SNP site for identifying freezing tolerance of holstein bull sperm

Abstract

The application discloses a SNP site for identifying the sperm freezing tolerance of Chinese Holstein bulls. The SNP site is the nucleotide at the 161th position from the 5' end of SEQ ID NO:1 in the genome of the bull. By detecting the genotype of the SNP site in the genome of the bull to be tested, the sperm freezing tolerance of the Chinese Holstein bull can be screened or assisted to be screened. The method is not only simple, rapid, sensitive, but also reliable, stable and accurate. The application is suitable for large population scale detection and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to SNP sites used to identify the freeze resistance of sperm from Chinese Holstein bulls. Background Technology

[0002] The dairy industry is a vital component of animal husbandry, not only closely related to people's ever-increasing material needs but also holding significant strategic importance for increasing farmers' and herders' income. Holstein cattle are the world's most important dairy cattle breed. Currently, artificial insemination using frozen semen is the primary method of dairy cattle breeding. With the development of frozen semen technology, improving the quality of bull frozen semen has received considerable attention, especially semen motility after thawing, which has a significant economic impact on cow conception rates and the dairy production system. Breeding bulls can contribute over 70% to the genetic improvement of dairy cattle populations. Therefore, breeding superior breeding bulls with excellent semen quality is the core work of dairy cattle breeding. However, a significant decrease in semen motility after freezing and poor semen freeze resistance are among the main problems currently facing bull frozen semen production in my country. Currently, the motility of Holstein bull frozen semen in my country lags significantly behind that of some developed dairy countries, urgently requiring further improvement.

[0003] Researchers have found that even under the same freezing procedures, the changes in semen response to cold stress vary among different individuals. Some bulls, while exhibiting good fresh semen motility, showed a significant decrease in motility after freezing. The marked differences in cold stress tolerance among different bulls' sperm are clearly partly due to differences in their genetic makeup. Therefore, in recent years, many studies have utilized multi-omics and other technologies to identify key genes and molecular markers influencing sperm cryoprotection at the genetic level, applying them to breeding programs to accelerate the selection and improvement of cryoprotection traits in bull semen.

[0004] Single nucleotide polymorphisms (SNPs) refer to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level. They are widely distributed, numerous, and exhibit high genetic stability, making genotyping easy. By identifying key functional genes and their SNP loci related to the cryoprotection of Holstein bull sperm and applying molecular marker-assisted selection to bull semen quality traits, the accuracy of selection can be further improved. Furthermore, early selection can lead to individual culling, reducing feeding costs. Therefore, identifying molecular markers related to cryoprotection in bull sperm has significant economic value for breeding Holstein bull semen quality traits and provides an important theoretical basis for elucidating the genetic basis of cryoprotection traits in bull semen and effectively conducting molecular breeding for frozen semen traits in bulls.

[0005] ATP5F1A, as one of the core subunits of the mitochondrial ATP synthase complex, plays a crucial role in various biological processes, including cellular energy metabolism, mitophagy, and cell survival. Studies have shown that ATP5F1A degradation is inhibited when mitochondrial membrane potential is decreased or mitochondria are damaged. Further research revealed that PHB2 (prohibitin 2) affects mitophagy by regulating ATP5F1A degradation, revealing the key role of ATP5F1A in mitophagy. Furthermore, TNK2 / ACK1 kinase can directly phosphorylate ATP5F1A and increase mitochondrial energy output, indicating that ATP5F1A plays an important role in cellular energy metabolism and survival. Summary of the Invention

[0006] The purpose of this invention is to identify the cryoprotective properties of Holstein bull sperm (especially Chinese Holstein bulls). In this application, the cryoprotective properties of Chinese Holstein bull sperm refer to the following: frozen semen produced according to the People's Republic of China Agricultural Industry Standard for the Production of Frozen Bovine Semen NY / T 1234-2018, with sperm motility tested after thawing and the following judgment made: if the sperm motility after low-temperature treatment is ≥0.40, then the sperm of the tested Chinese Holstein bull is cryoprotective; if the sperm motility after low-temperature treatment is ≤0.39, then the sperm of the tested Chinese Holstein bull is not cryoprotective.

