A kit for improving the quality and conception rate of frozen pig semen and its application

Abstract

The present invention provides a monoclonal antibody targeting recombinant porcine relaxin, an ELISA detection method, and its use in a kit for improving the quality and conception rate of frozen porcine semen. The recombinant porcine relaxin protein, obtained through analysis and optimization using bioinformatics software, and the monoclonal and polyclonal antibodies generated using this as an antigen exhibit strong specificity and high antigen binding affinity. The resulting ELISA kit offers enhanced sensitivity. This invention uniquely quantitatively detects relaxin in seminal plasma, effectively improving post-freeze semen quality and significantly enhancing various indicators.

Description

Technical Field

[0001] The present invention relates to the field of biological proteins, and more particularly to an antibody targeting relaxin in pig seminal plasma, a quantitative detection method for relaxin in pig seminal plasma, and an application of the antibody in a kit for improving the quality and conception rate of frozen pig semen. Background Art

[0002] Semen consists of sperm and seminal plasma. Sperm is the primary carrier of genetic material, while seminal plasma provides the necessary nutrients and environment for sperm. Seminal plasma, a crucial component of semen, is composed of secretions from various glands, including the testicles, epididymis, prostate, seminal vesicle, and bulbourethral glands. Approximately 70% of the fluid in seminal plasma is composed of seminal vesicle fluid, 20% is prostate secretions, and less than 10% is secreted by other glands. Seminal plasma maintains a pH of 7.35-7.5, providing buffering properties that protect sperm from the acidic vaginal environment during fertilization. Seminal plasma is rich in protein, with an average protein concentration of 35-55 g / L. During fertilization, it primarily provides sperm with the nutrients and energy they need to survive. Furthermore, the expression of fucose and sialylated residues on certain glycoproteins in seminal plasma can prevent microorganisms and other antigens from penetrating mucosal surfaces, thereby participating in both innate and adaptive immunity.

[0003] Porcine semen cryopreservation technology holds great promise for pig genetic resource conservation and pig breeding and production. However, frozen semen presents challenges such as low sperm motility, altered physicochemical properties, and low artificial insemination efficiency after thawing. Existing reports have demonstrated that adding seminal plasma from boars with high sperm freeze tolerance to the cryo-diluent significantly improves semen quality. Other studies suggest that adding seminal plasma to the thawing solution can improve acrosome integrity and fertilizing capacity of thawed sperm. However, sperm reproductive performance varies between boar breeds and even within individuals of the same breed, demonstrating that boar semen traits are significantly influenced by genetics. Furthermore, direct sperm analysis often disrupts sperm structure and reduces sperm storage, posing a significant challenge to scarce resources. Therefore, further research is needed to determine whether boars produce viable sperm and ensure effective artificial insemination.

[0004] Studies have confirmed a direct correlation between relaxin concentration in boar semen and sperm motility. Relaxin levels in semen from boar breeds such as Yorkshire, Landrace, Duroc, and Boar-Midland show a strong positive correlation with sperm motility across all breeds. However, there is no commercially available test kit for detecting relaxin in boar semen samples. Existing relaxin detection reagents often suffer from low sensitivity and specificity. The lack of a convenient and accurate method for rapid quantification makes it difficult to translate research findings into practical production. Summary of the Invention

[0005] To address the above problems, the present invention first provides a recombinant porcine relaxin protein, and further prepares polyclonal antibodies and monoclonal antibodies that specifically recognize the recombinant protein, which are used as capture antibodies and detection antibodies in an ELISA double-antibody sandwich kit, respectively. In addition, a method for using the detection kit for relaxin content in porcine seminal plasma and its application are established.

