Boar semen normal-temperature preservation diluent as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of a diluent for normal-temperature preservation of boar semen, and belongs to the technical field of biology. The normal-temperature preservation diluent comprises the following main components: glucose, citric acid, sodium citrate, sodium bicarbonate, tris (hydroxymethyl) aminomethane, disodium ethylene diamine tetraacetate, penicillin G sodium salt, streptomycin sulfate, arbutin, double distilled water and the like. The diluent can maintain high vitality of pig sperms in a short time, can be stored at normal temperature for about 10 days, and meets the requirements of artificial insemination. The arbutin of the diluent has good anti-inflammatory and anti-oxidation protection effects, effectively improves the preservation activity and effect of the pig sperms, and can be used for direct semen deposition after dilution or short-term preservation after dilution and semen deposition after short-distance transportation. According to the diluent, the preservation quality of the boar semen is improved with low production cost and a simple operation method, the utilization rate of excellent breeding stock is increased, the reproductive rate is increased, and the diluent is convenient to popularize and apply.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a pig semen room-temperature storage diluent and a preparation method and application thereof. Background Art

[0002] In the process of pig reproduction, artificial insemination and semen preservation technology are the most widely used biotechnologies. Globally, most semen is stored in liquid form at 15-18°C, and sperm metabolism is reduced by dilution and cooling to extend the semen preservation time. However, during the semen preservation process, the antioxidant capacity of the diluted seminal plasma decreases, reactive oxygen free radicals (ROS) gradually accumulate, the redox homeostasis in pig semen is destroyed, and the preservation quality is also reduced.

[0003] The plasma membrane of sperm is rich in unsaturated fatty acids, which are easily attacked by ROS and cause lipid peroxidation. However, the antioxidant capacity of sperm itself is limited, and ROS gradually accumulates during short-term transportation and storage. The accumulated ROS will damage lipids, proteins and DNA, leading to cell apoptosis, changing enzyme activity, reducing sperm vitality and motility, and ultimately affecting fertilization. In addition, pig semen is usually stored under suitable temperature, pH, osmotic pressure and other conditions, which is conducive to the growth and reproduction of microorganisms. In the process of their own growth and reproduction, bacteria and microorganisms will consume nutrients in the diluent and affect the normal metabolism of sperm; at the same time, the metabolites produced by the life activities of bacteria and microorganisms will have an adverse effect on the survival of sperm, and in severe cases will cause the death of sperm. In addition, there are many problems in the artificial insemination process, such as irregular operation and non-standard process of semen dilution, which can easily lead to poor quality of diluted semen, resulting in breeding failure, reducing the reproduction rate and utilization rate of excellent female livestock, and increasing production costs. At present, there are two main methods of artificial insemination, namely traditional insemination and deep intrauterine insemination. Traditional artificial insemination has high requirements for semen dilution and effective sperm count. Generally, 70-100mL of diluted semen is required, including effective sperm count of no less than 1.5 billion, while deep insemination only requires no less than 30mL of diluted semen with effective sperm count greater than 1 billion. Improving the quality of semen preservation by combining deep insemination technology can effectively improve the breeding utilization rate of excellent breeding male livestock, improve the utilization rate of excellent germplasm resources, reduce production costs, and improve economic benefits. At the same time, it is conducive to the cross-regional exchange of germplasm resources and the effective expansion of local protected varieties. However, there are many varieties of commercial diluents on the market, and there are many problems; foreign brand diluents have many formulas and are expensive, which reduces production efficiency; the current semen diluents have a short storage time, sperm are easily threatened by oxidative stress, and the utilization rate of excellent breeding livestock is not high.

[0004] Therefore, in order to effectively promote pig artificial insemination technology, it is urgent to develop a high-quality and low-cost pig semen diluent for storage at room temperature. Summary of the invention

[0005] In order to overcome the defects in the prior art, the present invention provides a high-quality and low-cost porcine semen room-temperature storage diluent and its preparation method and application. The diluent improves the preservation quality of porcine semen with low production cost and simple operation method, helps to improve the utilization rate of excellent breeding stock, improve the reproduction rate, and is easy to promote and apply.

[0006] The technical solution is as follows:

[0007] First, an embodiment of the present invention provides a porcine semen room temperature storage diluent, comprising double distilled water, 27.0-28.0 g / L of glucose, 3.1-3.3 g / L of citric acid, 7.8-7.9 g / L of sodium citrate, 2.2-2.4 g / L of ethylenediaminetetraacetic acid, 0.9-1.1 g / L of sodium bicarbonate, 5.5-5.7 g / L of tris(hydroxymethyl)aminomethane, 0.6-0.7 g / L of penicillin G sodium salt, 0.9-1.1 g / L of streptomycin sulfate and 60-70 mg / L of polymyxin B sulfate.