[0007] This invention first protects a kit for identifying the cryoprotectiveness of bull sperm, which may include a substance for detecting the genotype of the bull to be tested based on a SNP site; the SNP site is the nucleotide at position 161 from the 5' end of SEQ ID NO:1 in the bull genome.

[0008] The "reagent kit for identifying the cryoprotectiveness of bull sperm" can specifically be composed of substances that detect the genotype of the bull to be tested based on SNP loci.

[0009] The "substance for detecting the genotype of the bull to be tested based on SNP sites" mentioned above can specifically be a primer pair consisting of upstream primer F and downstream primer R;

[0010] The upstream primer F can be a single-stranded DNA molecule as shown in SEQ ID NO:2;

[0011] The downstream primer R can be a single-stranded DNA molecule as shown in SEQ ID NO:3.

[0012] The substance used to detect the SNP-based genotype of the bull under test, as described above, can also be used to determine the nucleotide types of SNP sites in the bull genome through at least one of the following methods: DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, and SNP microarrays. The SNP microarrays include microarrays based on nucleic acid hybridization reactions, microarrays based on single-base extension reactions, microarrays based on allele-specific primer extension reactions, microarrays based on one-step reactions, microarrays based on primer ligation reactions, microarrays based on restriction endonuclease reactions, microarrays based on protein-DNA binding reactions, and / or microarrays based on fluorescent molecule-DNA binding reactions.

[0013] This invention also protects the molecular marker shown in SEQ ID NO: 1.

[0014] The application of any of the above-described reagent kits or molecular markers in identifying the cryoprotective properties of bull sperm is also within the scope of protection of this invention.

[0015] The application of any of the above-described reagent kits or molecular markers in screening bulls with different sperm freeze resistance is also within the scope of protection of this invention.

[0016] The application of any of the above-described kits or molecular markers in identifying bull genotypes also falls within the scope of protection of this invention.

[0017] The application of any of the aforementioned reagent kits or molecular markers in bull breeding also falls within the scope of this invention. The goal of this bull breeding is to develop bull breeds with cryogenically resistant sperm.

[0018] This invention also protects a method for identifying the freeze resistance of bull sperm.

[0019] The method for identifying the cryogenic resistance of bull sperm protected by this invention can specifically be Method 1, which may include the following steps: detecting whether the genotype of the bull to be tested is homozygous CC or homozygous TT based on the SNP site, and then making the following judgment: the cryogenic resistance of the sperm of bulls with homozygous CC genotype based on the SNP site is greater than that of bulls with homozygous TT genotype based on the SNP site;

[0020] The SNP site is the nucleotide at position 161 from the 5' end of SEQ ID NO:1 in the bull genome.

[0021] The method for identifying the freeze resistance of bull sperm protected by this invention can specifically be Method Two, which may include the following steps:

[0022] (1) Using the genomic DNA of the bull to be tested as a template, PCR amplification was performed using a primer pair consisting of upstream primer F and downstream primer R to obtain PCR amplification products; the PCR amplification products were sequenced, and then the following judgments were made: if the 161st position of the PCR amplification product is only C, then the genotype of the bull to be tested based on the SNP site is CC homozygous; if the 161st position of the PCR amplification product is only T, then the genotype of the bull to be tested based on the SNP site is TT homozygous.

[0023] (2) Based on the results of (1), the following judgment is made: the sperm freezing resistance of bulls with the genotype CC based on the SNP site is greater than that of bulls with the genotype TT based on the SNP site.