[0006] Specifically, the present invention provides a recombinant porcine relaxin protein, the amino acid sequence of the recombinant porcine relaxin protein is shown in SEQ ID NO.1;

[0007] Furthermore, the present invention provides a monoclonal antibody 3B4 against the above-mentioned recombinant relaxin protein, wherein the heavy chain amino acid sequence of the monoclonal antibody 3B4 is shown in SEQ ID NO: 2, and the light chain amino acid sequence is shown in SEQ ID NO: 3;

[0008] Furthermore, the present invention provides a polyclonal antibody, which is obtained by immunizing animals with the above-mentioned recombinant relaxin protein and then screening by conventional means, and the effective titer of the polyclonal antibody is as high as 1:256000;

[0009] Furthermore, the present invention provides an ELISA kit for quantitatively detecting porcine relaxin protein, comprising the aforementioned monoclonal antibody 3B4, a recombinant relaxin polyclonal antibody, an ELISA plate, a blocking solution, an HRP-goat anti-mouse IgG secondary antibody, a developing solution, a stop solution, a coating solution, and a washing solution. The recombinant relaxin polyclonal antibody serves as a capture antibody, and the monoclonal antibody 3B4 serves as a detection antibody.

[0010] Furthermore, the present invention provides a kit for improving the quality and conception rate of frozen pig semen, wherein the kit comprises the above-mentioned ELISA detection kit and a semen protection diluent; wherein the ingredients of the semen protection diluent are: 0.5% sodium lauryl sulfate, 0.5% laurylamide, 0.2% anhydrous sorbitan fatty acid ester, 0.3% fatty acid monoethanolamide, 0.5% lauric acid, 0.5% triethanolamine, 1% fatty acid monoethanolamide sulfosuccinate salt, 0.2% EDTA, 0.2% epigallocatechin gallate, 10% citric acid buffer, and the balance is double distilled water.

[0011] Furthermore, the present invention provides the use of the above-mentioned kit in improving the quality and conception rate of frozen pig semen, wherein the use is:

[0012] 1) Isothermally dilute the semen protectant and the original semen in a 1:1 ratio, aliquot, and slowly cool down; centrifuge to obtain the supernatant of the seminal plasma and the sperm sediment in the lower layer;

[0013] 2) Quantitatively detect relaxin in the seminal plasma supernatant using an ELISA kit, and select the seminal plasma sample with the highest content for use;

[0014] 3) The sperm pellet was resuspended in porcine sperm cryopreservation solution, wherein the porcine sperm cryopreservation solution was prepared by adding 20% egg yolk and 2.5% homorelaxin seminal plasma sample to the above-mentioned protective diluent and filtering the mixture through a 0.22 μm sterilizing filter;

[0015] 4) The resuspended sperm solution is placed in a container and gradually cooled to 4°C, then sealed and frozen. After freezing, the semen tubes are stored in liquid nitrogen.

[0016] Among them, the quantitative detection method of the ELISA detection kit is:

[0017] 1) Purified polyclonal antibody coating: Dilute the above-mentioned polyclonal antibody against porcine relaxin to 1:500 with coating solution, incubate at 4°C overnight, add 100 μL per well, and store in a refrigerator at 4°C overnight;

[0018] 2) Blocking: Add 200 μL of 5% skim milk powder to each well and incubate at 37°C for 1.5 h;

[0019] 3) Add antigen: Add 100 μL of sample to each well and incubate at 37°C for 1-2 hours;

[0020] 4) Add purified monoclonal antibody: dilute monoclonal antibody 3B4 to 1:1000 with 5% skim milk powder, incubate at 37°C for 1-2 hours, 100 μL per well;

[0021] 5) Add enzyme-labeled secondary antibody: dilute HRP-goat anti-mouse IgG secondary antibody to 1:16000 with 5% skim milk powder, incubate at 37°C for 2 h, 100 μL per well;

[0022] 6) Substrate color development: Add 100 μL of color development solution to each well and allow to develop for 10 min;

[0023] 7) Stop the reaction: add 50 μL / well of 2 mol / L concentrated sulfuric acid to each well, measure the OD450nm reaction value, and substitute it into the standard curve formula to calculate the relaxin content.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1) The recombinant porcine relaxin of the present invention is obtained based on porcine relaxin protein data in the NCBI GenBank database, analyzed and optimized using bioinformatics software. The monoclonal and polyclonal antibodies obtained using this as an antigen have the advantages of strong specificity and high antigen binding affinity. The polyclonal antibody is used as the capture antibody and the monoclonal antibody is used as the detection antibody, which makes the established ELISA kit more sensitive during detection.

