A long-acting room-temperature pig semen preservation diluent and its application

Abstract

The present invention provides a long-acting room-temperature pig semen preservation diluent and its application. The present invention can maintain the sperm cell structure, reduce the lipid oxidation level, increase the sperm antioxidant enzyme activity and increase the sperm AMPKα phosphorylation level by adding methyl gallate to the semen, significantly improving the quality of sperm stored at room temperature and extending the storage time; pBD129 significantly improves the sperm quality and extends the storage time of semen by maintaining the sperm cell structure and increasing the sperm AMPKα phosphorylation level. The present invention realizes the long-term storage of pig semen diluent at room temperature, which is significantly improved compared with the existing conventional methods and conventional diluents, and does not require additional operations and environmental control, is low-cost and simple in method, and is conducive to popularization and application in industry.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a long-acting room-temperature pig semen preservation diluent and an application thereof. Background Art

[0002] In order to effectively carry out artificial insemination of pigs, the dilution and storage of semen is a key technology. The success of semen freezing technology has enabled semen to be stored for a long time, but due to the low conception rate of frozen semen in pigs, the difficulty of freezing technology and the high investment in freezing equipment, it cannot be widely used in production. The low-temperature storage of boar semen is not as good as the room temperature storage. Therefore, the room temperature storage of boar semen has great practical value in production. The international community attaches great importance to the liquid storage of pig semen. Over the years, researchers from various countries have also done a lot of research work.

[0003] Among the external environmental factors that affect sperm survival, storage temperature is crucial. Pig sperm is very sensitive to low temperatures and can only tolerate temperatures above 15°C, which involves the sensitivity of the membrane to temperature. Buhr (1990) explored this from the cellular molecular level and believed that the fluid state of the protoplasmic membrane of the head of pig sperm will change under certain conditions. This change reflects that the normal molecular rearrangement is affected by calcium ions and temperature. A large number of experiments on the effect of temperature are constantly being carried out. Studies have shown that although casein and the low-density lipoprotein-containing part of egg yolk are known to reduce the damaging effects of cold shock and cooling on sperm, if the storage temperature is above 15°C, the fertilization energy can be better preserved. Therefore, pig semen is generally stored at a temperature of 15-18°C. However, to date, the diluent formula that can store pig semen at room temperature for more than 3 days while maintaining a good fertilization rate has not achieved satisfactory results. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a long-acting room-temperature pig semen preservation diluent.

[0005] Another object of the present invention is to provide an application of the above-mentioned long-acting room-temperature pig semen preservation diluent.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A long-acting room-temperature pig semen preservation diluent comprises a basic diluent and an additive.

[0008] The formula of the basic diluent is that per 100 mL, it includes 2000-3000 mg of glucose, 400-600 mg of Tris, 600-800 mg of sodium citrate, 200-400 mg of citric acid, 50-200 mg of sodium bicarbonate, 100-300 mg of EDTA, 20-100 mg of penicillin, 50-200 mg of streptomycin, and the balance is water; preferably, per 100 mL, it includes 2750 mg of glucose, 565 mg of Tris, 690 mg of sodium citrate, 290 mg of citric acid, 100 mg of sodium bicarbonate, 235 mg of EDTA, 60 mg of penicillin, 100 mg of streptomycin, and the balance is water.

[0009] The additive is 5-50 μmol / L methyl gallate and / or 0.25-10 μg / mL pBD129 protein; preferably 5-50 μmol / L methyl gallate and 0.25-10 μg / mL pBD129 protein.

[0010] The diluent can preserve the sperm motility of pig semen at room temperature (16-18°C).

[0011] The above diluent is used for storing pig semen at room temperature.

[0012] A method for preserving pig semen at room temperature comprises the following steps:

[0013] Add basic diluent to the collected pig semen, then add additives and store at room temperature.

[0014] The normal temperature is 16-18°C.

[0015] The amount of the basic diluent added is to control the sperm density in the final solution to be 1×10 6～10 The data is based on the data of pieces / mL.

[0016] The additive is 5-50 μmol / L methyl gallate and / or 0.25-10 μg / mL pBD129 protein; preferably 5-50 μmol / L methyl gallate and 0.25-10 μg / mL pBD129 protein.