[0024] The SNP site is the nucleotide at position 161 from the 5' end of SEQ ID NO:1 in the bull genome;

[0025] The upstream primer F can be a single-stranded DNA molecule as shown in SEQ ID NO:2;

[0026] The downstream primer R can be a single-stranded DNA molecule as shown in SEQ ID NO:3.

[0027] The bull mentioned above may be a Holstein bull. The Holstein bull may be a Chinese Holstein bull.

[0028] In the above text, the term > can specifically refer to > in statistical terms.

[0029] Experiments have shown that the method provided in this invention, which detects the genotype of bulls based on SNP loci, can screen or assist in screening for the sperm cryoprotective trait in Chinese Holstein bulls. This method is not only simple, rapid, and sensitive, but also provides reliable, stable, and accurate results. This invention is suitable for large-scale population testing and has significant application value. Attached Figure Description

[0030] Figure 1 This describes the differential expression of ATP5F1A protein in step five of Example 1 in the high-sperm cryoprotection group and the low-sperm cryoprotection group of Chinese Holstein bulls.

[0031] Figure 2 Example 5 shows the Western Blot analysis of the expression level of ATP5F1A protein in bull sperm from different genotypes. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0034] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0035] Example 1: Differential Expression of ATP5F1A in Sperm Protein Groups of High and Low Sperm Freeze-Thaw Groups in Chinese Holstein Bulls I. Experimental Animals and Grouping

[0036] The fresh semen samples used in this embodiment were all from the Shandong Aux Bull Breeding Station. The bulls were of the Chinese Holstein breed, and all bulls were raised and managed in the same environment and were in good health. Fresh semen was collected from Chinese Holstein bulls using the artificial vagina method, and then transferred to the laboratory in a 19°C incubator. The semen was diluted with PBS buffer at a ratio of 1:10 to obtain a semen dilution. 10 μL of the semen dilution was dropped onto glass slides, and sperm motility was detected using a computer-assisted sperm analysis system (CASA) to obtain the phenotypic data of fresh semen motility from Chinese Holstein bulls. The pre-freezing sperm motility of each bull's semen was the average of the fresh semen motility recorded in the 10 tests prior to sampling (i.e., pre-freezing motility). The post-freezing sperm motility of each bull's semen was the average of the thawed sperm motility recorded in the 10 tests prior to sampling (i.e., post-freezing motility). For those with fewer than 10 records, the average of all valid sperm motility values ​​was calculated.

[0037] Based on multiple phenotypic records of fresh and frozen sperm motility, 130 Chinese Holstein bulls were screened, resulting in a high-sperm-tolerance freezing group (Group H) consisting of 8 Chinese Holstein bulls and a low-sperm-tolerance freezing group (Group L) consisting of 6 Chinese Holstein bulls. In Group H, the fresh sperm motility of the bull semen was ≥60%, and the thawed sperm motility was ≥40%. In Group L, the fresh sperm motility of the bull semen was ≥60%, and the thawed sperm motility was ≤27%. There was no significant difference in sperm motility between the two groups before freezing; however, the average sperm motility of Group H after freezing was 43.13±2.53%, significantly higher than that of Group L (22.67±3.88%). Therefore, there was a significant difference in frozen sperm motility between Group H and Group L (P-value <0.0001, see Table 1).

[0038] Table 1. Phenotypic information of individual experimental bulls

[0039]

[0040]

[0041] Note: Pre-freezing motility refers to the sperm motility before freezing, measured directly after collection; post-freezing motility refers to the sperm motility after thawing, which is the average of 10 recorded samples from an individual; individuals with "H" in their serial number belong to the high sperm freeze-resistance group, and individuals with "L" in their serial number belong to the low sperm freeze-resistance group.

[0042] II. Sperm separation

[0043] Fresh semen collected from Chinese Holstein bulls in step one was aliquoted into 1.5 mL centrifuge tubes and centrifuged at 19°C and 800 × g for 15 min to obtain sperm precipitate. The sperm precipitate was then washed twice with PBS buffer and placed into cryovials, flash-frozen in liquid nitrogen, and stored at -80°C for later use, thus obtaining sperm samples.

