Hainan pig semen preservation diluent as well as preparation method, application and detection method thereof

Abstract

According to the Hainan pig semen preservation diluent as well as the preparation method, the application and the detection method thereof disclosed by the invention, galangin and kaempferide are added into a basic diluent to develop a diluent formula capable of efficiently preserving semen at a low temperature (4 DEG C); the galangin and the kaempferol added into the diluent have a good sperm protection effect and a certain antibacterial ability and can replace antibiotics, and bovine serum albumin (BSA) is further combined as a low-temperature protective agent, so that sperm movement parameters are improved, and the low-temperature preservation time of the boar semen is effectively prolonged; the use amount of antibiotics in pig raising production is reduced, the problem of drug resistance of bacteria can be effectively relieved, and the use of the antibiotics and the generation of drug-resistant bacteria are greatly reduced. According to the method, the semen utilization rate of excellent breeding boars is increased, sow health is guaranteed, the conception rate of deep insemination is increased, and efficient and low-cost artificial insemination is achieved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a Hainan pig semen preservation diluent, and also relates to a preparation method of the Hainan pig semen preservation diluent, an application of the Hainan pig semen preservation diluent, and a detection method of the Hainan pig semen preservation diluent. Background Art

[0002] The Hainan pig is a superior local breed, cultivated over a long period of time under the high-temperature conditions of Hainan Island. Primarily produced in Lingao, Wenchang, Tunchang, and Ding'an counties of Hainan Province, they are also known as Lingao, Wenchang, Tunchang, and Ding'an, respectively, and collectively as the Hainan pig. Hainan pigs are beloved by locals for their early maturity, easy fattening, thin skin, fine bones, and delicious meat, but their current population is limited. Currently, Hainan pig breeding primarily relies on natural crosses, with artificial insemination rarely used. To strengthen seed conservation and breeding efforts and further meet the public's demand for high-quality Hainan pig meat, there is an urgent need to expand the Hainan pig population through artificial insemination.

[0003] Semen preservation involves treating collected semen to extend its viability, freeing artificial insemination from geographical and temporal constraints. There are three methods for semen preservation: room temperature (15-25°C), cryopreservation (0-5°C), and cryopreservation (-196°C). Cryopreservation, at lower temperatures, slows sperm metabolism, resulting in superior preservation results compared to room temperature storage, while requiring less stringent procedures and equipment than cryopreservation.

[0004] Although current porcine semen cryopreservation technology can prolong sperm survival in vitro and reduce the risk of disease transmission, its core diluent formula still faces the following technical bottlenecks: 1. Existing diluents generally inhibit bacterial proliferation by adding antibiotics such as penicillin and streptomycin. Long-term use can easily lead to antibiotic residues and increased bacterial resistance, posing a threat to food safety and public health. 2. During cryopreservation, sperm are susceptible to oxidative stress, which intensifies lipid peroxidation and significantly reduces sperm motility, acrosome integrity, and mitochondrial membrane potential. Existing formulas have limited metal ion chelation capabilities and lack effective ingredients that can stably increase ATP levels and total antioxidant capacity. Summary of the Invention

[0005] The first purpose of the present invention is to provide a Hainan pig semen preservation diluent, which is beneficial to improving the low-temperature preservation effect of Hainan pig semen.

[0006] The second object of the present invention is to provide a method for preparing a Hainan pig semen preservation diluent.

[0007] The third object of the present invention is to provide an application of Hainan pig semen preservation diluent.

[0008] The fourth object of the present invention is to provide a method for detecting Hainan pig semen preservation diluent.

[0009] The first technical solution adopted by the present invention is a Hainan pig semen preservation diluent, which is prepared by adding a plant extract to a basic diluent and mixing it. The plant extract includes galangin and kaempferol, and the basic diluent is composed of glucose, sodium citrate, ethylenediaminetetraacetic acid, sodium bicarbonate, potassium chloride, bovine serum albumin and ultrapure water.

