresh porcine semen.

[0018] In the above method for storing porcine semen at room temperature, the sperm motility rate of the fresh porcine semen is above 80%.

[0019] In the above method for storing porcine semen at room temperature, after the diluent stored at room temperature and porcine semen are mixed, mix them once every 8-12 hours during the storage period to prevent sperm sedimentation and affect the storage effect.

[0020] The beneficial effects of the present invention are as follows:

[0021] The present invention first proves that MA-5 plays an important role in improving the performance of porcine semen stored at room temperature. Based on this, the present invention provides a diluent for storing semen at room temperature. This diluent can make the vitality of porcine semen still reach more than 80% after being stored at room temperature for one week, effectively solving the timeliness problem of fresh semen artificial insemination. At the same time, the operation of storing porcine semen with this diluent is simple and convenient, facilitating large-scale popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Shows the effect of MA-5 on the mitochondrial function of porcine sperm (WB results); among them, Figure A is MT-ND1, and Figure B is MT-ND6;

[0023] Figure 2 Shows the effect of MA-5 on the mitochondrial transcription and translation of porcine sperm (qPCR results); among them, Figure A is MT-ND1, and Figure B is MT-ND6.

[0024] Note: MT-ND1 and MT-ND6 are genes related to respiratory chain complex I in the mitochondrial genome, and complex I participates in the oxidative phosphorylation pathway to supply energy for sperm. DETAILED DESCRIPTION OF THE INVENTION

[0025] I. Reagents

[0026] Mitochondrial acid 5 standard, purity ≥ 98%, glucose, disodium EDTA, sodium citrate, citric acid, sodium bicarbonate, Tris, penicillin, streptomycin, ATP content detection kit (Beijing Solarbio Science & Technology Co., Ltd.); FITC-PNA (Sigma); MitoSOX TM Mitochondrial superoxide indicator, enhanced mitochondrial membrane potential detection kit (Beyotime Biotechnology Co., Ltd.), etc. In the present invention, Tris refers to Tris(hydroxymethyl)aminomethane, i.e., Tris buffer solution.

[0027] II. Diluent

[0028] Formulation components and contents of the diluent: D-glucose at 27.5 g / L, disodium EDTA at 2.35 g / L, sodium citrate at 6.9 g / L, citric acid at 2.9 g / L, sodium bicarbonate at 1.0 g / L, Tris at 5.65 g / L, penicillin at 1 million IU / L, streptomycin at 1 million IU / L, polymyxin at 400,000 IU / L, and MA-5 at 0.01 nmol / L, 0.1 nmol / L, 1 nmol / L, or 10 nmol / L. Weigh each component of the above diluent formulation according to the content and mix well with double-distilled water, then make up the volume to 1 L to obtain the diluent for pig semen preservation at room temperature, with a pH value of about 7.2.

[0029] In the present invention, glucose serves as an energy source to supply energy for the life activities of sperm to maintain a relatively high sperm motility and sperm viability during the preservation of pig semen. Disodium EDTA, sodium citrate, citric acid, sodium bicarbonate, and Tris act as buffer substances (among them, disodium EDTA is a chelating agent, and sodium citrate, citric acid, sodium bicarbonate, and Tris are non-toxic and have pH regulation and good buffer stability), enabling pig semen to maintain a constant pH value and osmotic pressure during preservation, thereby maintaining the integrity rate of the plasma membrane and acrosome of pig sperm. On this basis, MA-5 added in the diluent of the present invention according to the ratio has the effects of anti-aging, preventing cell apoptosis, and enhancing sperm energy metabolism, etc., which can significantly improve the sperm motility after preservation at room temperature, reduce the concentration of reactive oxygen species (ROS) in pig sperm, improve sperm motility performance and sperm energy metabolism, and thus improve sperm viability. At the same time, in addition to the two antibiotics penicillin and streptomycin, the diluent of the present invention also contains polymyxin as an active ingredient that destroys the bacterial cell wall and exerts a bactericidal effect during the reproduction period of bacterial cells, which can effectively control the colony number of bacteria in pig semen and has no toxic effect on sperm, thereby improving the sperm viability.