[0044] III. Extraction and sequencing of total sperm protein

[0045] 1. Extraction of total sperm protein

[0046] Add 400 μL of lysis buffer (solute and concentration of 7M urea, 2M thiourea, 0.1% PMSF protease inhibitor and 65mM DTT, solvent is water) to the bull sperm samples obtained in step 2, and sonicate under ice bath conditions (70W; 5s on, 10s off, 3-5 times), and place on ice for 40 min; then centrifuge at 4℃, 14000×g for 30 min, and collect the supernatant, which is the total sperm protein sample.

[0047] 2. Protein concentration determination

[0048] The concentration of total sperm protein in the sperm protein sample obtained in step 1 was determined using the Bradford assay (Beyotime, Shanghai, China).

[0049] The steps for determining total sperm protein concentration using the Bradford assay are as follows:

[0050] (1) Mix 10 μl of total sperm protein sample with 300 μl of staining solution to obtain sperm staining solution; mix BSA solutions of different concentrations (obtained by diluting BSA standard with water) with 300 μl of staining solution to obtain BSA staining solution;

[0051] (2) The sperm staining solution and BSA staining solution were kept at room temperature in the dark for 10 min, and then the absorbance at 562 nm was detected by an enzyme-linked immunosorbent assay (ELISA) reader.

[0052] (3) Plot a standard curve with the concentration of BSA solution as the abscissa and the corresponding absorbance as the ordinate; then, based on the absorbance of the sperm staining solution and the standard curve, obtain the concentration of total sperm protein in the sperm total protein sample.

[0053] 3. SDS-PAGE electrophoresis

[0054] 20 μg of total sperm protein was collected from each Chinese Holstein bull, and the proteins were separated using 12% SDS-PAGE. The separated gel was then stained with Coomassie Brilliant Blue. After staining, the gel was precisely scanned using an ImageScanner scanner to obtain detailed image information.

[0055] 4. In-gel enzyme digestion

[0056] (1) Using a scalpel, cut the film, after Coomassie brilliant blue staining and gel destaining, into pieces approximately 1-2 mm in size. 2 Small pieces of the same size were then placed into the EP tube.

[0057] (2) After completing step (1), add 500 μl of decolorizing solution (a mixture of acetonitrile and 50 mM NH4HCO3 aqueous solution at a volume ratio of 1:1) to the EP tube to fully immerse the film in the liquid; then shake thoroughly for 20 minutes and discard the waste liquid. Repeat this step 1-2 times until the film is decolorized to colorless. Finally, add 500 μl of acetonitrile and discard the waste liquid again.

[0058] (3) After completing step (2), add 50-100 μL of DTT (concentration of 10 mmol / L) reducing solution to the EP tube and keep it at 56°C for 30 minutes. Then, discard the waste liquid and add 500 μL of acetonitrile for dehydration treatment for 5-10 minutes.

[0059] (4) After completing step (3), add 50-100 μl of iodoacetamide (concentration of 55 mmol / L) to the film and let it stand in the dark for 30 minutes (for the purpose of alkylation).

[0060] (5) After completing step (4), first add 500 μl of decolorizing solution and wash for 5-10 minutes; then add 500 μL of acetonitrile and discard the waste liquid; next, dry the film under freezing conditions for 20 minutes.

[0061] (6) After completing step (5), add 50-100 μL of trypsin (concentration of 0.01 μg / μl) to the gel and incubate at 4°C for 30 minutes to allow the gel to completely absorb the enzyme solution. Then add 50-100 μl of enzyme digestion buffer (i.e., 25 mM NH4HCO3 aqueous solution) to ensure that the gel is completely immersed in the liquid and incubate at 37°C for at least 15 hours.