[0010] The first technical solution of the present invention is also characterized in that: Each liter of basic diluent contains the following raw materials by mass: glucose 24g-50g, sodium citrate 4g-8g, ethylenediaminetetraacetic acid 1.0g-1.5g, sodium bicarbonate 1.0g-1.5g, potassium chloride 0.5g-1.0g, bovine serum albumin 2g-6g, and the rest is ultrapure water; Add 0.014g-0.040g of galangin and 0.0015g-0.0045g of kaempferol to each liter of basic dilution solution.

[0011] The basic diluent contains the following raw materials by mass: 37 g of glucose, 6 g of sodium citrate, 1.25 g of ethylenediaminetetraacetic acid, 1.25 g of sodium bicarbonate, 0.75 g of potassium chloride, 4 g of bovine serum albumin, and the remainder is ultrapure water; 0.027 g of galangin and 0.003 g of kaempferol are added to each liter of the basic diluent.

[0012] The second technical solution adopted by the present invention is a method for preparing a Hainan pig semen preservation diluent, comprising the following steps: Glucose, sodium citrate, ethylenediaminetetraacetic acid, sodium bicarbonate, potassium chloride, and bovine serum albumin were weighed in proportion and fully dissolved in ultrapure water to obtain a basic diluent; galangin and kaempferol were added to the basic diluent in proportion and mixed uniformly to obtain a diluent.

[0013] The third technical solution adopted by the present invention is to use a Hainan pig semen preservation diluent, place the Hainan pig semen in the diluent, and preserve it at a low temperature of 0°C to 5°C.

[0014] The third technical solution of the present invention is also characterized in that: The volume ratio of Hainan pig semen to diluent is 1:5.

[0015] The fourth technical solution adopted by the present invention is a method for detecting Hainan pig semen preservation diluent, comprising the following steps: Hainan pig semen was collected by hand-holding method, and semen with normal color and odor and sperm motility above 80% was selected; 2 mL of semen was taken into a sterile 10 mL centrifuge tube, and then the diluent was slowly added to the 10 mL scale line. The centrifuge tube was wrapped with a towel, and water of the same temperature was added to the foam box. The diluted semen was placed into the foam box, covered with a lid, and stored at a low temperature of 0℃ to 5℃, and then dropped to 4℃ for 24 hours; stored for 15 days, and the semen was slowly turned over once every 12 hours. The sperm motility was checked at 3, 6, 9, 12, and 15 days respectively; the semen quality indicators and bacterial content were tested and evaluated at 3, 6, and 9 days after storage.

[0016] The fourth technical solution of the present invention is also characterized in that: Indicators include sperm motility, acrosome integrity, and plasma membrane integrity.

[0017] The beneficial effects of the present invention are: This invention adds galangin (Gal) and kaempferide (Kae) to a basic diluent to develop a diluent formula that can efficiently preserve semen at low temperatures (4°C). This diluent, supplemented with galangin and kaempferide, exhibits excellent sperm protection and antibacterial properties, making it an alternative to antibiotics. Furthermore, the use of bovine serum albumin (BSA) as a cryoprotectant improves sperm motility parameters, effectively extending the low-temperature storage time of pig semen. It also reduces the use of antibiotics in pig production, effectively alleviating bacterial resistance and significantly reducing antibiotic use and the development of resistant bacteria. This invention helps increase the utilization of semen from high-quality boars, safeguarding sow health, and improving conception rates through deep insemination, enabling efficient and cost-effective artificial insemination. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the change in Hainan pig sperm motility of the present invention; Figure 2 This is a schematic diagram of the change in Hainan pig sperm motility of the present invention; Figure 3 Schematic diagram of the acrosome integrity rate of Hainan pig sperm of the present invention; Figure 4 Schematic diagram of the plasma membrane integrity rate of Hainan pig sperm of the present invention; Figure 5 It is a schematic diagram of the bacterial content of Hainan pig semen of the present invention.