[0017] Compared with the prior art, the present invention has the following advantages and effects:

[0018] 1. Adding methyl gallate to semen can significantly improve the quality of sperm stored at room temperature and prolong the storage time by maintaining sperm cell structure, reducing lipid oxidation levels, increasing sperm antioxidant enzyme activity and increasing sperm AMPKα phosphorylation levels; pBD129 can significantly improve sperm quality and prolong the storage time of semen by maintaining sperm cell structure and increasing sperm AMPKα phosphorylation levels.

[0019] 2. The present invention achieves long-term storage of pig semen diluent at room temperature by adding methyl gallate and / or pBD129 protein, which is significantly improved compared with the existing conventional methods and conventional diluents, and does not require additional operations, has low cost and simple method, which is conducive to promotion and application in industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the result of the effect of MG on the motility parameters of pig sperm during storage at room temperature.

[0021] Figure 2 This is a fluorescence image of pig sperm plasma membrane staining using 6-CFDA.

[0022] Figure 3 This is a fluorescence image of pig sperm acrosome staining using PNA-FITC.

[0023] Figure 4 This is a fluorescence image of sperm mitochondrial membrane potential stained with JC-1.

[0024] Figure 5 This is a graph showing the effect of MG on the mitochondrial membrane potential level of sperm during room temperature storage.

[0025] Figure 6 This is a graph showing the effect of MG on the T-AOC level of pig sperm during room temperature storage.

[0026] Figure 7 This is the result of the effect of MG on the MDA level of pig sperm during room temperature storage.

[0027] Figure 8 This is the result of the effect of MG on the SOD activity of pig sperm during storage at room temperature.

[0028] Fig. 9 This is the result of the effect of MG on the GSH-Px activity of pig sperm during room temperature storage.

[0029] Fig.10 This is the result of the effect of MG on the CAT activity of pig sperm during storage at room temperature.

[0030] Fig.11 This is the result of the effect of MG on the phosphorylation level of AMPK protein in pig sperm during room temperature storage.

[0031] Fig.12 This is a diagram showing the results of Western blotting and Coomassie Brilliant Blue staining to verify the prokaryotic expressed pBD129 protein.

[0032] Fig.13 This is a graph showing the effect of pBD129 on the motility parameters of pig sperm during storage at room temperature.

[0033] Fig.14 This is a graph showing the effect of pBD129 on the phosphorylation level of AMPK protein in pig sperm during storage at room temperature.

[0034] Fig.15 This is a graph showing the effects of MG and pBD129 on the motility parameters of pig sperm during storage at room temperature. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0036] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0037] Example 1 Effect of methyl gallate (MG) on the preservation of pig semen at room temperature

[0038] 1. Collection of pig semen samples

[0039] This study used semen from healthy adult Landrace boars. Semen was collected by hand-holding at fixed intervals in a thermos cup lined with three layers of filter paper to filter out colloid and other impurities. When the semen had normal odor and color, the semen was quickly returned to the laboratory for preliminary sperm evaluation, which required that the motility of semen under microscopic examination be greater than 80%.

[0040] 2. Dilution and storage of pig semen

[0041] Preparation of basic diluent: Use an electronic analytical balance to accurately weigh 2750 mg of glucose, 565 mg of Tris, 690 mg of sodium citrate, 290 mg of citric acid, 100 mg of sodium bicarbonate, 235 mg of EDTA, 60 mg of penicillin, and 100 mg of streptomycin into a clean 100 mL beaker. Add double distilled water to the beaker to dissolve and adjust the pH to 7.2. Pour it into a 100 mL volumetric flask to make up the volume. Filter with a 0.22 μm filter membrane and then divide it into portions. Seal it with sealing film for later use and store it at 4°C.

[0042] Preparation of MG storage solution: weigh 500μmol / L of MG in a 50mL clean beaker, add an appropriate amount of diluent to the beaker, and after complete dissolution, dilute to 50mL to make the final concentration of MG 500μmol / L, filter with a 0.22μm filter membrane and dispense, seal with sealing film for later use, and store at -20℃. Put the semen screened in step 1 into a sterile beaker, add an appropriate amount of isothermal basic diluent to the beaker, and make the final sperm density 1×10 8 / mL. Add methyl gallate at final concentrations of 5μmol / L, 10μmol / L, 20μmol / L, and 50μmol / L to the semen. Wrap the semen with multiple layers of towels and place it in a 17℃ constant temperature box. Invert and mix the sample every 12 hours.