[0030] III. Semen quality assessment

[0031] Determination of sperm viability, motility, and other motility parameters:

[0032] Take 1 mL of the sample (for example, semen stored at room temperature), incubate it at 37 °C for 10 min, then take 10 μL of the sample and use a computer-aided analysis system (CASA) to measure sperm motility rate, vitality, and other sperm motility parameters (mainly including the straight-line motility rate and curvilinear motility rate) under a 250-fold microscope field of view; when measuring, select more than 6 different fields of view, with the number of sperm in each field of view being more than 200 and evenly distributed for detection.

[0033] Detection of sperm plasma membrane integrity:

[0034] The SYBR-14 / PI fluorescence dye method is used for detection. Take 1 mL of semen into a 1.5 mL centrifuge tube, incubate it in a 37 °C incubator for 15 min, take 100 μL of the sample into a new centrifuge tube, add 0.1 μL of SYBR-14 working solution, mix well and incubate it in a 37 °C incubator for 5 min, then add 0.5 μL of PI working solution, mix well, and incubate it in the dark for 10 min. Take 5 μL of the sample to be tested on a glass slide, cover it with a coverslip, randomly take more than 5 fields of view under a 200-fold upright fluorescence microscope, with the number of sperm in each field of view being more than 200, count the integrity of the sperm plasma membrane, and statistically analyze the sperm plasma membrane integrity rate.

[0035] Detection of sperm acrosome integrity:

[0036] The FITC-PNA / PI staining method is used for detection. Take 20 μL of the sample and smear it on a glass slide, air-dry it naturally, fix it in absolute methanol for 10 min, and air-dry it naturally in the air; soak and wash the glass slide twice with PBS, take 20 μL of FITC-PNA working solution, evenly smear it on the glass slide to ensure coverage of the sample, then place the glass slide in a wet box and incubate it in a 37 °C incubator for 30 min, take out the glass slide, soak and wash the glass slide twice with PBS; then add 20 μL of PI and continue to incubate it in a 37 °C incubator for 5 min, soak and wash the glass slide twice with PBS. After washing off the floating color, randomly take more than 5 fields of view for photography under a 200-fold fluorescence microscope in the dark, with the number of sperm in each field of view being more than 200, count the integrity of the sperm acrosome, and statistically analyze the sperm acrosome integrity rate.

[0037] Detection of sperm mitochondrial membrane potential:

[0038] The mitochondrial activity of sperm was detected using the kit fluorescent probe JC-1. 10 μL of JC-1 (200×) was added to 2 mL of JC-1 staining buffer for preparation. 100 μL of sperm sample was stained with 0.5 mL (1×) staining working solution, and pipetted several times to mix evenly, then incubated in the cell culture incubator at 37 °C in the dark for 20 min. After incubation at 37 °C, centrifuge at 600 g, 4 °C for 3 min and discard the supernatant. Subsequently, wash the sample 2 times with 1× staining buffer, and resuspend the sample with 1× staining buffer. After resuspension, use flow cytometry to evaluate the mitochondrial activity.

[0039] Detection of ATP content in sperm:

[0040] Detection was carried out using an ATP content detection kit. Take 100 μL of the sample, centrifuge at 3000 g / min for 5 min, discard the supernatant, resuspend with Modena diluent and repeat the centrifugation operation twice. Add a certain volume of ATP extraction solution, and perform ultrasonic fragmentation for 1 min (ice bath, intensity 20% or 200 W, ultrasound for 2 s, pause for 1 s), centrifuge at 10000 g, 4 °C for 10 min; take the supernatant to another EP tube, add 500 μL of chloroform, shake well, centrifuge at 10000 g, 4 °C for 3 min, take the supernatant, add a certain volume of ATP detection working solution and a certain volume of the supernatant after sperm lysis and centrifugation to the 96-well plate, use a multi-functional microplate reader to detect the ATP concentration, and detect the BCA concentration. The whole experiment was carried out under dark conditions; ATP content = ATP concentration / BCA concentration.

[0041] Detection of the level of reactive oxygen species (ROS) in sperm:

[0042] Detection was carried out using a ROS content detection kit. Take 100 μL of the sample, add 200 μL of Modena diluent, mix evenly, centrifuge at 800 g / min for 5 min, discard the supernatant, add a certain volume of ROS working solution (DCFH-DA) according to the kit instructions, incubate in a water bath at 37 °C for 20 min, mix evenly every 3 - 5 min, centrifuge at 1000 g / min for 5 min after incubation, discard the supernatant, resuspend with Modena diluent and repeat the centrifugation operation twice, and use flow cytometry for detection. The whole experiment was carried out under dark conditions.