[0008] Preferably, 13.6-54.5 mg / L of arbutin is also included. The amount of arbutin is related to the preservation of sperm motility, forward motility, acrosome integrity, and plasma membrane integrity.

[0009] Preferably, the pH of the diluent for storage at room temperature is 7.0 to 7.2.

[0010] Preferably, it comprises double distilled water, 27.5 g / L glucose, 3.2 g / L citric acid, 7.85 g / L sodium citrate, 2.3 g / L ethylenediaminetetraacetic acid, 1.0 g / L sodium bicarbonate, 5.6 g / L tris(hydroxymethyl)aminomethane, 0.65 g / L penicillin G sodium salt, 1.0 g / L streptomycin sulfate, 66.7 mg / L polymyxin B sulfate and 27.2 mg / L arbutin.

[0011] Second, an embodiment of the present invention provides a method for preparing a porcine semen diluent for storage at room temperature, comprising the following steps:

[0012] Step 1. Use a clean beaker with a capacity of 1L, pour 800mL of double distilled water into it and place it on a magnetic stirrer. Add clean magnetic beads to facilitate the next step of stirring.

[0013] Step 2, prepare the solution, turn on the magnetic stirrer to stir the solution, use an electronic balance to weigh the reagents, weigh 27.5 g of glucose, 3.2 g of citric acid, 7.85 g of sodium citrate, 2.3 g of ethylenediaminetetraacetic acid, 1.0 g of sodium bicarbonate, 5.6 g of tris(hydroxymethyl)aminomethane, 0.65 g of penicillin G sodium salt, 1.0 g of streptomycin sulfate, 66.7 mg of polymyxin B sulfate and 27.2 mg of arbutin in sequence, add them into a beaker, and stir until the solution is clear;

[0014] Step 3, adjusting the pH, using a pH meter to adjust the solution pH to 7.0-7.2;

[0015] Step 4. Use a clean 1L volumetric flask to dilute the prepared solution to 1L. Mix by inverting the flask upside down and transfer to the corresponding container for standby use.

[0016] Third, an embodiment of the present invention provides a method for preserving semen using a porcine semen room temperature preservation diluent, comprising the following steps:

[0017] Step 1: Select healthy adult Duroc boars for semen collection, store fresh semen at 37°C, and mix with diluent of the same volume and temperature in a ratio of 1:1;

[0018] Step 2: Gently invert the tube to mix well, and then count the sperm density using a sperm counting plate;

[0019] Step 3: Calculate the volume of diluent required based on sperm density to make the final sperm density 0.5-1.5×10 8 Pieces / mL;

[0020] Step 4: Mix the 37°C diluent with the semen and equilibrate at room temperature for 2 hours;

[0021] Step 5: Divide the semen diluent into smaller pieces according to actual needs and store them in an incubator. During storage, gently mix the semen by turning it upside down every 12 or 24 hours to prevent sperm from settling and dying.

[0022] Step 6: The preserved semen diluent can be used for artificial insemination after quality inspection.

[0023] Furthermore, in step 3, the sperm density was diluted to 1.0×10 8 Pieces / mL.

[0024] Furthermore, in step 5, the temperature of the incubator is 15-18°C.

[0025] Furthermore, in step 5, the temperature of the incubator is 17°C.

[0026] Furthermore, in step 6, the semen is stored for no more than 240 hours.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The porcine semen room temperature preservation diluent of the present invention takes the problem of porcine semen preservation as the starting point, and uses glucose-citric acid-sodium citrate-sodium bicarbonate as the basic components to develop a diluent formula that can efficiently preserve semen at room temperature. The diluent of the present invention has a good room temperature liquid preservation effect on porcine semen, solves the problem of porcine semen preservation in artificial insemination technology during large-scale pig breeding, reduces boar breeding, and improves the utilization rate of high-quality boar semen; at the same time, the diluent has a simple preparation method, low production cost, is easy to use, and is convenient for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of preparing a semen diluent according to an embodiment of the present invention;

[0030] Figure 2 This is a schematic diagram of semen storage and application in an implementation case of the present invention;