[0043] 3. Sperm motility test

[0044] The CASA system was used to detect sperm motility on days 0, 2, 4, 6, and 8 after the start of storage. The detection steps were performed according to the instructions. The water bath was opened in advance and heated to 37°C. The semen was taken out of the 17-degree Celsius incubator, 100 μL of semen was put into a 200 μL centrifuge tube, and the semen was put into the preheated water bath. After heating for 5 minutes, 10 μL of semen was taken from it and scored into the preheated disposable sperm counting plate. The focus of the microscope was adjusted for motility detection. Five fields of view were taken for each sample. The number of sperm in each field of view was more than 200. The results were averaged. The results are shown in Tables 1 and Figure 1 As shown in A~D.

[0045] Table 1 Effect of methyl gallate on pig sperm motility

[0046]

[0047] Note: Data with different letters in the same row are significantly different (p<0.05). n=5, representing semen from 5 different boars.

[0048] 4. Detection of sperm plasma membrane integrity

[0049] The 6-CFDA / PI fluorescent staining method was used to detect the integrity of the sperm plasma membrane at 0, 2, 4, 6, and 8 days of storage at room temperature. Take 100μL of semen and put it into a 200μL centrifuge tube. Add 1μL of 6-CFDA concentrated storage solution and PI concentrated storage solution and incubate it in a 37℃ water bath for 15 minutes. After the incubation, take 10μL of semen and drop it on a glass slide, cover it with a coverslip, and place it under a fluorescent inverted microscope to detect the integrity of the sperm plasma membrane and take pictures. Green fluorescence indicates that the sperm head plasma membrane is intact, and red fluorescence or no fluorescence indicates that the sperm head plasma membrane is damaged. The experimental results are as follows: Figure 2 When taking photos, each sample was required to take 5 fields of view, and there were more than 200 sperm in each field of view. The experiment was conducted in a dark environment throughout the whole process. The results are shown in Table 2.

[0050] Table 2 Effect of methyl gallate on sperm plasma membrane integrity

[0051]

[0052] Note: Data with different letters in the same row are significantly different (p<0.05). n=5, representing semen from 5 different boars.

[0053] 5. Sperm acrosome integrity test

[0054] Fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) and Hoechst 33342 fluorescent dyes were used to stain sperm stored at room temperature for 0, 2, 4, 6, and 8 days, and then detected and photographed using a fluorescence microscope. 10 μL of semen was dropped on a glass slide, and evenly spread with a cover glass, and air-dried naturally; after air-drying, 10 μL of anhydrous methanol was dropped on the glass slide and fixed at room temperature for 10 minutes; after the methanol was air-dried, 20 μL of FITC-PNA working solution was evenly dropped on the glass slide, and after ensuring that the sample was covered, the glass slide was incubated at 37°C in the dark for 30 minutes, and 10 μL of Hoechst dye solution was dropped on the sample, and incubated for 10 minutes. After the incubation, the glass slide was gently rinsed 2-3 times with PBS to wash off the floating color; after air-drying, it was sealed with glycerol, covered with a cover glass, and detected and photographed under a fluorescence microscope. Five fields of view were taken for each sample, and each field of view had more than 200 sperm. The experiment was conducted in a dark environment. FITC-PNA made the sperm acrosome emit green fluorescence, while Hoechst33342 made the sperm nucleus emit blue fluorescence. Figure 3 , the results are shown in Table 3.

[0055] Table 3 Effect of methyl gallate on the acrosome integrity rate of pig sperm

[0056]

[0057] Note: Data with different letters in the same row are significantly different (p<0.05). n=5, representing semen from 5 different boars.

[0058] 6. Detection of sperm mitochondrial membrane potential level

[0059] The mitochondrial membrane potential of sperm stored at room temperature on days 0, 2, 4, 6, and 8 was detected using the Bio-Tech Mitochondrial Membrane Potential Detection Kit (JC-1) according to the instructions of the kit. The confocal fluorescence microscopy photos are shown in the figure. Figure 4 The experimental results are as follows Figure 5 shown.