[0026] 2) The present invention uniquely performs quantitative detection of relaxin in seminal plasma, and uses the relaxin content as a standard to screen the source of seminal plasma added to the semen cryopreservation reagent, thereby avoiding the uncertainty and blindness of previous seminal plasma additions, and can more effectively improve the quality of frozen semen, with significant improvements in indicators such as sperm motility, acrosome integrity, catalase, glutathione peroxidase, and glutathione reductase activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Electrophoresis of PCR-amplified recombinant porcine relaxin, where M stands for maker and 1 stands for the amplified PCR product of approximately 546 bp in length;

[0028] Figure 2 The figure shows the SDS-PAGE electrophoresis results of the induced expression of recombinant porcine relaxin.

[0029] Figure 3 This is the western blot result after the target protein is expressed and purified, where M is the protein Maker, 1 is the negative control, and 2 is the recombinant protein;

[0030] Figure 4 Western blotting of recombinant porcine relaxin monoclonal antibody, M: protein molecular weight standard; 1: negative control; 2: recombinant porcine relaxin protein;

[0031] Figure 5 Standard curve for sandwich ELISA assay of porcine relaxin protein. Example

[0032] Although the present invention can be implemented in many different forms, what is disclosed here is its specific illustrative embodiment that proves the principle of the present invention. It should be emphasized that the present invention is not limited to the specific embodiment illustrated. In addition, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0033] Generally, terms relating to, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification.

[0034] Example 1. Preparation of Relaxin Monoclonal Antibody

[0035] The amino acid and nucleotide sequences of porcine relaxin protein were downloaded from the NCBI GenBank database. Sequence alignment was performed using DNAStar MegAlign software, and conserved regions were selected. Hydrophilicity analysis was performed using DNAMAN, DNAstar, and ExPASy software. Synthetic peptides were designed by selecting conserved hydrophilic regions while avoiding sites with amino acid differences. The final antigenic peptide sequence was as follows: MPRLFSYLLGVWLLASQLPREIPGQSTNDFIKACGRELVRLWVEICGSVSWGRTALSLEEPQLETGPPAETMPSFINKDA EILKMMLEFVPNLPQELKLTLSEMQPALRELQQSALKDSNLNFEEFRKIILNRQNEAEDKSLLELKNLGLDKHSRKHRLF RMALSEKCCQVGCIRKDIARLC (SEQ ID NO. 1). This peptide was synthesized by Jier Biochemical (Shanghai) Co., Ltd.

[0036] According to the sequence provided by the biological company, the specific upstream primer RLX-F (5'-CCGGATGCATGACCGATGTCAT-3') and downstream primer RLX-R (5'-CTTGGGATCGCCAAACCGGGCACTTCTTTACTGC-3') were designed to amplify the recombinant porcine relaxin. The PCR product was separated by agarose gel electrophoresis and recovered ( Figure 1 The recovered fusion protein gene and the expression plasmid vector pMD20-T were digested with HindIII and BamHI, respectively, recovered again by electrophoresis, and ligated with T4 DNA ligase. The ligation products were transformed into competent E. coli BL21 cells, and colonies were selected from the plates for inoculation. Plasmid DNA was extracted and identified by PCR. Clones positive for the fusion protein gene by PCR were sequenced and analyzed, and clones with completely correct sequences were used.

[0037] Transfer the overnight bacteria from a single colony culture to 100 ml LB medium at a ratio of 1:100, add kanamycin at a final concentration of 50 μg / ml, and culture at 37°C with shaking until the OD600 is 0.6-0.8. Add 0.1 mol / L IPTG and culture at 25°C with shaking for 8 hours. After harvesting, the bacteria are ultrasonically disrupted ( Figure 2 The recombinant protein carries a histidine tag and is affinity purified using a nickel column. After elution with different concentrations of imidazole solution, each fraction and flow-through are loaded separately for SDS-PAGE separation and detection. The purity of the recombinant protein is above 90%, and the concentration is approximately 1-1.5 mg / mL, which can meet the requirements of immunized animals and antibody screening and identification ( Figure 3 ).