[0019] Figure 6 It is a schematic diagram of the Hainan pig sperm quality related indicators of the present invention. DETAILED DESCRIPTION

[0020] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0021] Example 1 The Hainan pig semen preservation diluent disclosed in this embodiment is prepared by adding a plant extract to a basic diluent, wherein the plant extract includes galangin and kaempferol. The basic diluent is composed of glucose, sodium citrate, ethylenediaminetetraacetic acid (EDTA), sodium bicarbonate, potassium chloride, bovine serum albumin (BSA), and ultrapure water.

[0022] In this embodiment, the diluent does not add antibiotics, which can not only effectively inhibit the proliferation of bacteria in semen and ensure the in vitro storage of sperm, but also reduce the use and residue of antibiotics in animal husbandry and reduce the probability of drug-resistant bacteria. The diluent has the characteristics of inhibiting bacterial growth and chelating metal ions, which can effectively reduce lipid peroxidation and increase the Ca content in sperm. 2+ concentration, ATP content. Therefore, it has the effects of improving sperm motility and motility, plasma membrane integrity, acrosome integrity, DNA fragmentation index and mitochondrial membrane potential level, total antioxidant capacity, etc. After the various components are fully mixed in proportion, they are isothermally diluted with fresh semen and sealed for storage. Isolating the semen from the air is beneficial to protecting the sperm from damage by the external environment. When stored at low temperature for 9 days, the motility rate can still reach more than 60%, which fully meets the needs of breeding pig farms for deep insemination. The present invention utilizes the antibacterial properties of galangin and kaempferol to achieve the effect of extending the storage time of semen and reducing the cost of semen storage.

[0023] Example 2 Based on Example 1, each liter of the basic diluent contains the following raw materials by mass: glucose 24g-50g, sodium citrate 4g-8g, ethylenediaminetetraacetic acid 1.0g-1.5g, sodium bicarbonate 1.0g-1.5g, potassium chloride 0.5g-1.0g, bovine serum albumin 2g-6g, and the rest is ultrapure water; Add 0.014g-0.040g of galangin and 0.0015g-0.0045g of kaempferol to each liter of basic dilution solution.

[0024] The present invention uses galangin and kaempferol as main ingredients, and screens the optimal compatibility concentration of galangin and kaempferol by adding different concentrations of galangin and kaempferol together, taking the vitality and viability of diluted sperm as evaluation indicators, and then detects the acrosome integrity rate, plasma membrane integrity rate, DNA fragmentation index, MDA and Ca in semen after dilution. 2+, ATP content, mitochondrial membrane potential level, total antioxidant capacity and bacterial content to ensure that pig semen maintains good biological activity during low-temperature storage.

[0025] Example 3 Based on Example 2, each liter of the basic diluent contains the following raw materials by mass: 24 g glucose, 4 g sodium citrate, 1.0 g EDTA, 1.0 g sodium bicarbonate, 0.5 g potassium chloride, 2 g bovine serum albumin, and the remainder is ultrapure water; Add 0.014 g of galangin and 0.0015 g of kaempferol to each liter of basic dilution solution.

[0026] Example 4 Based on Example 2, the basic diluent contained the following raw materials by mass: 37 g glucose, 6 g sodium citrate, 1.25 g EDTA, 1.25 g sodium bicarbonate, 0.75 g potassium chloride, 4 g bovine serum albumin, and the remainder was ultrapure water; 0.027 g galangin and 0.003 g kaempferol were added per liter of the basic diluent.