[0060] 7. Total Antioxidant Capacity of Sperm

[0061] The total antioxidant capacity of sperm stored at room temperature on days 0, 2, 4, 6, and 8 was tested using the Bio-Tian Total Antioxidant Detection Kit. The operation was performed according to the instructions of the kit. The results are as follows: Figure 6 .

[0062] 8. Sperm malondialdehyde content detection

[0063] The MDA content in sperm stored at room temperature on days 0, 2, 4, 6, and 8 was detected using the Bio-Tech MDA content detection kit. The operation was performed according to the kit instructions. The results were as follows: Figure 7 .

[0064] 9. Sperm superoxide dismutase level detection

[0065] The superoxide dismutase level in sperm stored at room temperature on days 0, 2, 4, 6, and 8 was detected using the Bio-Tech Superoxide Dismutase Detection Kit. The procedure was performed according to the kit instructions. The results were as follows: Figure 8 .

[0066] 10. Sperm glutathionease activity detection

[0067] The glutathionease activity in sperm stored at room temperature on days 0, 2, 4, 6, and 8 was detected using the Bio-Tech Glutathionease Activity Detection Kit. The operation was performed according to the instructions of the kit. The results were as follows: Fig. 9 .

[0068] 11. Sperm catalase activity detection

[0069] The catalase activity in sperm stored at room temperature on days 0, 2, 4, 6, and 8 was detected using the Bio-Tian Peroxidase Activity Detection Kit. The results were as follows: Fig.10 .

[0070] 12. Western blot assay to detect the effect of MG on sperm AMPKα phosphorylation level

[0071] Protein was extracted from sperm stored for 5 days. Sperm protein was extracted using the Keygen Biotech Whole Protein Extraction Kit, and the concentration of the extracted protein was detected using the Keygen BCA Protein Content Detection Kit. The operation was performed according to the instructions of the kit. The protein level of the selected protein was verified by SDS-PAGE and Western blot. According to the size of the protein, a 12% polyacrylamide gel separation gel was selected; the electrophoresis splint was installed, and ddH2O was added to test for leaks for 10 minutes. After the leak test, the separation gel was slowly added to it, and then ddH2O was slowly and evenly added to it, and waited for 25 minutes. When a clear dividing line between the gel and the water appeared, the upper layer of gel was poured out and the compressed gel was slowly added to it, and then the comb was immediately inserted, and the comb was pulled out after waiting for 25 minutes, and the sample was added. Add electrophoresis solution to the electrophoresis tank and perform SDS-PAGE gel electrophoresis. Set the program to 80V, 35min, 120V, 100min. After the electrophoresis, take out the gel and gently place it in the transfer solution for 10min. Cut out the PVDF membrane of the corresponding size, activate it in methanol for 10sec, and then place it in the transfer solution for equilibration. Use wet transfer and place the sponge pad, filter paper, SDS gel and membrane in a sandwich manner. Place the SDS gel on the negative pole of the transfer splint, place the membrane on the gel, and then place the filter paper and sponge pad. Use a triangular glass rod to carefully drive away the bubbles. After clamping the splint, place the splint in the transfer tank according to the positive and negative poles. Pour the transfer solution into the transfer tank so that the transfer solution completely immerses the splint. Set the program to 90v, 80min. Open the splint carefully. After observing that the maker pre-stained band has been completely transferred to the PVDF membrane, carefully take out the PVDF membrane without leaving marks on the membrane. Use TBST solution Wash once, 5min; pour out TBST, cover the membrane with the prepared blocking solution to block, place it on a shaker at room temperature for 1h, wash it 3 times with TBST, 5min each time; dilute the antibody with the primary antibody diluent according to the ratio written in the antibody manual, pour it into the incubation box, cover the PVDF membrane, incubate it at 4℃ overnight, wash it 5 times with TBST, 5min each time; dilute the secondary antibody with TBST according to the ratio written in the secondary antibody manual, pour the diluted secondary antibody into the incubation box, cover the PVDF membrane, incubate it at room temperature for 1h, wash it 5 times with TBST after the incubation, 5min each time; turn on the exposure instrument of the fully automatic digital gel imaging analysis system in advance to precool; prepare ECL luminescent solution, mix the same volume of solution A and solution B, quickly and evenly drop it on the PVDF membrane, react for about 10sec, absorb the excess luminescent solution with filter paper, and expose it for analysis in the fully automatic digital gel imaging analysis system. Protect from light during the exposure process. AMPKα1 / 2 antibody (D-6) was purchased from Santa Cruz Biotechnology, USA; the results are shown in Fig.11 .

