Normal-temperature preservation diluent for pig sperm and preparation method and application thereof

Abstract

The application discloses a preparation method of a normal-temperature preservation diluent for pig semen, and belongs to the technical field of biology.The main components of the normal-temperature preservation diluent are glucose, citric acid, sodium citrate, sodium bicarbonate, tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid disodium salt, penicillin G sodium salt, streptomycin sulfate, arbutin and double-distilled water.The diluent can maintain high activity of pig sperm in a short period, and the normal-temperature preservation time can reach about 10 days and meet the requirements of artificial insemination.The arbutin of the diluent has good anti-inflammatory and antioxidant protection effects, effectively improves the preservation activity and effect of pig sperm, and can be directly used for insemination after dilution or used for insemination after short-term preservation and short-distance transportation after dilution.The diluent improves the preservation quality of pig semen by using a low production cost and a simple operation method, is helpful to improve the utilization rate of excellent breeding stock, improve the reproduction rate, and is convenient for popularization and application.

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] Artificial insemination and semen preservation are the most widely used biotechnologies in porcine reproduction. Globally, most semen is stored in liquid form at 15-18°C, using dilution and cooling to reduce sperm metabolism and prolong semen storage. However, during semen storage, the antioxidant capacity of diluted semen decreases, leading to the accumulation of reactive oxygen species (ROS), disrupting redox homeostasis in pig semen and reducing its storage quality.

[0003] Sperm plasma membranes are rich in unsaturated fatty acids, making them susceptible to ROS, which can lead to lipid peroxidation. However, sperm have limited antioxidant capacity, and ROS accumulate during short-term transportation and storage. Accumulated ROS can damage lipids, proteins, and DNA, leading to cell apoptosis, altering enzyme activity, and reducing sperm motility and motility, ultimately impairing fertilization. Furthermore, porcine semen is typically stored under optimal temperature, pH, and osmotic pressure conditions, which favor the growth and reproduction of microorganisms. During their growth and reproduction, bacteria and microorganisms consume nutrients in the diluent, impacting normal sperm metabolism. Furthermore, the metabolic products produced by these activities can adversely affect sperm survival and, in severe cases, lead to sperm death. Furthermore, artificial insemination processes are subject to numerous challenges, such as improper semen dilution procedures and substandard processes, which can lead to poor semen quality, resulting in breeding failures, reduced reproductive rates, reduced utilization of high-quality females, and increased production costs. Currently, there are two main methods of artificial insemination: traditional insemination and deep intrauterine insemination. Traditional artificial insemination (AI) requires high semen dilution volumes and high sperm counts, typically requiring 70-100 mL of diluted semen with a minimum of 1.5 billion sperm. Deep insemination, on the other hand, only requires at least 30 mL of diluted semen with a sperm count exceeding 1 billion. Improving semen preservation quality through deep insemination can effectively increase the reproductive utilization rate of high-quality breeding stock, improve the utilization rate of high-quality germplasm resources, reduce production costs, and enhance economic benefits. It can also facilitate the cross-regional exchange of germplasm resources and the effective propagation of locally protected breeds. However, the current commercial diluents available on the market are numerous and present numerous challenges. Foreign brands have numerous formulas and are expensive, reducing production efficiency. Current semen diluents have short shelf lives, making sperm susceptible to oxidative stress and limiting the utilization rate of high-quality breeding stock.

[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 that can be stored at room temperature. Summary of the Invention

[0005] In order to overcome the defects in the prior art, the application provides a pig semen normal-temperature preservation diluent, a preparation method and application thereof, which improves the pig semen preservation quality at a low production cost and with a simple operation method, helps to improve the utilization rate of excellent breeding stock, improve the reproduction rate, and facilitate popularization and application.

[0006] The technical scheme is as follows:

[0007] Firstly, the embodiment of the application provides a pig semen normal-temperature preservation diluent, which comprises 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 further included. The amount of arbutin is related to the motility, forward motility, acrosome integrity and plasma membrane integrity of the preserved sperm.

[0009] Preferably, the pH of the normal-temperature preservation diluent is 7.0-7.2.

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

[0011] Secondly, the embodiment of the application provides a preparation method of the pig semen normal-temperature preservation diluent, which comprises the following steps:

[0012] Step 1, a clean beaker with a capacity of 1 L is used, 800 mL of double-distilled water is poured into the beaker, and a clean magnetic bead is placed on a magnetic stirrer to facilitate the next step of stirring;

[0013] Step 2: Prepare the solution by turning 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 order, add them to a beaker, and stir until the solution is clear.