[0031] Figure 3 The effect of the diluent of the present invention on the motility of pig sperm after being stored at room temperature for 10 days, wherein CON is the control group, ARB is the diluent group of the present invention, and the data are expressed in the form of mean ± standard error, and the data are derived from samples of five boars, and ** indicates extremely significant difference (P<0.01);

[0032] Figure 4 The sperm plasma membrane integrity was detected by SYBR-14 and PI immunofluorescence double staining method for 10 days of storage at room temperature, wherein CON is the control group, ARB is the diluent group of the present invention, SYBR-14 (green) indicates sperm with intact plasma membrane, PI red indicates sperm with damaged plasma membrane, scale = 100 μm;

[0033] Figure 5 The effect of the diluent of the present invention on the integrity of the plasma membrane of pig sperm after 10 days of storage at room temperature, CON is the control group, ARB is the diluent group of the present invention, the data are expressed in the form of mean ± standard error, the data are from samples of five boars, ** represents extremely significant difference (P < 0.01);

[0034] Figure 6 The sperm acrosome integrity was detected by PNA and DAPI immunofluorescence double staining method for 10 days of storage at room temperature. CON was the control group, ARB was the diluent group of the present invention, PNA (green) indicated the acrosome morphology, DAPI (blue) indicated the sperm position, and the scale bar = 100 μm;

[0035] Figure 7The effect of the diluent of the present invention on the acrosome integrity of pig sperm stored at room temperature for 10 days, wherein CON is the control group, ARB is the diluent group of the present invention, and the data are expressed in the form of mean ± standard error, and the data are derived from samples of five boars, and ** represents extremely significant difference (P<0.01);

[0036] Figure 8 The effect of the diluent of the present invention on the ATP content of pig sperm after being stored at room temperature for 10 days, wherein CON is the control group, ARB is the diluent group of the present invention, and the data are expressed in the form of mean ± standard error, and the data are derived from samples of five boars, and * indicates significant difference (P<0.05);

[0037] Fig. 9 The effect of the diluent of the present invention on the mitochondrial membrane potential of pig sperm stored at room temperature for 10 days, wherein CON is the control group, ARB is the diluent group of the present invention, the data are expressed in the form of mean ± standard error, the data are from samples of five boars, * represents significant difference (P < 0.05);

[0038] Fig.10 The figure shows the effect of the diluent of the present invention on the reactive oxygen species (ROS) level of pig sperm stored at room temperature for 10 days. CON is the control group, ARB is the diluent group of the present invention, and the data are expressed as mean ± standard error. The data are derived from samples of five boars. * indicates significant difference (P<0.05). DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0040] (I) Formula for diluent for room temperature storage of semen in deep insemination of pigs

[0041] The present invention takes the semen preservation problem in deep insemination of pigs as the starting point, takes glucose-citric acid-sodium citrate-sodium bicarbonate as the basic components, adds the antioxidant arbutin, and develops a diluent formula that can efficiently preserve semen at room temperature (15-18°C). After adding arbutin to the diluent, it has a good antioxidant protection effect on sperm and has a certain antibacterial ability, which effectively alleviates the problem that sperm is easily subjected to oxidative stress during the room temperature storage of pig semen. The present invention is conducive to expanding the semen utilization rate of excellent boars, ensuring the health of sows and improving the conception rate of deep insemination, and realizing efficient and low-cost deep insemination.

[0042] The semen room-temperature preservation diluent for deep insemination of pigs disclosed by the invention comprises the following formula: 13.6-54.5 mg / L of arbutin is added to glucose, citric acid, sodium citrate, ethylenediaminetetraacetic acid, sodium bicarbonate, tris(hydroxymethyl)aminomethane, sodium salt of penicillin G, streptomycin sulfate and polymyxin B sulfate, the mixture is mixed with double distilled water, and the pH value is adjusted to 7.0-7.2, so as to form the room-temperature preservation diluent.

[0043] Arbutin is a natural antioxidant extracted from plant leaves. It has strong anti-inflammatory, antioxidant and antibacterial effects and has been widely used in the skin care industry. Glucose in the diluent provides an energy source for sperm movement and is essential for the long-term survival of sperm. Citric acid, sodium citrate, sodium bicarbonate, and tris(hydroxymethyl)aminomethane are used as buffering agents in the diluent to maintain the osmotic pressure of the diluent and adjust the pH to maintain the stability of the solution. Ethylenediaminetetraacetic acid is an important metal ion chelator that can combine with divalent metal ions such as magnesium ions, zinc ions, and divalent iron ions to reduce the damage of metal ions to sperm, and can also play a buffering role. Penicillin G sodium salt, streptomycin sulfate, and polymyxin B sulfate can inhibit the growth of bacteria in the diluent. The invention is characterized in that: ① the addition of the antioxidant arbutin significantly enhances the antioxidant capacity of semen, and also has a certain antibacterial effect, thereby improving the preservation quality of pig semen diluent, and improving the preservation vitality, forward vitality, plasma membrane integrity, acrosome integrity and antioxidant enzyme activity of pig sperm; ② the price of arbutin is cheap, the addition amount is small, the cost of the diluent is low, and the operation is simple, which is conducive to the promotion and use of pig artificial insemination technology.