[0062] (7) After completing step (6), add 100 μl of extraction solution I (5% TFA aqueous solution) to the EP tube, treat in a 40°C water bath for 1 hour (during which, sonication is performed every 30 minutes for 3 minutes each time), and collect liquid phase I.

[0063] (8) After completing step (7), add 100 μl of extraction solution II (an aqueous solution containing 2.5% TFA and 50% acetonitrile) to the gel block of the EP tube, keep it at 30°C for 1 hour (during which time, sonication is performed once every 30 minutes, each time for 3 minutes), and collect liquid phase 2.

[0064] (9) Combine liquid phase 1 and liquid phase 2 to obtain the extract, and then dry it to obtain the sample.

[0065] 5. Liquid chromatography and mass spectrometry

[0066] The sample obtained in step 4 (9) was dissolved in 0.1% formic acid (FA) and loaded into LC-MS / MS liquid mass tandem mass spectrometry.

[0067] LC conditions are as follows: flow rate: 600 nL / min; column: C18 pre-column (PepMap C18). 100μm×2cm, 5μm), analytical column (PepMap C18, The mobile phase consisted of buffer B and buffer A (75 μm × 50 cm, 2 μm); buffer A was ACN-H2O-FA (1.9:98:0.1, v / v / v); buffer B was ACN-H2O-FA (98:1.9:0.1, v / v / v). Gradient elution conditions: 6-12% buffer B, 0-10 min; 12-30% buffer B, 10-95 min; 30-40% buffer B, 95-113 min; 40-95% buffer B, 133-134 min; 95% buffer B, 114-1130 min; 3% buffer B, 2 min, followed by equilibration for 3 min.

[0068] The mass spectrometry conditions were as follows: For mass spectrometry analysis, the mass resolution of the first-stage MS was first fine-tuned to 35000, and the automatic gain control value was calibrated to 1e6. To ensure data accuracy, the maximum injection time was limited to 50 milliseconds. A full scan mode was selected for mass spectrometry scanning, with the mass-to-nuclear ratio (m / z) range set between 400 and 1500. To further investigate key components in the sample, the top 10 most intense peaks in the scan results were subjected to MS / MS depth scanning. During MS / MS spectrum acquisition, a data-dependent positive ion mode was used, and high-energy collision fragmentation technology was applied, with the collision energy set to 30. Furthermore, to improve the precision of MS / MS analysis, the MS / MS resolution was set to 17500, and the automatic gain control value was adjusted to 2e5, with the maximum injection time also limited to 50 milliseconds. Simultaneously, to avoid duplicate scans, a dynamic exclusion time of 30 seconds was set to ensure the accuracy and reliability of the results.

[0069] IV. Search Databases and Software

[0070] Database used: The bovine Uniprot protein sequence library (https: / / www.uniprot.org / proteomes?query=Bos+taurus) was used as the database. The search software was Maxquant (1.5.2.8). The analysis parameters were as follows: 'Enzyme' was Trypsin / P; 'Max Missed Cleavages' was 2; 'Fixed modifications' was Carbamidomethyl (C); 'Variable modifications' were Oxidation (M) and Acetyl (Protein N-term); 'Min.peptide length' was 7; 'Min.razor+unique peptides' was 1. The result filtering parameters were: Peptide FDR ≤ 0.01, Protein FDR ≤ 0.01.

[0071] V. Identification of Differentially Expressed Proteins

[0072] Differentially expressed proteins were screened using a significance threshold of |log2FC| > 0.5 and P < 0.05. The results showed that 147 differentially expressed proteins were identified in Chinese Holstein bull sperm, with 93 upregulated proteins identified in group H and 54 upregulated proteins identified in group L. The identification results of ATP5F1A protein in the differentially expressed protein results are shown in Table 2. The expression of ATP5F1A protein in Chinese Holstein bull sperm is shown in... Figure 1 .