[0027] The diluent of the present invention primarily supplements sperm with nutrients, maintains semen osmotic pressure, reduces the harmful effects of semen bacteria, reduces the harmful effects of sperm metabolites, and reduces the harmful effects of reactive oxygen species in semen on sperm membrane structure. Experiments have demonstrated that the diluent formula disclosed in the present invention has a favorable effect on the cryopreservation of Hainan pig semen (for example, 0.027g of galangin and 0.003g of kaempferol do not affect semen osmotic pressure and effectively control the bacterial count in semen; after nine days of cryopreservation, sperm motility reaches over 60%, meeting production requirements). It effectively inhibits bacterial growth in semen, maintains sperm motility and acrosome integrity, and can effectively improve pig artificial insemination conception rates and in vitro fertilization efficiency, thereby enhancing artificial insemination production efficiency.

[0028] Example 5 Based on Example 2, each liter of the basic diluent contains the following raw materials by mass: 50 g glucose, 8 g sodium citrate, 1.5 g EDTA, 1.5 g sodium bicarbonate, 1.0 g potassium chloride, 6 g bovine serum albumin, and the remainder is ultrapure water; Add 0.040 g of galangin and 0.0045 g of kaempferol to each liter of basic dilution solution.

[0029] Example 6 This embodiment discloses a method for preparing the Hainan pig semen preservation diluent according to Examples 1-2, comprising the following steps: Glucose, sodium citrate, ethylenediaminetetraacetic acid, sodium bicarbonate, potassium chloride, and bovine serum albumin were weighed in proportion and fully dissolved in ultrapure water to obtain a basic diluent; galangin and kaempferol were added to the basic diluent in proportion and mixed uniformly to obtain a diluent.

[0030] Furthermore, the Hainan pig semen is placed in a diluent and stored at a low temperature of 0°C to 5°C.

[0031] Specifically, the collected semen was mixed evenly after being checked by a computer sperm analyzer. The semen was diluted with the prepared optimal concentration of galangin and kaempferol diluent, wrapped in multiple layers of towels, and stored in a refrigerator at 4°C. During the storage period, the sperm motility, vitality, plasma membrane integrity, acrosome integrity, DNA fragmentation index, mitochondrial membrane potential level, malondialdehyde content, total antioxidant capacity, ATP, Ca 2+ The content and colony count in semen (sperm motility, functional integrity rate, energy metabolism parameters, and antibacterial effect) were tested to clarify the role of this plant extract (galangin and kaempferol) in the preservation of Hainan pig semen.

[0032] Furthermore, the volume ratio of Hainan pig semen to diluent was 1:5, which was used to adjust the sperm density to 2.5×10 7 Too high a sperm density will increase competition and energy consumption among sperm, leading to a decrease in survival rate; too low a density may not be able to break through the cervical mucus barrier, reducing the pregnancy rate. 7 The density balances the sperm survival rate and effective penetration, ensuring the success rate of artificial insemination.

[0033] Example 7 This embodiment is based on the detection method of the Hainan pig semen preservation diluent of Example 1-2, comprising the following steps: Hainan pig semen was collected by hand-holding method, and semen with normal color and odor and sperm motility above 80% was selected; 2 mL of semen was taken into a sterile 10 mL centrifuge tube, and then the diluent was slowly added to the 10 mL scale line. The centrifuge tube was wrapped with a towel, and water of the same temperature was added to the foam box. The diluted semen was placed into the foam box, covered with a lid, and stored at a low temperature of 0℃ to 5℃, and then dropped to 4℃ for 24 hours; stored for 15 days, and the semen was slowly turned over once every 12 hours. The sperm motility was checked at 3, 6, 9, 12, and 15 days respectively; the semen quality indicators and bacterial content were tested and evaluated at 3, 6, and 9 days after storage.

[0034] Furthermore, the indicators include sperm motility, acrosome integrity, and plasma membrane integrity.