[0072] The primary antibodies were AMPKα1 / 2 antibody (D-6) purchased from Santa Cruz Biotechnology, USA, and AMPKα1 / 2 (phospho Thr183 / 172) Polyclonal Antibody purchased from ImmunoWay Biotechnology, USA; the secondary antibodies were horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) and horseradish peroxidase-labeled goat anti-mouse IgG (H+L), both purchased from Shanghai Biotime.

[0073] The experimental results show that adding 10 μmol / L methyl gallate to the semen diluent has the best effect on improving the room temperature storage of semen. Compared with the control group, it significantly improved sperm motility, motility rate, plasma membrane integrity and acrosome integrity (p<0.05); significantly reduced lipid oxidation level (p<0.05), and significantly increased sperm total antioxidant capacity and SOD, GPx, and CAT activities (p<0.05), as well as AMPKα phosphorylation level (p<0.05); and extended the room temperature storage time of semen to the 8th day.

[0074] Example 2 Effect of pBD129 on the Storage of Swine Semen at Normal Temperature

[0075] 1. Preparation of pBD129

[0076] RNA was extracted from the boar epididymis and reverse transcribed into cDNA. The CDS region without signal peptide and terminator was amplified by PCR using pBD129-F / R. The internal reference was set at the same time. The band with the expected target gene size was found to be amplified by gel electrophoresis, with a length of about 492bp. After successfully amplifying pBD129 without signal peptide and terminator, it was recovered and connected to the pMD-18T vector and transformed into Escherichia coli DH5α. The positive bacteria with correct sequencing were amplified in large quantities. After the plasmid was extracted, the target fragment was double-digested and connected to the pET-28a expression vector, and then transformed into Escherichia coli BL21 (DE3). After the monoclonal colony was picked and expanded for culture, a PCR test was performed to identify the positive colony. It was sent for sequencing and found to be consistent with the PDB129 gene (GenBank accession number: NM_001129975.1), indicating that the pET-28a-pBD129 plasmid was successfully constructed.

[0077] pBD129-F: CGCCATATGATGGAATACTTTGGCTTGGGAAG;

[0078] pBD129-R: CCGCTCGAGAGTCATGCTGTCGGCAGGGT;

[0079] Nucleotide sequence of pBD129:

[0080] ATGGAATACTTCGGTCTGGGCCGTTGCGTTATGGGCCTGGGTCGTTGCAAAGATCACTGCGCGGTTAACGAAAAAGAAATCGATAAATGCAAAAAGAAAAAATGCTGCATCGGCCCGAAAGGTATCCAGCTGATCAAAAGCTACCTGCAGAACGAAATGATCCGTACCCTGGAAGAAGTTGCGAAAAAGAACATCACCAAAAACAGCAAAGTTGTTACCCCGAGCAAATACCG TCCGCTGAGCCACCTGCCGGAAATCAAAAGCACCAACCCGTTCGCGAGCATCAACACCACCATCATCCCGAACGGCACCACCGTTAACAGCACCACCACCAACAGCACCACCAGCCGTCGTAGCACCCACCCGGCGACCAGCACCAAAAGCGATACCAAAAAACGTCGTGATAGCCCGACCGAAAGCCCGCCGGCGGATTCTAGCCCGATCGATCCGGCGGATAGCATGACC;

[0081] Amino acid sequence of pBD129:

[0082] MEYFGLGRCVMGLGRCKDHCAVNEKEIDKCKKKKCCIGPKGIQLIKSYLQNEMIRTLEEVAKKNITKNSKWVVTPSKYRPLSHLPEIKSTNPFASINTTIIPNGTTVNSTTTNSTTSRRSTHPATSTKSDTKKRRDSPTESPPADSSPIDPADSMT;