[0073] Table 2. Differential expression of ATP5F1A protein in the high sperm cryopreservation group and the low sperm cryopreservation group.

[0074] Record number Gene name Group H mean Group L mean <![CDATA[log2FC]]> P-value P19483 ATP5F1A 31.20 30.68 0.52 0.01

[0075] The results showed that in Chinese Holstein bulls, the content of ATP5F1A protein was higher in the high sperm cryoresistance group compared to the low sperm cryoresistance group. This indicates that ATP5F1A protein is closely related to sperm cryoresistance.

[0076] The proteomic experiments revealed significant differential expression of the ATP5F1A protein in the sperm of Chinese Holstein bulls from the high-sperm cryogenic tolerance group and the low-sperm cryogenic tolerance group, particularly in the high-sperm cryogenic tolerance group. Therefore, the gene encoding the ATP5F1A protein (i.e., the ATP5F1A gene) is inferred to be an important candidate gene for cryogenic tolerance in Chinese Holstein bull sperm.

[0077] Example 2: Discovery of SNP sites in the genome of Chinese Holstein bulls and acquisition of primer pairs for identifying these SNP sites.

[0078] I. Discovery of SNP loci in the genome of Chinese Holstein bulls

[0079] The inventors of this application conducted extensive sequence analysis, alignment, and preliminary experiments on the gene encoding the ATP5F1A protein (i.e., the ATP5F1A gene) in the genome of Chinese Holstein bulls. They discovered a single SNP site on this gene, which causes a missense mutation in the amino acid. Specifically, this SNP site is the 161st nucleotide from the 5' end of SEQ ID NO:1 in the Chinese Holstein bull genome, and the genotype is CC homozygous, TT homozygous, or CT heterozygous.

[0080] SEQ ID NO:1 is:

[0081] (Y represents C / T)

[0082] Since genomic DNA is a double-stranded DNA molecule composed of two single-stranded DNA molecules that are antisense complements, the DNA molecule encoding the protein is generally named the sense DNA molecule, and the DNA molecule that is antisense complement to the sense DNA molecule is named the antisense DNA molecule. In this patent, the genotype at all loci is the antisense DNA genotype.

[0083] II. Obtaining primer pairs for identifying SNP sites

[0084] Based on the aforementioned SNP sites and their preceding and following nucleotide sequences, primer pairs for identifying these SNP sites were designed and synthesized. Each primer pair consists of an upstream primer F and a downstream primer R, and is used to amplify the target sequence of the aforementioned SNP sites.

[0085] The nucleotide sequences of upstream primer F and downstream primer R are shown in Table 3.

[0086] Table 3

[0087] Primer name Nucleotide sequence (5'-3') and its position in the sequence listing upstream primer F TGATGGATGGTGCTCATG(SEQ ID NO:2) Downstream primer R TTTCCATCGGTCCTATTT(SEQ ID NO:3)

[0088] Example 3: Establishment of a SNP-based genotyping method for Chinese Holstein bulls

[0089] 1. Genomic DNA was extracted from the semen of Chinese Holstein bulls to be tested.

[0090] 2. Using the genomic DNA of the semen of Chinese Holstein bulls to be tested as a template, PCR amplification was performed using a primer pair consisting of upstream primer F and downstream primer R to obtain the PCR amplification product.

[0091] The reaction system consisted of 25 μl of genomic DNA from Chinese Holstein bull semen (concentration 100 ng / μL), 1 μL of upstream primer F aqueous solution (concentration 10 pmol / μL), 1 μL of downstream primer R aqueous solution (concentration 10 pmol / μL), and 22 μL of PCR mix (a product of Qingke Biotechnology Co., Ltd., product code cat.TES101).

[0092] The reaction conditions were: 98℃ for 2 min; 98℃ for 10 s, 56℃ for 10 s, 72℃ for 15 s, 35 cycles; 72℃ for 5 min.