[0035] In conjunction with Example 4, the effect of the Hainan pig semen preservation diluent prepared by the present invention is verified by experiment: As shown in Table 1, 37 g of glucose, 6 g of sodium citrate, 1.25 g of ethylenediaminetetraacetic acid, 1.25 g of sodium bicarbonate, 0.75 g of potassium chloride, and 4 g of bovine serum albumin were weighed. Galangin and kaempferol were prepared at concentrations of 0.001 µM, 0.01 µM, 0.1 µM, 1 µM, 10 µM, 100 µM, and 1000 µM, respectively. According to the formulation in Table 1, 10 mg of each of galangin and kaempferol were weighed. Under a sterile operating hood, galangin was dissolved in 37.004 µL of DMSO to prepare a 1 M stock solution, and kaempferol was dissolved in 33.304 µL of DMSO to prepare a 1 M stock solution. 10 µL of each stock solution was added to 10 mL of sperm diluent to obtain a 1000 µM initial working solution. Subsequently, seven working solutions were prepared by serial dilution. After preparation, these solutions were stored at 4°C until use.

[0036] Table 1

[0037] The porcine semen used in this experiment came from 20-month-old Hainan boars. Semen collected by hand-grip was filtered, placed in an insulated container, and quickly brought back to the laboratory. Semen with normal color and odor, and a sperm motility of over 80%, was selected for use.

[0038] Place 2 mL of semen into a sterile 10 mL centrifuge tube and slowly add diluent to the 10 mL mark. Wrap the centrifuge tube with four layers of towels. Simultaneously, add water of the same temperature to a foam box. Place the diluted semen into the foam box, cover it, and place it in a 4°C constant-temperature refrigerator. Allow approximately 24 hours to cool to 4°C. Store for 15 days, slowly stirring the semen every 12 hours. Sperm motility was checked at 3, 6, 9, 12, and 15 days. Semen quality indicators and bacterial counts were assessed at 3, 6, and 9 days. Each treatment was repeated three times.

[0039] The main measurement indicators are as follows: 1. Sperm motility and vitality. Use computer-assisted sperm analyzer (CASA) to detect sperm motility and vitality. Preheat the computer in advance and set the stage temperature to 37°C. Dilute the sperm to a concentration of 5×10 6 Then put it in a 37℃ constant temperature water bath to preheat for 15 minutes, draw 2μL of semen and slowly drip it into the micropores of the matching glass slide, adjust the field of view to clearly capture the sperm count, and take at least 200 sperms. Repeat the test at least 3 times for one test group. Figure 1 and Figure 2As shown, Hainan pig sperm motility and vitality gradually decreased with prolonged storage. During cryopreservation for 6-15 days, the motility and vitality of the galangin-kaempferol combination in both the antibiotic and non-antibiotic groups were significantly higher than those of the blank and negative controls (P < 0.05). The sperm motility and vitality of the antibiotic blank and negative controls were slightly higher than those of the non-antibiotic control groups (P > 0.05). Furthermore, the sperm motility and vitality of the galangin-kaempferol combination in the non-antibiotic system were slightly better than those of the same antibiotic-treated group (P > 0.05). These results suggest that this combination system has the potential to replace antibiotics in antimicrobial analysis. Based on these results, subsequent experiments will uniformly utilize a non-antibiotic storage system for sperm analysis, focusing on sperm quality measurements on days 6 and 9 of cryopreservation.

[0040] 2. Acrosome integrity rate. The Coomassie brilliant blue staining method was used to detect the sperm acrosome integrity rate. The specific operation was as follows: 100 μL of liquid-stored semen was placed in a 1.5 mL centrifuge tube, and the diluent was added to 1 mL and mixed to keep the sperm density at 5×10 5 , place it in a 37℃ water bath and incubate for 15 minutes to restore sperm motility. After the incubation, centrifuge the incubated sperm (800g, 5min), pour out the supernatant after centrifugation, add 1mLPBS to resuspend the sperm, centrifuge at 1500r / min for 4min, repeat the operation twice to wash away impurities in the sperm. Add 1mL4% paraformaldehyde to the washed sperm for fixation, centrifuge at 1500r / min for 10min after 10min, discard the supernatant, add 1mLPBS to wash the sperm, and repeat the operation twice. After centrifugation, add an appropriate amount of PBS to resuspend the sperm, make a semen smear, air dry naturally, and then put it in a Coomassie brilliant blue dyeing tank for staining for 30min. Tilt the slide, slowly rinse the slide with deionized water in the sink, completely rinse off the dye on the slide, tilt the sperm slide to dry for 5-10min, and finally use a microscope to check the condition of the sperm acrosome. Figure 3 As shown in the results, on the 6th and 9th days of cryopreservation, the sperm acrosome integrity rate in the galangin-kaempferol complex group was significantly higher than that in the blank control and negative control group (P < 0.05).