[0083] The BL21-pET28-pBD129 cells were activated overnight, and 1 mL of uninduced BL21 cell protein was extracted as a control. After overnight induction with the addition of IPTG at a final concentration of 1 mmol / L, the BL21 cells were ultrasonically disrupted and subjected to SDS-PAGE followed by Coomassie Brilliant Blue staining and Western blot to verify protein expression. The results showed that a clear band was found at about 24 kDa ( Fig.12), which is consistent with the expected size, and it can be preliminarily determined that the induced protein is the target protein. The BL21 cells after induction and crushing were separated into soluble proteins and inclusion bodies. After Western blot, it was found that the expression of the target protein could be detected in both non-inclusion bodies and inclusion bodies, indicating that the pBD129 protein was expressed in the form of soluble protein and inclusion body protein. After that, the recombinant protein in the supernatant was purified using a Ni-NTA Agarose resin HP column, and the target protein obtained by eluting with a non-denaturing eluent was collected. The concentration of the recombinant protein after purification was detected using a Keygen BCA kit, and the results showed that the purified porcine defensin pBD129 protein was obtained (the specific preparation method of the pBD129 protein can be referred to CN114891819A).

[0084] 2. Collection of pig semen samples

[0085] Refer to step 1 of Example 1.

[0086] 3. Dilution and storage of pig semen

[0087] Referring to step 2 of Example 1, pBD129 was added to the semen diluted with the basic diluent at final concentrations of 0.25 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, and 10 μg / mL, and then the subsequent storage steps were performed.

[0088] 4. Sperm motility test

[0089] Referring to step 3 of Example 1, the sperm motility parameters were detected using the CASA system. The results are shown in Table 4 and Fig.13 A~D.

[0090] Table 4 Effect of pBD129 on pig sperm motility

[0091]

[0092] Note: Data with different letters in the same row are significantly different (p<0.05). n=5, representing semen from 5 different boars.

[0093] 5. Sperm plasma membrane integrity test

[0094] Referring to step 4 of Example 1, the 6-CFDA / PI fluorescent staining method was used to detect the integrity of the sperm plasma membrane at 0, 2, 4, 6, 8, and 10 days of storage at room temperature. The results are shown in Table 5.

[0095] Table 5 Effect of pBD129 on the integrity of pig sperm plasma membrane

[0096]

[0097] Note: Data with different letters in the same row are significantly different (p<0.05). n=5, representing semen from 5 different boars.

[0098] 6. Sperm acrosome integrity test

[0099] Referring to step 5 of Example 1, sperm stored at room temperature on days 0, 2, 4, 6, 8, and 10 were stained with fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) and Hoechst 33342 fluorescent dyes and examined and photographed using a fluorescence microscope. The results are shown in Table 6.

[0100] Table 6 Effect of pBD129 on the acrosome integrity rate of pig sperm

[0101]

[0102] Note: Data with different letters in the same row are significantly different (p<0.05). n=5, representing semen from 5 different boars.

[0103] 7. Western blot assay to detect the effect of pBD129 on sperm AMPKα phosphorylation level

[0104] The operation is as shown in step 12 of Example 1. Fig.14 .

[0105] The experimental results showed that adding 1.25μg / mL and 2.5μg / mL of pBD129 to the semen diluent had the best effect on improving the storage of semen at room temperature, significantly improving sperm motility, motility, plasma membrane integrity and acrosome integrity (p<0.05); and significantly increasing the AMPKα phosphorylation level (p<0.05); and significantly extending the storage time of semen at room temperature to the 10th day, which was significantly higher than the control group (p<0.05).

[0106] Example 3

[0107] 1. Effect of MG on sperm in vitro fertilization

[0108] Pig ovaries were collected from slaughterhouses, placed in thermos bottles containing 37°C saline, and transported back to the laboratory within 3 hours; the ovaries were thoroughly washed 2 to 3 times with saline containing 1000 IU penicillin and streptomycin, and then placed in a beaker and placed in a water bath preheated at 37°C; a 10 mL syringe with a No. 12 needle was inserted into the side of the needle into a follicle with a diameter of 3 to 6 mm to extract follicular fluid, and then the follicular fluid was collected in a 50 mL centrifuge tube placed in a 37°C water bath; the follicular fluid was allowed to stand for 20 minutes, the supernatant was discarded, and the ovaries were resuspended with DPBS-PVA egg washing solution preheated at 37°C and allowed to stand for 20 minutes. After 20 minutes, repeat 3 times; resuspend the precipitate and pour a small amount into a clean culture dish, select the cumulus-oocyte complexes (COCs) with uniform cytoplasm and wrapped by a large number of cumulus cells under a stereomicroscope; wash the selected cumulus-oocyte complexes 3 times with DPBS-PVA egg washing solution, and then wash them 3 times with balanced oocyte maturation culture medium; place the washed cumulus-oocyte complexes in oocyte maturation culture medium balanced for 6-12 hours in advance, and mature and culture them in an incubator for 44-48 hours, and the culture conditions are 38.5°C, 5% CO2, and 100% relative humidity;