[0093] 3. After completing step 2, sequence the PCR amplification products. Based on the sequencing results, determine the genotype of the Chinese Holstein bulls to be tested based on the SNP sites. The specific determination principles are as follows:

[0094] If the PCR amplification product of the Chinese Holstein bull to be tested has only C at position 161, then the genotype of the Chinese Holstein bull to be tested based on the SNP site is CC homozygous.

[0095] If the PCR amplification product of the Chinese Holstein bull to be tested has only T at position 161, then the genotype of the Chinese Holstein bull to be tested based on the SNP site is TT homozygous.

[0096] If the PCR amplification product of the Chinese Holstein bull to be tested contains C and T at position 161, then the genotype of the Chinese Holstein bull to be tested based on the SNP site is CT heterozygous.

[0097] Example 4: Association analysis of SNP-based genotype and sperm cryoprotection in Chinese Holstein bulls

[0098] The fresh semen samples from 130 Chinese Holstein bulls in this embodiment were all provided by the Shandong Aux Bull Breeding Station and came from different Chinese Holstein bulls. All the Chinese Holstein bulls that provided the fresh semen samples were raised to sexual maturity under normal nutritional levels and feeding conditions.

[0099] I. Testing the fresh semen and frozen semen motility of 130 Chinese Holstein bulls

[0100] 1. The production process of frozen semen from 130 Chinese Holstein bulls was carried out entirely in accordance with the People's Republic of China Agricultural Industry Standard for Technical Specifications for the Production of Frozen Bovine Semen (NY / T 1234-2018). The specific steps are as follows:

[0101] (1) Take a dilution bottle containing 30 mL of diluent (formula can be found in Appendix C of the Agricultural Industry Standard of the People's Republic of China, Technical Specification for Production of Frozen Bovine Semen (NY / T 1234-2018)) that has been preheated in a 34°C water bath, dilute and mix the semen, and then temporarily store it in a 34°C water bath for 10 minutes before adding more diluent to the final dilution volume. Then proceed as follows: a) or b)

[0102] a) After standing for another 10 minutes, the semen can be filled, sealed, and labeled on a laboratory workbench at room temperature (below 20℃). Place the filled tubes into opaque plastic boxes, ideally holding 300 tubes per box. Place the plastic boxes in a 3℃-5℃ low-temperature cabinet for 3-4 hours to equilibrate and assess motility.

[0103] b) After adding the diluent, fill a cup with an appropriate amount of 34°C water and place the dilution bottle in a 3°C-5°C low-temperature chamber to cool and equilibrate. After 2 hours, add ice to the cup to rapidly cool it to 3°C-5°C (the capillary tube should also be cooled to 3°C-5°C) and assess its viability. Fill, seal, and label the capillary tubes in the low-temperature chamber.

[0104] (2) After completing step (1), use a programmable cryostat to freeze the semen. Set the optimal freezing program and strictly follow the operating procedure of the programmable cryostat for freezing. After freezing, open the lid of the programmable cryostat and put the frozen semen into a container filled with liquid nitrogen to quickly immerse it in the liquid nitrogen and obtain a frozen semen sample.

[0105] 2. The experiment was repeated three times, and the average result was taken. Each semen sample (fresh or frozen) was tested according to the following steps each time:

[0106] (1) Take a fresh semen sample and dilute it with physiological saline in an appropriate ratio to obtain a fresh semen diluent; take a frozen semen sample out of liquid nitrogen, quickly immerse it in 37°C water and shake it. After the frozen semen in the capillary tube has dissolved, take it out, wipe off the water droplets, cut off one end of the capillary tube, and use a special pusher to squeeze the semen into a small test tube to obtain a frozen semen diluent.

[0107] (2) Drop 10 μL of semen diluent (fresh semen diluent or frozen semen diluent) onto a glass slide, then place it on the microscope stage connected to a computer-assisted sperm analysis (CASA) system to measure the motility of sperm, expressed as a percentage;

[0108] Sperm motility = (number of progressively motile sperm / total number of sperm) × 100%.