[0038] Eight 8-week-old SPF-grade BALB / c female mice (purchased from the Laboratory Animal Research Center of the Chinese Academy of Sciences) were immunized with recombinant porcine relaxin as an antigen. The antigen was mixed with equal volumes of complete Freund's adjuvant (first immunization) and incomplete Freund's adjuvant (boost immunization) and emulsified. The mixture was thoroughly mixed to a water-in-oil state and multiple subcutaneous immunizations were performed. 2-3 booster immunizations were performed with a 2-week interval between each immunization. Afterwards, the titer was tested. If the titer was higher than >1:10,000, intraperitoneal perfusion was performed within 1 week. The immunization dose of antigen was directly dissolved in 250 μL of PBS. The specific immunization times and immunization doses are shown in Table 1:

[0039] Table 1 Immunization schedule

[0040] Number of immunizations Immunization time (d) Immunization dose First immunization 1 200 μg / head (emulsified with equal amount of complete Freund's adjuvant) Second immunization 15 200 μg / head (emulsified with an equal amount of incomplete Freund's adjuvant) The third immunization 30 200 μg / head (emulsified with an equal amount of incomplete Freund's adjuvant) Fourth immunization 3 days before fusion 150 μg antigen

[0041] The serum collected before immunization was used as a negative serum control. On the tenth day after each immunization, blood was collected from the tail vein of the immunized mice. The antibody titer of the collected serum was tested by indirect ELISA. When the serum titer of the mouse reached 105 or above, the antigen without adjuvant was used for booster immunization, and cell fusion could be performed 3 days later.

[0042] Three days after the last shock, positive control blood was collected, spleens were taken, and single-cell suspensions were prepared; SP2 / 0 cells in the logarithmic phase were treated and mixed with spleen cells in a certain ratio (1:5-1:10), and 50% PEG1450 was used for 1 minute, and the suspension was terminated by dilution with basal medium DMEM. After low-speed centrifugation, the suspension was gently suspended and mixed with HAT medium containing 20% fetal bovine serum, and plated onto pre-prepared feeder cell plates at 2×107 / plate, and cultured at 5% CO2 and 37°C.

[0043] After the cells in the fusion plate are replaced with medium and grow to a medium size of about 10,000 cells or more, the test is started. 450 <0.2, positive control OD 450 >1.0) and then select the positive wells (generally OD 450 ≥0.5) for subcloning.

[0044] The wells with high positive test values (OD450>2.0) in the fusion plate were picked for limiting dilution, and 60% of the monoclonal wells on each plate were counted for subcloning. Each time, the monoclonal wells with higher positive values were picked for limiting dilution. ELISA testing could be performed 5-7 days after each subcloning until a monoclonal cell line that could stably secrete positive antibodies was finally screened for expanded culture.

[0045] The cell lines that stably secreted positive antibodies screened in the subcloning stage were expanded and cultured in 24-well plates. After expansion, the supernatant was collected for antigen detection. ELISA gradient dilution and western-blotting were used to verify its stability. The hybridoma cell line 3B4 (3B4) with a monoclonal antibody specific for porcine relaxin protein was screened and obtained. Figure 4 ), and had no positive reaction with relaxin from other species (horse, dog, rat, human).

[0046] First, pristane or liquid paraffin was injected into the mouse peritoneal cavity. One week later, the hybridoma cell line 3B4 was inoculated into the mouse peritoneal cavity. After the cells were established, 10% fetal bovine serum medium was used for expansion culture. When the cell density reached 1×10 6 -2×10 6 When the protein was 100 μg / mL, centrifugation at 800 rpm was performed. The precipitate was collected, resuspended in PBS, and injected intraperitoneally into mice (liquid paraffin). After 7-10 days, ascites was collected and pretreated before purification using a Protein A-agarose affinity chromatography column. The starting buffer was 20 mM phosphate buffer, pH 7.0; the elution buffer was 0.1 mM glycine-HCl, pH 2.7. A sufficient amount of starting buffer (8-10 mL) was used to equilibrate the Protein A-agarose affinity chromatography column (HiTrap Protein A 1 mL, Pharmacia Biotech). 15-25 mL of the sample to be purified (10.2-21.1 mg protein per mL) was loaded onto the column at a flow rate of 0.5 mL / min. The column was then washed sequentially with 7-8 mL of starting buffer, 6-7 mL of elution buffer, and 5 mL of starting buffer at the same flow rate. 1 mL of the eluate was collected per tube. The purified antibody was tested for titer using recombinant porcine relaxin protein as antigen using indirect ELISA. The titer of the purified 3B4 monoclonal antibody was 1:920,000 as determined by ELISA.