[0109] After 44-48h of maturation culture, the cells are taken out of the incubator, and the expansion degree of the cumulus cells around the oocytes is observed under a stereomicroscope. The oocytes with good expansion of the surrounding cumulus cells are taken out and placed in a centrifuge tube, and the de-cumulus fluid is added to the centrifuge tube, and the mixture is blown and mixed for 2-3min, and then the cumulus cells are removed by gentle blowing about 20 times with a pipette; the oocyte and the de-cumulus fluid mixture are taken out and placed in a culture dish, and then the oocytes are transferred to the HN operating solution to select the mature oocytes, which are washed 3 times with the HN operating solution, and then washed 3 times with the fertilization solution, and then placed in the fertilization droplet balanced for 12h in advance, and returned to the incubator to be fertilized;

[0110] Take out the semen diluted with the MG-treated diluent prepared in step 2 of Example 1 and stored for 5 days from a 17°C incubator, mix gently and transfer to a centrifuge tube, centrifuge at 1300rpm for 9 minutes, and discard the supernatant; wash and resuspend the sperm with room temperature semen washing solution, centrifuge at 1300rpm for 4 minutes, and discard the supernatant; repeat 3 times; add room temperature mTBM to the centrifuge tube to resuspend the sperm, centrifuge at 1300rpm for 4 minutes, and discard the supernatant; resuspend the sperm with a small amount of mTBM, transfer to a new centrifuge tube, add 2mL of capacitation solution, tilt at 45°, put into an incubator, and capacitation by flotation method. The incubator is set to 38.5°C, 5% CO2, and 100% relative humidity for 40-60 minutes; take the upper sperm, adjust the sperm density to 10 6 / mL, take out the fertilized drop with oocytes from the incubator, add 20μL of diluted semen to each fertilized drop, and then put it in the incubator for incubation for 6-8h;

[0111] After the incubation, the fertilized eggs were transferred to the HN operating solution to wash away the sperm on the surface of the oocytes; the oocytes were washed three times with PZM-3 embryo culture medium, and then the washed oocytes were transferred to the equilibrated PZM-3 droplets and placed in an incubator for culture. The incubator was set at 38.5°C, 5% CO2, and 100% relative humidity;

[0112] After 48 h of culture, the cleavage rate was observed. Cleavage rate = number of cleavage cells / total number of oocytes*100%. The results are shown in Table 7.

[0113] Table 7 Effect of adding methyl gallate to semen on the cleavage rate of in vitro fertilization

[0114]

[0115] Note: Data marked with different capital letters have extremely significant differences (p<0.01). n=5, representing semen from 5 different boars.

[0116] 2. Effect of pBD129 on sperm in vitro fertilization

[0117] The steps are similar to those in step 1, except that the semen diluent treated with pBD129 prepared in Example 2 is used. The results are shown in Table 8.

[0118] Table 8 Effect of pBD129-added semen on in vitro fertilization cleavage rate

[0119]

[0120] Note: Data marked with different capital letters have extremely significant differences (p<0.01). n=5, representing semen from 5 different boars.

[0121] The experimental results showed that adding 5-20 μmol / L MG or 1.25-10 μg / mL pBD129 to the semen diluent could significantly increase the cleavage rate of sperm stored for 5 days in vitro fertilization (p<0.05).

[0122] Example 4 Effect of co-administration of MG and pBD129 on the storage of porcine semen at room temperature

[0123] In Examples 1 and 2, it was experimentally verified that when MG concentration was 5 μmol / L or 10 μmol / L and pBD129 concentration was 1.25 μg / mL or 2.5 μg / mL in the pig semen diluent stored at room temperature, the preservation effect was the best. Therefore, the two substances were further compounded to further verify the optimal preservation formula. The specific experimental steps are as follows:

[0124] 1. Collection of pig semen samples

[0125] Refer to step 1 of Example 1.