[0043] Sperm Western Blot:

[0044] This experiment can verify whether MA-5 has an impact on the expression level of mitochondrial proteins. The WB experiment was conducted as described in previous literature (Li et al., 2022). Briefly, sperm cell proteins were lysed in RIPA buffer containing a protease inhibitor mixture, and after completion, a BCA protein concentration assay kit was used. Total proteins were separated by 10% SDS-PAGE gels. Then the proteins were transferred to PVDF membranes (GE Bioscience, Newark, NJ, USA), blocked with 5% BSA (dissolved in TBST) for 1 hour, and the membranes were incubated with primary antibodies overnight at 4°C. The next day, the PVDF membranes were washed with TBST and incubated with secondary antibodies (1:1000 in TBST) for 1 h at room temperature. Finally, the protein bands were visualized and analyzed.

[0045] Sperm quantitative real-time PCR (qPCR):

[0046] To verify the results of the WB experiment and clarify whether MA-5 has an impact on the transcription and translation of mitochondrial genes, sperm quantitative real-time PCR was performed, and the specific steps are as follows:

[0047] (1) Prepare samples: Use an animal cell total RNA extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing) to extract RNA samples for reverse transcription and amplification to obtain the cDNA samples to be tested.

[0048] (2) Prepare the reaction solution: Mix the PCR enzyme, primers, template DNA, and fluorescent probes in the PCR kit evenly, and add an appropriate volume of reaction buffer and deionized water to prepare sufficient reaction solution.

[0049] (3) Plate: Distribute the reaction solution evenly into a 96-well plate, avoiding the generation of bubbles.

[0050] (4) Place the samples: Add the samples to be tested into the 96-well plate, taking care to avoid sample contamination.

[0051] (5) Put into the PCR instrument: Place the 96-well plate into the PCR instrument and perform the PCR amplification reaction according to the temperature and time program set in the experiment.

[0052] (6) Export data: Export the data corresponding to the 96-well plate from the computer connected to the PCR instrument.

[0053] Other materials used in the present invention, unless otherwise stated, can be obtained through commercial channels. Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below with reference to specific examples and data. The following examples are only for illustrative purposes of the present invention and do not limit the scope of the present invention in any way.

[0054] Example 1

[0055] The boar semen used in the experiment was all from a certain pig farm in Binzhou City, Shandong Province. The semen of a total of 25 healthy adult (1 - 3 years old) Landrace boars was collected by the hand - holding method. The sperm motility, vitality and density of the freshly collected semen were detected at 37°C, so as to evaluate the quality of the semen of each boar. The results are shown in Table 1. The sperm density of the freshly collected semen of the boars was in the range of 3.0 - 5.0×10 8 per mL, and the sperm motility was in the range of 80 - 92%. The sperm motility was greater than 80% and above, and the sperm density ≥2×10 8 per mL could be used for subsequent experiments. According to the volume of the freshly collected semen (at a temperature of 37°C) and the sperm density, the prepared diluent (pre - heated to 37°C) was added until the sperm density was 1.5 - 2.0×10 8 per mL, and then it was sub - packed and stored in an environment of 17°C for more than one week. During the storage period, it was inverted and mixed evenly every 12 hours. Taking the Modena diluent as the experimental control, the preservation quality of the freshly collected semen was compared.

[0056] The formula of the Modena diluent is as follows: D - glucose at 27.5 g / L, disodium EDTA at 2.35 g / L, sodium citrate at 6.9 g / L, citric acid at 2.9 g / L, sodium bicarbonate at 1.0 g / L, Tris at 5.65 g / L, penicillin at 1 million IU / L, streptomycin at 1 million IU / L, polymyxin at 400,000 IU / L.

[0057] The test results are shown in Table 1:

[0058] Table 1

[0059]

[0060] As can be seen from Table 1, after adding MA - 5 to the semen diluent and storing it at room temperature for one week, the sperm motility in the pig semen could still reach more than 80% (significantly higher than 70%), the plasma membrane integrity rate reached more than 80%, the acrosome integrity rate reached more than 76%, the mitochondrial membrane potential and ATP content were significantly higher than those of the experimental control, and the ROS level was significantly lower than that of the Modena diluent except for the 10 nmol / L group.