[0047] Total RNA from the hybridoma cell line 3B4 was extracted and reverse transcribed into cDNA using universal primers. The light and heavy chains of the antibody were amplified and separated, cloned into a standard cloning vector for expression, and identified by single colony PCR. 5-10 single colonies with the correct light and heavy chain lengths were selected for sequencing. If the results of 5 sequencing runs were nearly identical, the sequence was considered to be the authentic sequence of the antibody (the sequencing process and large-scale antibody expression were performed by BGI Genomics (Beijing) Co., Ltd.).

[0048] Among them, the heavy chain amino acid sequence of 3B4 is:

[0049] EVQLEESGPQLLKPGASVKISCKADYVIHTRYWIHWVKQRPPGGSTWIGMIDPSASESLNQKYMQLSSTLTEDTAM QLSSPTSEDSAYCVRRYFGYWGQGGTTLTVSS(SEQ ID NO.2)

[0050] The light chain variable region sequence is as follows:

[0051] DIVLTQSPASLAVGLGQRASISCKASQSVDYDGDSYLQKPGAQKPGQPPKLLYAASGVPDRFSGSGTDFTLNIHPV EEEDVATHYCYQGSHVPLTFGAGSKLELK (SEQ ID NO. 3).

[0052] Example 2: Preparation of Relaxin Monoclonal Antibody

[0053] New Zealand white rabbits were immunized with the recombinant porcine relaxin prepared in Example 1 as an immunogen. An equal amount of complete Freund's adjuvant was added for emulsification during the initial immunization, followed by an equal amount of incomplete Freund's adjuvant for the subsequent three immunizations. Four-month-old male New Zealand white rabbits were immunized subcutaneously at multiple sites on the neck and back with the emulsified immune complexes at a dose of 800 μL per rabbit. The second immunization was performed on day 14, the third on day 28, and the fourth on day 42. On day 45, blood was collected from the jugular vein and stored overnight at 4°C. The blood was centrifuged at 5000 rpm for 10 minutes, and serum was collected. Polyclonal antibodies were purified from a portion of the serum using the caprylic acid-ammonium sulfate salting-out method conventional in the art (see the Handbook of Molecular Cloning).

[0054] Indirect ELISA was used to detect the titer of polyclonal antibodies in another part of the collected serum. When the sample absorbance value / negative absorbance value was greater than 2.1, it was considered positive. The results are shown in Table 2. The results showed that the effective titer of the polyclonal antibody obtained by screening was as high as 1:256000, which also had good specificity.

[0055] Table 2. Titer of polyclonal antibodies detected by indirect ELISA

[0056]

[0057]

[0058] Example 3: Establishment of a sandwich ELISA method for porcine relaxin protein

[0059] Based on the applicant's previous research results, the basic reaction steps for establishing an ELISA for relaxin in pig seminal plasma are as follows:

[0060] 1) Purified polyclonal antibody coating: Dilute the above-mentioned polyclonal antibody against porcine relaxin to 1:500 with coating solution, incubate at 4°C overnight, add 100 μL per well, and store in a refrigerator at 4°C overnight;

[0061] 2) Blocking: Add 200 μL of 5% skim milk powder to each well and incubate at 37°C for 1.5 h;

[0062] 3) Add antigen: Add 100 μL of sample to each well and incubate at 37°C for 1-2 hours;

[0063] 4) Add purified monoclonal antibody: dilute monoclonal antibody 3B4 to 1:1000 with 5% skim milk powder, incubate at 37°C for 1-2 hours, 100 μL per well;