[0041] Sperm acrosome integrity rate = sperm with intact acrosome / total number of sperm counted * 100% 3. Plasma membrane integrity rate. First, prepare a hypotonic solution, weigh 0.735g sodium citrate and 1.351g fructose and add them to 100mL distilled water. Take 100µL of semen in a 1.5mL centrifuge tube, incubate in a 37℃ water bath for 15min to restore sperm motility, then add 1mL of hypotonic solution and continue incubating in a 37℃ water bath for half an hour. While incubating, turn on the computer-assisted sperm analyzer to preheat. After the incubation is completed, take 2µL of semen and drop it into the matching glass slide micropore. Through the display screen of the computer-assisted sperm analyzer, you can clearly observe whether the sperm tail is bent. Figure 4 As shown in the results, on the 6th and 9th days of cryopreservation, the sperm plasma membrane integrity rate in the galangin-kaempferol complex group was significantly higher than that in the blank control and negative control groups (P < 0.05).

[0042] Sperm plasma membrane integrity rate = sperm with intact plasma membrane / total number of sperm counted * 100% 4. Total bacterial colony count. Dilute the pig sperm diluent 100 times and then draw 1 ml into LB medium. Spread the plate and mix well. Incubate in a 37℃ biochemical incubator for 24 hours. Take out and count the colonies. Detect the bacterial content in the pig sperm diluent after 3, 6, and 9 days of storage. Repeat each treatment 3 times. Figure 5 As shown, the total bacterial count in semen samples showed an accumulative trend with prolonged storage. On days 3, 6, and 9 of cryopreservation, the total bacterial count in the galangin-kaempferol combination group was consistently significantly higher than that in the blank and negative control groups (P < 0.05). In particular, on day 6 of storage, the bacterial count in the control group significantly exceeded that in the combination-treated group (P < 0.05), and this difference continued to increase with prolonged storage.

[0043] 5. Detect sperm quality. The specific operation is as follows: (1) Sample pretreatment: Accurately transfer 100µL of semen sample to a 1.5mL centrifuge tube, add BeltLsville diluent (BTS) to adjust the final volume to 1mL, and place in a water bath (37℃) for 15min of activity recovery treatment. (2) Gradient centrifugation purification: Use a centrifuge to perform 600g centrifugation for 4min, discard the supernatant and wash twice with PBS (600g×4min×2 times). After effectively removing impurities, detect the ATP content and Ca in the semen according to the reagent instructions. 2+ Content, malondialdehyde (MDA), mitochondrial membrane potential level, total antioxidant capacity and DNA fragmentation index, and each treatment was repeated 3 times. Figure 6 As shown, the ATP content and Ca 2+The content, mitochondrial membrane potential level and total antioxidant capacity of the galangin-kaempferol compound group were significantly higher than those of the blank control and negative control group (P<0.05); the MDA level and DNA fragmentation index of the galangin-kaempferol compound group were significantly lower than those of the blank control and negative control group (P<0.05). Therefore, the combination of 0.027g galangin and 0.003g kaempferol can effectively maintain the low-temperature storage quality of Hainan pig sperm. The present invention improves the low-temperature preservation effect of Hainan pig semen by adding a combination of galangin and kaempferol. During the low-temperature storage of semen, compared with the control group, the sperm motility, vitality, plasma membrane integrity rate and acrosome integrity rate of the added plant extracts (galangin and kaempferol) decreased slowly. The mitochondrial membrane potential level, ATP content, total antioxidant capacity and Ca of the sperm in the group with added galangin and kaempferol decreased significantly. 2+ The content was significantly higher than that in the control group, and the semen quality was the highest when semen was preserved with a combination of 0.027g galangin and 0.003g kaempferol.