[0014] Step 3: Adjust the pH of the solution to 7.0-7.2 using a pH meter.

[0015] Step 4. Use a clean 1L volumetric flask to dilute the prepared solution to 1L. Mix thoroughly by inverting the flask and transfer it to the corresponding container for aliquoting.

[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 at a ratio of 1:1;

[0018] Step 2: Gently invert the tube upside down to mix thoroughly, and then count the sperm density using a sperm counting chamber.

[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 / mL;

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

[0021] Step 5: Divide the semen diluent into smaller portions according to actual needs and store in an insulated container. Gently invert the semen every 12 or 24 hours to mix it thoroughly to prevent sperm from settling and dying.

[0022] Step 6: The semen diluent is stored and 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 insulated box 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 described in this invention addresses the issue of porcine semen preservation. Using glucose, citric acid, sodium citrate, and sodium bicarbonate as its basic ingredients, a diluent formula has been developed that efficiently preserves semen at room temperature. This diluent exhibits excellent room-temperature liquid preservation properties for porcine semen, resolving the semen preservation challenges associated with artificial insemination in large-scale pig farming, reducing boar breeding and increasing the utilization rate of high-quality boar semen. Furthermore, this diluent is simple to prepare, offers low production costs, and is easy to use, facilitating large-scale application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the preparation of 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 embodiment of the present invention;

[0031] Figure 3 The effect of the diluent of the present invention on the motility of pig sperm after 10 days of storage at room temperature is shown. CON is the control group, and ARB is the diluent group of the present invention. The data are expressed as mean ± standard error. The data are from samples of five boars. ** indicates a very significant difference (P < 0.01).

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

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

[0034] Figure 6 The sperm acrosome integrity was detected by PNA and DAPI double fluorescence staining 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, scale bar = 100 μm;

[0035] Figure 7The effect of the diluent of the present invention on the acrosome integrity of porcine sperm after 10 days of storage at room temperature is shown. CON is the control group, and ARB is the diluent group of the present invention. The data are expressed as mean ± standard error. The data are from samples of five boars. ** indicates 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 10 days of storage at room temperature, wherein CON is the control group and ARB is the diluent group of the present invention. The data are expressed as mean ± standard error. The data are from samples of five boars. * indicates significant difference (P < 0.05).

[0037] Figure 9 The effect of the diluent of the present invention on the mitochondrial membrane potential of pig sperm after 10 days of storage at room temperature, wherein CON is the control group and ARB is the diluent group of the present invention. The data are expressed as mean ± standard error. The data are derived from samples of five boars. * indicates significant difference (P < 0.05).

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

[0039] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0040] (1) Formula for semen dilution solution for deep insemination of pigs at room temperature

[0041] This invention addresses the issue of semen preservation during deep insemination in pigs. Using glucose, citric acid, sodium citrate, and sodium bicarbonate as the base ingredients, and adding the antioxidant arbutin, a diluent formula has been developed that can efficiently preserve semen at room temperature (15-18°C). The addition of arbutin to this diluent provides excellent antioxidant protection for sperm and possesses certain antibacterial properties, effectively alleviating the susceptibility of semen to oxidative stress during room-temperature storage. This invention is beneficial for increasing semen utilization from high-quality boars, protecting sow health, and improving conception rates through deep insemination, achieving efficient and cost-effective deep insemination.