[0064] 5) Add enzyme-labeled secondary antibody: dilute HRP-goat anti-mouse IgG secondary antibody to 1:16000 with 5% skim milk powder, incubate at 37°C for 2 h, 100 μL per well;

[0065] 6) Substrate color development: Add 100 μL of color development solution to each well and allow to develop for 10 min;

[0066] 7) Stop the reaction: add 50 μL / well of 2 mol / L concentrated sulfuric acid and measure the OD450nm reaction value;

[0067] Wash the plate 3 to 5 times with washing solution between each step 1) to 5), each time for 8 minutes.

[0068] 8) Establishment of standard curve

[0069] The purified recombinant porcine relaxin was diluted to 300, 250, 125, 62.5, 31.25, 15.63, 7.82, 3.9, 1.95, and 0.975 pg / mL, and three replicates were performed for each concentration. The standard curve results are shown in the table. Figure 5 .

[0070] As can be seen from the figure, when the concentration of recombinant porcine relaxin is between 1.95 and 300 pg / mL, the natural logarithm of protein concentration has a good linear relationship with OD value, and the linear regression equation is y = 0.2304x - 0.1507, R 2 =0.991, where x is the natural logarithm of the concentration of recombinant porcine relaxin protein. The detection range of the kit is 1.95-300 pg / mL, and the detection limit is 1.95 pg / mL.

[0071] Example 4: Detection of pig frozen semen quality and conception rate

[0072] Based on the applicant's previous research results, a semen protection diluent was prepared and maintained at 37°C for later use; the ingredients of the semen protection diluent were: 0.5% sodium lauryl sulfate, 0.5% laurylamide, 0.2% sorbitan fatty acid ester, 0.3% fatty acid monoethanolamide, 0.5% lauric acid, 0.5% triethanolamine, 1% fatty acid monoethanolamide sulfosuccinate salt, 0.2% EDTA, 0.2% epigallocatechin gallate, 10% citric acid buffer, and the balance was double-distilled water.

[0073] Routine semen collection is performed on the boars for semen collection. After being numbered, the fresh semen collected is subjected to routine semen examination. Normal semen is milky white in color, free of impurities, odor, and a slight bloody smell. The live rate is above 90%, the vitality is above 80%, and the sperm density is weighed and measured.

[0074] Isothermally dilute the semen with the semen protectant at a 1:1 ratio. Allow the diluted semen to stand at room temperature for 1 hour. Then, aliquot the semen into 50mL centrifuge tubes. Place the tubes in a beaker filled with isothermal water and transfer them to a 17°C incubator to slowly cool. After approximately 2-3 hours, cool the semen to 17°C. After equilibration, cool the centrifuge to 17°C while idling. Centrifuge at 800g for 12 minutes at 17°C. Collect the supernatant and sperm pellet.

[0075] Among them, the seminal plasma supernatant was quantitatively analyzed using the sandwich ELISA method to screen out the two samples with the highest and lowest relaxin levels (S5 and S24), and placed in a 17°C environment for later use.

[0076] The sperm sediment in the lower layer was resuspended with porcine sperm cryopreservation solution (6 times the volume), wherein the porcine sperm cryopreservation solution was prepared by adding 20% of the total volume of egg yolk to the above-mentioned protective diluent and filtering it with a 0.22 μm sterilizing filter.

[0077] At the same time, in order to verify the effect of adding seminal plasma, 2.5% (v / v) of the total volume of S5 and S24 seminal plasma supernatants were added to the pig sperm cryopreservation solution, and the same volume of semen protection diluent was set up as a negative control.

[0078] The resuspended sperm solution is placed at 4°C for 2-3 hours, gradually cooled to 4°C at a rate of 5-8°C / h, and then filled and sealed with 0.5ml straws; the sealed straws are stacked on a rack and placed in a program freezer for freezing according to the set curve; after freezing, the straws are stored in liquid nitrogen.

[0079] Thawing procedure: Gently shake the cryovials stored in liquid nitrogen for 1 to 3 minutes in a water bath at 37°C until completely thawed, allowing them to revive and perform sperm motility testing.