[0126] 2. Dilution and storage of pig semen

[0127] Referring to step 2 of step example 1, final concentrations of 10 μmol / L MG, 2.5 μg / mL pBD129, 5 μmol / L MG + 1.25 μg / mL pBD129, 5 μmol / L MG + 2.5 μg / mL pBD129, 10 μmol / L MG + 1.25 μg / mL pBD129, and 10 μmol / L MG + 2.5 μg / mL pBD129 were added to the semen diluent, and then the subsequent storage step was performed.

[0128] 3. Sperm motility test

[0129] Referring to step 3 of step Example 1, the sperm motility parameters were detected using the CASA system. The results are shown in Table 9 and Fig.15 A~D.

[0130] Table 9 Effects of methyl gallate and pBD129 on sperm motility

[0131]

[0132] Note: The data with different letters on the same row are significantly different (p<0.05). n=5, representing the semen from 5 different boars.

[0133] 4. Detection of sperm plasma membrane integrity

[0134] Referring to step 4 of step example 1, the integrity of the sperm plasma membrane at 0, 2, 4, 6, 8, and 10 days of storage at room temperature was detected using 6-CFDA / PI fluorescent staining. The results are shown in Table 10.

[0135] Table 10 Effects of methyl gallate and pBD129 on the integrity of pig sperm plasma membrane

[0136]

[0137] Note: Data with different letters in the same row are significantly different (p<0.05). n=5, representing semen from 5 different boars.

[0138] 5. Sperm acrosome integrity test

[0139] Referring to step 5 of step example 1, fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) and Hoechst33342 fluorescent dyes were used to stain sperm stored at room temperature on days 0, 2, 4, 6, 8, and 10, and the sperm were detected and photographed using a fluorescence microscope. The results are shown in Table 11.

[0140] Table 11 Effects of methyl gallate and pBD129 on the acrosome integrity rate of pig sperm

[0141]

[0142] Note: Data with different letters in the same row are significantly different (p<0.05). n=5, representing semen from 5 different boars.

[0143] 6. In vitro fertilization

[0144] Each group of semen was subjected to in vitro fertilization according to step 1 of step example 3. The results are shown in Table 12.

[0145] Table 12 Effect of adding methyl gallate and pBD129 to semen on the cleavage rate of in vitro fertilization

[0146]

[0147] Note: Data marked with different capital letters have extremely significant differences (p<0.01). n=5, representing semen from 5 different boars.

[0148] The experimental results show that the semen diluent with the addition of 10μmol / L MG+1.25μg / mL pBD129 has the best comprehensive preservation effect. Compared with diluents of other concentrations, it has a better preservation effect at room temperature and still has good fertilization activity after being stored at room temperature for 10 days.

[0149] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A long-acting room temperature pig semen preservation diluent, characterized in that: Includes basal diluent, methyl gallate, and / or pBD129 protein; The formula of the basic diluent is that per 100 mL, it includes 2750 mg of glucose, 565 mg of Tris, 690 mg of sodium citrate, 290 mg of citric acid, 100 mg of sodium bicarbonate, 235 mg of EDTA, 60 mg of penicillin, 100 mg of streptomycin, and the balance is water; The added amount of methyl gallate is 5 to 50 μmol / L; The added amount of the pBD129 protein is 0.25-10 μg / mL.

2. Use of the diluent according to claim 1 in preserving pig semen at room temperature.

3. A method for preserving pig semen at room temperature, characterized in that The steps include: Adding a basic diluent to the collected pig semen, then adding methyl gallate and / or pBD129 protein, and storing at room temperature; The formula of the basic diluent is that per 100 mL, it includes 2750 mg of glucose, 565 mg of Tris, 690 mg of sodium citrate, 290 mg of citric acid, 100 mg of sodium bicarbonate, 235 mg of EDTA, 60 mg of penicillin, 100 mg of streptomycin, and the balance is water; The added amount of methyl gallate is 5 to 50 μmol / L; The added amount of the pBD129 protein is 0.25-10 μg / mL.

4. The method according to claim 3, characterized in that: The normal temperature is 10-27°C.

5. The method according to claim 3, characterized in that: The amount of the basic diluent added is to control the sperm density in the final solution to be 1×10 6～10 The data is based on the data of pieces / mL.

Images