[0042] The invention discloses a semen room-temperature storage diluent for deep insemination of pigs. The diluent comprises the following formula: glucose, citric acid, sodium citrate, ethylenediaminetetraacetic acid, sodium bicarbonate, tris(hydroxymethyl)aminomethane, penicillin G sodium salt, streptomycin sulfate, and polymyxin B sulfate, 13.6-54.5 mg / L of arbutin is added to the mixture; the mixture is mixed with double-distilled water; and the pH value is adjusted to 7.0-7.2 to form a room-temperature storage 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. The glucose in the diluent provides an energy source for sperm movement and is crucial for the long-term survival of sperm. Citric acid, sodium citrate, sodium bicarbonate, and tris(hydroxymethyl)aminomethane act as buffering agents in the diluent to maintain the osmotic pressure of the diluent and adjust the pH, thereby maintaining the stability of the solution. Ethylenediaminetetraacetic acid is an important metal ion chelator that can bind to divalent metal ions such as magnesium ions, zinc ions, and divalent iron ions to reduce the damage of metal ions to sperm and also act as a buffer. Penicillin G sodium salt, streptomycin sulfate, and polymyxin B sulfate can inhibit the growth of bacteria in the diluent. The characteristics of the present invention are: 1. The addition of the antioxidant arbutin significantly enhances the antioxidant capacity of semen, also has a certain antibacterial effect, improves the preservation quality of the pig semen diluent, and improves the preservation vitality, forward vitality, plasma membrane integrity, acrosome integrity and antioxidant enzyme activity of pig sperm; 2. 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, and reagents such as glucose are weighed in sequence according to the present invention and added to a beaker in sequence. The solution is stirred with the help of a magnetic stirrer to prepare the solution, and the pH of the solution is measured using a pH meter and adjusted to 7.0-7.2. A 1 L 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 breeding 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 the 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 pig artificial insemination after quality inspection within 240 hours.

[0046] The preparation of this semen diluent for deep insemination in pigs is simple. After preparing the ingredients according to the required dosage, add the appropriate amount of diluent based on the fresh semen concentration at 37°C. After equilibration at room temperature for 2 hours, store in a 17°C incubator. Before artificial insemination, place in a 37°C water bath for 10 minutes before use. The semen can be stored for up to 240 hours, and the viability and forward motility meet the needs of artificial insemination farms.

[0047] The optimal composition of a porcine semen storage diluent for deep insemination at room temperature is as follows: 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. Thoroughly mix the above ingredients with 1 L of double-distilled water to obtain the porcine semen storage diluent for deep insemination at room temperature. The pH of the diluent is 7.0-7.2.

[0048] (2) Preservation methods of fresh pig semen

[0049] Fresh semen collection and quality assessment: Semen was collected manually, filtered through gauze to remove colloid, and the mid-ejaculate was collected. The semen was aliquoted into clean 50mL plastic bottles, which were purged of air and sealed. Fresh semen density was measured, and fresh semen motility was determined using a computer-assisted sperm analysis (CASA) system. Samples with motility above 0.8 were further diluted and stored based on the measured sperm density.

[0050] Room temperature storage method: according to the volume and density of the fresh semen (temperature 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 up and down to mix, and divide into 80mL semen bottles. After equilibration at room temperature for 2 hours, transfer to a 17℃ semen incubator. The storage time can be up to 240 hours. During this period, 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: Before insemination, semen was incubated in a 37°C water bath for 10 minutes. An 8 μL sample was taken and sperm motility, progressive motility, and other motility parameters were measured using a computer-assisted sperm analysis (CASA) system. Sperm motility parameters primarily include linear velocity, curvilinear velocity, and average path velocity.

[0053] Sperm plasma membrane integrity was assessed using a dual fluorescent staining method (SYBR-14 / PI). SYBR-14 (green) indicates sperm with intact plasma membranes, while PI (red) indicates sperm with damaged plasma membranes. Sperm were stained with the dye, and 10 μL of semen was immediately observed and photographed under a 400x fluorescence microscope. Three randomly selected fields of view (each containing more than 200 sperm) were used to count the plasma membrane integrity and calculate the sperm plasma membrane integrity rate.

[0054] Sperm acrosome integrity testing: 20 μL of sperm sample was fixed and prepared into a smear, which was then tested using a dual fluorescent staining method (FITC-PNA / DAPI). FITC-PNA (green) indicates acrosome morphology, and DAPI (blue) indicates the location of the sperm nucleus. Sperm were stained with dye and then observed and photographed under a fluorescence microscope. Three randomly selected fields of view (each containing more than 200 sperm) were used to count the acrosome integrity and calculate the sperm acrosome integrity rate.