[0080] The methods for detecting sperm motility, acrosome integrity, and seminal plasma antioxidant enzyme activity after freezing are as follows:

[0081] (1) Immediately after thawing, sperm motility is tested at a temperature of 35°C to 37°C. To assess motility, use a glass rod to dip a drop of semen onto a glass slide. Place a cover slip over the slide, ensuring the space between the cover slip and the slide is filled with semen. The slide is then placed under a microscope at 400x to 600x magnification. Within the microscope's field of view, sperm motility is assessed by comprehensively observing the sperm motility in the upper, middle, and lower layers of the semen.

[0082] (2) Place a drop of thawed semen on a clean glass slide. Use another glass slide at a 45-degree angle to spread the semen to the other side to make a smear. After air-drying, fix the slide with 1-2 ml of formalin phosphate buffer for 15 minutes, rinse and dry, and stain with Giemsa for 90 minutes. Remove the stain from the smear, air-dry, and observe 300 sperm under a biological microscope at 1000x magnification. Record the total number of sperm and the number of sperm with intact acrosomes.

[0083] (3) The hypoosmotic swelling test (HOST) was used to calculate the sperm tail bending rate to determine the sperm plasma membrane integrity. Sperm plasma membrane integrity = number of sperm with bent tails / number of sperm counted × 100%.

[0084] (4) The activity of seminal plasma antioxidant enzymes was determined using relevant enzyme determination kits (all enzyme determination kits were from Nanjing Jiancheng Biotechnology Co., Ltd.) in strict accordance with the operating procedures.

[0085] The test results are shown in Table 3.

[0086] Table 3 Results of frozen semen sperm quality test

[0087]

[0088] The above results demonstrate that adding seminal plasma to a cryoprotectant effectively protects sperm and reduces physical and chemical damage to sperm during freezing and thawing. Furthermore, seminal plasma with high relaxin expression, as screened using the quantitative method described above, significantly improved the protective capacity of the diluent for frozen semen in indicators such as sperm motility, acrosome integrity, and catalase, glutathione peroxidase, and glutathione reductase activities, compared to seminal plasma with low relaxin expression. These results are consistent with existing findings and provide guidance for the development of more effective cryoprotectants.

[0089] The above description of the embodiments is intended to facilitate understanding and use of the present invention by those skilled in the art. Those skilled in the art will readily be able to make various modifications to these embodiments and apply the general principles described herein to other embodiments without resorting to creative effort. Therefore, the present invention is not limited to the above-described embodiments. Any improvements or modifications made by those skilled in the art based on the principles of the present invention that do not depart from the scope of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody 3B4 that specifically recognizes porcine relaxin protein, wherein the heavy chain amino acid sequence of the monoclonal antibody 3B4 is shown in SEQ ID NO: 2, the light chain amino acid sequence is shown in SEQ ID NO: 3, and the amino acid sequence of the porcine relaxin protein is shown in SEQ ID NO.

1.

2. An ELISA kit for quantitatively detecting porcine relaxin protein, comprising the monoclonal antibody 3B4 according to claim 1, a recombinant relaxin polyclonal antibody, an ELISA plate, a blocking solution, an HRP-goat anti-mouse IgG secondary antibody, a color development solution, a stop solution, a coating solution, and a washing solution; wherein, Recombinant relaxin polyclonal antibody was used as the capture antibody, and the monoclonal antibody 3B4 was used as the detection antibody.

3. A kit for improving the quality and conception rate of frozen pig semen, comprising the ELISA test kit according to claim 2 and a semen protection diluent; wherein: The semen protection diluent comprises the following ingredients: 0.5% sodium lauryl sulfate, 0.5% laurylamide, 0.2% sorbitan fatty acid ester, 0.3% fatty acid monoethanolamide, 0.5% lauric acid, 0.5% triethanolamine, 1% fatty acid monoethanolamide sulfosuccinate salt, 0.2% EDTA, 0.2% epigallocatechin gallate, 10% citric acid buffer, and the balance is double-distilled water.

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