[0044] like Figure 1 As shown, during the preparation of the semen diluent, an electronic balance is used to weigh the reagents, glucose and other reagents are weighed in sequence according to the present invention, added to the beaker in sequence, the solution is stirred with the aid of a magnetic stirrer to prepare the solution, a pH meter is used to measure the pH of the solution and the pH is adjusted to 7.0-7.2, a 1L volumetric flask is used to make the volume, and finally, the solution is dispensed into containers of appropriate sizes for use.

[0045] like Figure 2 As shown, the specific process of semen preservation and application is as follows: select excellent boars for semen collection, measure sperm motility and sperm density, and then use the diluent of the present invention to dilute the semen. After dilution to a specified density, equilibrate at room temperature for 2 hours, and store it in a semen incubator at 17°C. The semen diluent can be used for artificial insemination of pigs after quality inspection within 240 hours.

[0046] The preparation of the semen room temperature storage diluent for deep insemination of pigs is simple and easy to operate. After the formula components are prepared according to the dosage, the corresponding dose of diluent is added at 37°C according to the concentration of fresh semen. After equilibration at room temperature for 2 hours, it is placed in a 17°C incubator for storage and can be put into use after being bathed in a 37°C water bath for 10 minutes before artificial insemination. The storage time can reach 240 hours, and the vitality and forward vitality meet the needs of farms for artificial insemination.

[0047] The best content of the semen diluent for deep insemination of pigs at room temperature is as follows: 27.5g / L glucose, 3.2g / L citric acid, 7.85g / L sodium citrate, 2.3g / L ethylenediaminetetraacetic acid, 1.0g / L sodium bicarbonate, 5.6g / L tris(hydroxymethyl)aminomethane, 0.65g / L sodium salt of penicillin G, 1.0g / L streptomycin sulfate, 66.7mg / L polymyxin B sulfate and 27.2mg / L arbutin. The above ingredients are fully mixed with 1L double distilled water to obtain the semen diluent for deep insemination of pigs at room temperature. The pH value of the diluent is 7.0-7.2.

[0048] (II) Methods for preserving fresh pig semen

[0049] Fresh semen collection and quality assessment: Semen was collected by hand-holding method, and the colloid in the semen was filtered out with gauze. The semen in the middle of the boar ejaculation was collected and divided into 50mL clean plastic bottles, and the air in the bottles was exhausted and sealed. The fresh semen density was measured, and the fresh semen motility was measured with the help of computer-assisted sperm analysis system (CASA). Samples with motility higher than 0.8 were further diluted and stored according to the measured sperm density.

[0050] Room temperature storage method: according to the volume and density of the fresh semen (temperature is 37°C), add the isothermal pig semen room temperature storage diluent (under the optimal formula content) to make the sperm density 1×10 8 / mL, gently invert upside down to mix, and dispense into 80mL semen bottles. After equilibration at room temperature for 2 hours, transfer to a 17℃ semen incubator. It can be stored for up to 240 hours, during which time it can be used for deep insemination of pigs according to production needs.

[0051] 3. Quality assessment of pig semen after storage

[0052] Sperm motility measurement: Before insemination, the semen was placed in a 37°C water bath for 10 minutes, and 8 μL of the sample was taken to measure sperm motility, forward motility and other movement parameters using a computer-assisted sperm analysis system (CASA). Sperm movement parameters mainly include sperm linear movement rate, curvilinear movement rate and average path rate.

[0053] Sperm plasma membrane integrity detection: Double fluorescence staining (SYBR-14 / PI) method was used for detection. SYBR-14 (green) indicates sperm with intact plasma membrane, and PI (red) indicates sperm with damaged plasma membrane. Sperm was stained with dye, 10 μL of semen was immediately observed and photographed under a 400x fluorescence microscope, and 3 fields of view (more than 200 sperm in each field of view) were randomly selected to count the plasma membrane integrity and calculate the sperm plasma membrane integrity rate.