[0055] Sperm ATP content was determined using the ATP Assay Kit (Biyuntian, China). One mL of semen to be tested was centrifuged at 800 g for 5 min. The supernatant was discarded and the pellet was rinsed twice with PBS to remove impurities. 200 μL of ATP lysis buffer was added to the sperm pellet and lysed on ice. After lysis, the pellet was centrifuged at 12,000 g for 15 min at 4°C. The supernatant (i.e., the test sample) was stored on ice. ATP standard solution and ATP working solution were prepared in an ice bath. 100 μL of ATP working solution was added to the test wells of a black 96-well plate in the dark. After standing at room temperature for 3 min, 20 μL of the standard solution or the test sample was added to the test wells. Autofluorescence intensity was measured using a multifunctional microplate reader (BioTek, Synergy H1, USA). 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 level of sperm mitochondrial membrane potential. JC-1 staining solution and JC-1 staining buffer were prepared and added to 100 μL of sperm sample (1×10 8 150 μL of JC-1 staining solution was added to the culture medium (100 μL / mL), incubated at 37°C in the dark for 30 min, centrifuged at 800 g for 5 min, and the supernatant discarded. The cells were rinsed three times with pre-chilled JC-1 staining buffer to thoroughly remove any unincorporated fluorescent probe. The red / green fluorescence intensity of the samples was measured using a multifunctional microplate reader (BioTek, Synergy H1, USA). At least three technical replicates were performed for each sample.

[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 the culture medium (100 μL / mL) and incubated at 37°C in the dark for 20 minutes. The cells were centrifuged at 800 g for 5 minutes, the supernatant discarded, and the cells were rinsed three times with a 37°C Modena to thoroughly remove any DCFH-DA that had not entered the cells. Fluorescence intensity of the samples was measured using a multifunctional microplate reader (BioTek, Synergy H1, USA) at excitation / emission = 488 / 520 nm. Sperm counts were performed for each sample, and the mean fluorescence intensity of the corresponding sperm was calculated. At least three technical replicates were performed for each sample.

[0058] (IV) Specific experimental examples

[0059] Semen was collected from five healthy adult Duroc boars (1.2 to 2 years old) using the hand-grip method. Sperm density was calculated using a sperm counting chamber, and semen motility was assessed using a computer-assisted sperm analysis (CASA) system. Semen was stored using the aforementioned diluent and procedure for the indicated time period. Traditional Modena semen was used as a control. Semen quality was assessed for each boar. Results showed that the semen density of the collected boar semen samples ranged from 9.67 to 12.56 × 10 8 / mL range; activity ranged from 86.64% to 89.98%, with an average 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 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 reactive oxygen in semen to sperm membrane structure. Experiments have shown 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 of arbutin does 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 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 embodiment 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 this technical field within the technical scope disclosed in the present invention falls within the protection scope of the present invention.

Claims

1. A porcine 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, and also includes 13.6~54.5 mg / L arbutin.

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

2.

3. The porcine semen room temperature storage diluent according to claim 2, characterized in that: The solution includes 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.

4. A method for preparing the porcine semen diluent for storage at room temperature according to claim 1, characterized in that: The following steps are involved: Step 1: Pour 800 mL of double-distilled water into a clean 1 L beaker and place it on a magnetic stirrer. Add clean magnetic beads to facilitate stirring in the next step. Step 2: Prepare the solution by turning 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 order, add them to a beaker, and stir until the solution is clear. Step 3: Adjust the pH of the solution to 7.0-7.2 using a pH meter. Step 4. Use a clean 1 L volumetric flask to dilute the prepared solution to 1 L, invert it upside down to mix, and transfer it to the corresponding container for standby use.

5. A method for preserving semen using the porcine semen room temperature preservation diluent according to claim 1, 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 at a ratio of 1:1; Step 2: Gently invert the tube upside down to mix thoroughly, and then count the sperm density using a sperm counting chamber. Step 3: Calculate the volume of diluent required based on sperm density to make the final sperm density 0.5-1.5×10 8 / mL; Step 4: Mix the 37°C diluent with semen and equilibrate at room temperature for 2 h. Step 5: Divide the semen diluent into smaller portions according to actual needs and store them in an incubator. During storage, gently invert the semen every 12 or 24 hours to mix it thoroughly to prevent sperm from settling and dying. Step 6: The semen diluent is stored and used for artificial insemination after quality inspection.

6. The method for preserving semen using a porcine semen diluent at room temperature according to claim 5, characterized in that: In step 3, the final sperm density was 1.0×10 8 pieces / mL.

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

8. The method for preserving semen using a porcine semen diluent at room temperature according to claim 7, characterized in that: In step 5, the temperature of the incubator is 17°C.

9. The method for preserving semen using a porcine semen diluent at room temperature according to claim 5, characterized in that: In step 6, the semen should be stored for no more than 240 hours.

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