[0061] The results of WB and qPCR showed that, as Figure 1As shown, MA-5 can affect the expression level of mitochondrial proteins. In particular, the effect of 1 nmol / L MA-5 on the expression level of sperm mitochondrial proteins is significant. Thus, it can be seen that MA-5 improves the energy supply of mitochondria by affecting the oxidative phosphorylation pathway. Since it is known that MA-5 can affect the expression level of mitochondrial proteins, it is necessary to determine whether MA-5 affects the expression of sperm mitochondrial proteins through the transcription and translation of mitochondrial genes. As Figure 2 shown, MA-5 can affect the transcription and translation of mitochondrial genes. In particular, the effect of 1 nmol / L MA-5 on the transcription and translation of sperm is significantly higher than that of other groups. The above results show that MA-5 regulates the expression level of mitochondrial proteins by affecting the transcription and translation of mitochondria, thereby affecting the energy supply of the oxidative phosphorylation pathway. By increasing the energy supply of mitochondria and the content of ATP, the sperm motility can be improved.

[0062] The above results indicate that MA-5 significantly increases the ATP content of sperm during storage through the oxidative phosphorylation pathway. In addition, MA-5 can significantly improve the antioxidant capacity of sperm during the storage of porcine semen, scavenging excessive oxygen free radicals generated during semen storage, ensuring sufficient energy supply for sperm during room temperature storage, and extending the storage time of semen. It can be seen that adding MA-5 to the diluent not only solves the problem of oxidative damage of sperm during the storage of porcine semen, reducing the degree of oxidative damage of porcine sperm, but also provides energy guarantee for sperm during storage.

[0063] In summary, the diluent formula of the present invention has a good effect on the room temperature storage of boar semen, with a long semen storage time of more than one week. It can effectively improve sperm motility, plasma membrane integrity rate, acrosome integrity rate, mitochondrial membrane potential, and ATP content, and reduce the sperm ROS level. After storing fresh porcine semen at room temperature for one week, the sperm motility can still reach more than 80%, fully meeting the requirements of long-distance transportation of porcine semen and the needs of breeding livestock stations to expand the radiation range of artificial insemination, which is conducive to improving the production efficiency of artificial insemination, increasing the utilization rate of excellent boars and obtaining a high conception rate, reducing the spread of diseases, realizing the sharing of improved breed resources, and protecting the continuation of local breed pigs. At the same time, the diluent formula is simple and easy to prepare, facilitating large-scale popularization and application to improve the room temperature storage effect of porcine semen, making pig production more large-scale, professional, and intensive.

[0064] The above is only a preferred embodiment of the present invention and is not a limitation of the present invention in other forms. Any person skilled in the art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still belong to the protection scope of the technical solution of the present invention.

Claims

1. A room temperature preservation diluent for boar semen, characterized in that, It consists of the following components: D-glucose at 27.5 g / L, disodium EDTA at 2.35 g / L, sodium citrate at 6.9 g / L, citric acid at 2.9 g / L, sodium bicarbonate at 1.0 g / L, Tris at 5.65 g / L, penicillin at 1 million IU / L, streptomycin at 1 million IU / L, polymyxin at 400,000 IU / L, and MA-5 at 0.01 - 10 nmol / L.

2. The dilution solution for normal temperature preservation of boar semen according to claim 1, wherein The dosage of the said MA-5 is selected from 0.01 nmol / L, 0.1 nmol / L, 1 nmol / L, or 10 nmol / L.

3. A method for storing pig semen at room temperature, characterized in that, It includes the following steps: Mix the diluent for room temperature storage as described in any one of claims 1 - 2 with porcine semen, and store it at 15 - 20 °C.

4. The method according to claim 3, characterized in that, The mixing specifically includes the following steps: diluting the freshly collected porcine semen with the above-mentioned room temperature preservation diluent, and diluting the density of porcine sperm to 1.5 - 2.0×10 8 cells / mL.

5. The method according to claim 3, wherein Before mixing, preheat the diluent for room temperature storage to 37 - 38.5 °C to keep it isothermal with fresh porcine semen.

6. The method according to claim 3, wherein The sperm motility of the said fresh porcine semen is above 80%.

7. The method according to claim 3, characterized in that, After the mixing of the diluent for room temperature storage and porcine semen is completed, mix it once every 8 - 12 h during storage to prevent sperm sedimentation and affect the storage effect.

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