[0054] Detection of sperm acrosome integrity rate: 20 μL sperm sample was fixed to make a smear, and double fluorescence staining (FITC-PNA / DAPI) was used for detection. FITC-PNA (green) indicates the acrosome morphology, and DAPI (blue) indicates the position of the sperm nucleus. The sperm was stained with dye, then observed and photographed under a fluorescence microscope, and 3 fields of view (more than 200 sperm in each field of view) were randomly selected to count the acrosome integrity and calculate the sperm acrosome integrity rate.

[0055] Detection of sperm ATP content: The ATP Assay Kit (Biyuntian, China) was used to detect the ATP content of sperm. Take 1 mL of the semen to be tested, centrifuge at 800g for 5 minutes, discard the supernatant, rinse twice with PBS to wash away impurities, add 200 μL of ATP lysis solution to the sperm pellet, lyse on ice, centrifuge at 12000g at 4℃ for 15 minutes after lysis, and take the supernatant (i.e., the sample to be tested) and store on ice. Prepare ATP standard solution and ATP working solution in an ice bath, add 100 μL of ATP working solution to the detection well of a black 96-well plate in the dark, let stand at room temperature for 3 minutes, then add 20 μL of standard solution or the sample to be tested to the detection well, and use a multifunctional enzyme label reader (BioTek, Synergy H1, USA) to detect the intensity of autofluorescence. At least three technical replicates were performed for each sample.

[0056] Detection of sperm mitochondrial membrane potential (MMP): The mitochondrial membrane potential detection kit JC-1 (Biyuntian, China) was used to detect the sperm mitochondrial membrane potential level. JC-1 staining solution and JC-1 staining buffer were prepared and added to 100 μL sperm sample (1×10 8 150 μL JC-1 staining solution was added to 100 μL of 1% ...

[0057] Detection of sperm reactive oxygen species (ROS): The ROS level in semen was detected using a reactive oxygen species detection kit (Biyuntian, China). 8 0.1 μL DCFH-DA was added to 100 μL of culture medium (100 μL / mL), incubated at 37°C in the dark for 20 min, centrifuged at 800 g for 5 min, the supernatant was discarded, and the cells were rinsed 3 times with 37°C Modena to fully wash away the DCFH-DA that did not enter the cells. The fluorescence intensity of the samples was detected by a multifunctional enzyme label detector (BioTek, Synergy H1, USA) at excitation / emission = 488 / 520 nm. Sperm counts were performed for each sample, and the average fluorescence intensity of the corresponding sperm was calculated. At least three technical replicates were performed for each sample.

[0058] (IV) Specific experimental examples

[0059] The semen of five healthy adult Duroc boars (1.2 to 2 years old) was collected by hand-gripping method. The sperm density was calculated using a sperm counting plate, and the semen motility was tested with the help of a computer-assisted sperm analysis system (CASA). The semen was stored for a specified time using the above diluent and operation method, and the traditional Modena fluid was used as an experimental control. The quality of semen from each boar was evaluated. The results showed that the density of the fresh semen samples collected from the boars was between 9.67 and 12.56×10 8 / mL range; activity ranged from 86.64% to 89.98%, with an average value of 88.09%; after being stored at room temperature for 240 hours using the diluent of the present invention, activity ranged from 69.62% to 73.42%, with an average value of 70.68%; the average acrosome integrity rate was 80.89%; the average plasma membrane integrity rate was 76.67%, the average sperm ATP content was 3.76 μmol / g, the average mitochondrial membrane potential (MMP) was 2.95, and the average reactive oxygen species level (ROS) was 8.80. The data results are shown in Table 1 and Figures 3 to 10 .

[0060] The diluent of the present invention can mainly supplement nutrients for sperm, maintain the osmotic pressure of semen, reduce the harm of bacteria in semen to sperm, reduce the harm of sperm metabolites to sperm, and reduce the harm of active oxygen in semen to sperm membrane structure. Experiments have proved that the diluent formula disclosed in the present invention has a good effect on the room temperature storage of pig semen (for example, 27.2 mg / L arbutin will not affect the osmotic pressure of semen, and can effectively control the number of bacteria in semen; sperm motility meets production needs after 240 hours of room temperature storage), can effectively inhibit bacterial growth in semen, maintain sperm motility and acrosome integrity, reduce sperm deformity rate, and can effectively improve pig artificial insemination conception rate and in vitro fertilization efficiency. At the same time, the arbutin in the diluent is a harmless and safe substance without drug residues.