[0044] Finally, it should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of additional identical elements in the process, method, article, or device comprising the element.

[0045] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0046] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present application. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Hainan pig semen preservation diluent, characterized in that, The diluent is prepared by adding a plant extract into a basic diluent and mixing the mixture. The plant extract comprises galangin and kaempferol. The basic diluent consists of glucose, sodium citrate, ethylenediaminetetraacetic acid, sodium bicarbonate, potassium chloride, bovine serum albumin and ultrapure water.

2. The Hainan pig semen preservation diluent according to claim 1, characterized in that Each liter of basic diluent contains the following raw materials by mass: glucose 24g-50g, sodium citrate 4g-8g, ethylenediaminetetraacetic acid 1.0g-1.5g, sodium bicarbonate 1.0g-1.5g, potassium chloride 0.5g-1.0g, bovine serum albumin 2g-6g, and the rest is ultrapure water; Add 0.014g-0.040g of galangin and 0.0015g-0.0045g of kaempferol to each liter of basic dilution solution.

3. The Hainan pig semen preservation diluent according to claim 1 or 2, characterized in that The basic diluent contains the following raw materials by mass: 37 g of glucose, 6 g of sodium citrate, 1.25 g of ethylenediaminetetraacetic acid, 1.25 g of sodium bicarbonate, 0.75 g of potassium chloride, 4 g of bovine serum albumin, and the remainder is ultrapure water; 0.027 g of galangin and 0.003 g of kaempferol are added to each liter of the basic diluent.

4. The method for preparing the Hainan pig semen preservation diluent according to any one of claims 1 to 2, characterized in that: The following steps are involved: Glucose, sodium citrate, ethylenediaminetetraacetic acid, sodium bicarbonate, potassium chloride, and bovine serum albumin were weighed in proportion and fully dissolved in ultrapure water to obtain a basic diluent; galangin and kaempferol were added to the basic diluent in proportion and mixed uniformly to obtain a diluent.

5. The use of the Hainan pig semen preservation diluent according to any one of claims 1-2, characterized in that, The Hainan pig semen is placed in the diluent and stored at a low temperature of 0°C to 5°C.

6. The use of the Hainan pig semen preservation diluent according to claim 5, characterized in that: The volume ratio of the Hainan pig semen to the diluent is 1:

5.

7. The method for detecting the Hainan pig semen preservation diluent according to any one of claims 1-2, characterized in that: The following steps are involved: Hainan pig semen was collected by hand-holding method, and semen with normal color and odor and sperm motility of more than 80% was selected; 2 mL of semen was taken into a sterile 10 mL centrifuge tube, and the diluent was slowly added to the 10 mL scale line, and the centrifuge tube was wrapped with a towel. At the same time, water of the same temperature was added into a foam box, and the diluted semen was placed into the foam box, which was covered and stored at a low temperature of 0°C to 5°C, and then dropped to 4°C for 24 hours; the semen was stored for 15 days, and the semen was slowly turned over once every 12 hours, and the sperm motility was checked when the semen was stored for 3, 6, 9, 12, and 15 days respectively; the semen quality indicators and bacterial content were detected and evaluated when the semen was stored for 3 days, 6 days, and 9 days.

8. The method for detecting semen according to claim 7, characterized in that: Indicators include sperm motility, acrosome integrity, and plasma membrane integrity.

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