[0061] Table 1. Effect of the diluent of the present invention on the liquid storage of pig semen

[0062]

[0063] The above description is only a preferred specific implementation manner of the present invention, and the protection scope of the present invention is not limited thereto. Any simple change or equivalent replacement of the technical solution that can be obviously obtained by any technician familiar with the technical field within the technical scope disclosed in the present invention falls within the protection scope of the present invention.

Claims

1. A pig semen diluent for storage at room temperature, characterized in that: It includes double distilled water, 27.0-28.0 g / L glucose, 3.1-3.3 g / L citric acid, 7.8-7.9 g / L sodium citrate, 2.2-2.4 g / L ethylenediaminetetraacetic acid, 0.9-1.1 g / L sodium bicarbonate, 5.5-5.7 g / L tris(hydroxymethyl)aminomethane, 0.6-0.7 g / L penicillin G sodium salt, 0.9-1.1 g / L streptomycin sulfate and 60-70 mg / L polymyxin B sulfate.

2. The porcine semen room temperature storage diluent according to claim 1, characterized in that: It also includes 13.6-54.5 mg / L of arbutin.

3. The porcine semen room temperature storage diluent according to claim 1 or 2, characterized in that: The pH of the diluent for storage at room temperature is 7.0 to 7.

2.

4. The porcine semen room temperature storage diluent according to claim 2, characterized in that: It includes double distilled water, 27.5g / L glucose, 3.2g / L citric acid, 7.85g / L sodium citrate, 2.3g / L ethylenediaminetetraacetic acid, 1.0g / L sodium bicarbonate, 5.6g / L tris(hydroxymethyl)aminomethane, 0.65g / L penicillin G sodium salt, 1.0g / L streptomycin sulfate, 66.7mg / L polymyxin B sulfate and 27.2mg / L arbutin.

5. A method for preparing the pig semen room temperature storage diluent according to claim 1, characterized in that: The following steps are involved: Step 1. Use a clean beaker with a capacity of 1L, pour 800mL of double distilled water into it and place it on a magnetic stirrer. Add clean magnetic beads to facilitate the next step of stirring. Step 2, prepare the solution, turn on the magnetic stirrer to stir the solution, use an electronic balance to weigh the reagents, weigh 27.5 g of glucose, 3.2 g of citric acid, 7.85 g of sodium citrate, 2.3 g of ethylenediaminetetraacetic acid, 1.0 g of sodium bicarbonate, 5.6 g of tris(hydroxymethyl)aminomethane, 0.65 g of penicillin G sodium salt, 1.0 g of streptomycin sulfate, 66.7 mg of polymyxin B sulfate and 27.2 mg of arbutin in sequence, add them into a beaker, and stir until the solution is clear; Step 3, adjusting the pH, using a pH meter to adjust the solution pH to 7.0-7.2; Step 4. Use a clean 1L volumetric flask to dilute the prepared solution to 1L, invert it upside down to mix, and transfer it to the corresponding container for standby use.

6. A method for preserving pig semen by using a room temperature storage diluent, characterized in that: The following steps are involved: Step 1: Select healthy adult Duroc boars for semen collection, store fresh semen at 37°C, and mix with diluent of the same volume and temperature in a ratio of 1:1; Step 2: Gently invert the tube to mix well, and then count the sperm density using a sperm counting plate; Step 3: Calculate the volume of diluent required based on sperm density to make the final sperm density 0.5-1.5×10 8 Pieces / mL; Step 4: Mix the 37°C diluent with the semen and equilibrate at room temperature for 2 hours; Step 5: Pack the semen diluent according to actual needs and store it in an incubator. During storage, gently mix the semen by turning it upside down every 12 or 24 hours to prevent sperm from settling and dying. Step 6: The semen diluent is stored and used for artificial insemination after quality inspection.

7. The method for preserving semen by using a porcine semen room temperature preservation diluent according to claim 6, characterized in that: In step 3, the final sperm density is 1.0×10 8 Pieces / mL.

8. The method for preserving semen by using a porcine semen room temperature preservation diluent according to claim 6, characterized in that: In step 5, the temperature of the incubator is 15-18°C.

9. The method for preserving semen by using a porcine semen room temperature preservation diluent according to claim 8, characterized in that: In step 5, the temperature of the incubator is 17°C.

10. The method for preserving semen by using a room temperature preservation diluent of pig semen according to claim 6, characterized in that: In step 6, the semen is stored for no more than 240 hours.

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