[0109] Fresh sperm motility and frozen sperm motility were tested for each individual. The average values ​​of at least six fresh and frozen sperm motility phenotypes were statistically analyzed and grouped according to the following criteria:

[0110] Semen samples with fresh sperm motility ≥60% and frozen sperm motility ≥40% after thawing were classified into the high semen freeze resistance group, i.e., group H.

[0111] Semen samples with fresh sperm motility ≥60% and frozen sperm motility ≤39% after thawing were classified into the low semen freezing tolerance group, i.e., group L.

[0112] The results showed that Group H consisted of semen samples from 55 Chinese Holstein bulls. The sperm motility of the fresh semen samples in Group H was (68.13±1.23)%, and the sperm motility of the frozen semen samples after thawing was (41.62±1.48)%. Group L consisted of semen samples from 75 Chinese Holstein bulls. The sperm motility of the fresh semen samples in Group L was (64.64±4.94)%, and the sperm motility of the frozen semen samples after thawing was (31.58±5.42)%.

[0113] II. Genotyping of 130 Chinese Holstein bulls based on SNP loci

[0114] Following the method in Example 3, the semen samples of Chinese Holstein bulls to be tested were replaced with fresh semen samples from 130 Chinese Holstein bulls, with all other steps remaining unchanged. A total of 101 samples were successfully tested, thus obtaining the genotypes of 101 Chinese Holstein bulls based on SNP loci.

[0115] Table 4 shows the statistical results and genotype frequencies of 101 Chinese Holstein bulls based on SNP loci. The allele frequency of C was 0.223, and the allele frequency of T was 0.777.

[0116] Table 4

[0117] Genotype of SNP locus Quantity (heads) Genotype frequency CC homozygous 10 0.10 CT heterozygote 25 0.25 TT homozygous 66 0.65

[0118] III. Association Analysis of SNP-Based Genotype and Sperm Freeze-Thaw Resistance in Chinese Holstein Bulls

[0119] Association analysis based on SNP loci was performed on the genotypes of 101 Chinese Holstein bulls obtained in step two using SPSS 25 software, specifically a chi-square test. The test results are shown in Table 5.

[0120] Table 5

[0121]

[0122] Note: P-value represents the significance level of the chi-square test. *This indicates a significant difference in sperm cryoprotection among different genotypes (P < 0.05).

[0123] The results are as follows:

[0124] SNP sites were significantly associated with the cryoprotective properties of sperm from Chinese Holstein bulls;

[0125] The sperm cryoprotection of Chinese Holstein bulls with the genotype CC homozygous based on SNP sites is greater than that of Chinese Holstein bulls with the genotype TT homozygous based on SNP sites. For the sperm cryoprotection trait in Chinese Holstein bulls, based on SNP sites, the dominant allele is C, and the superior genotype is CC homozygous.

[0126] The above ">" all refer to statistically significant values ​​(i.e., significantly higher than at the 0.05 level).

[0127] The above results indicate that detecting the genotype of Chinese Holstein cattle based on SNP loci can rapidly and efficiently identify the sperm freeze resistance trait of Chinese Holstein bulls, which has important application value in breeding superior Chinese Holstein bulls, improving the selection of semen quality traits in Chinese Holstein bulls, and accelerating the breeding process of Chinese Holstein bulls.

[0128] Example 5: Verifying the function of SNP sites by detecting the expression level of ATP5F1A protein using Western blotting.

[0129] The expression levels of ATP5F1A protein in Chinese Holstein bulls with homozygous CC genotype and homozygous TT genotype based on SNP loci were detected by Western blotting. The specific steps are as follows:

[0130] 1. Frozen semen samples were collected from three Chinese Holstein bulls with a genotype of CC based on SNP loci and three Chinese Holstein bulls with a genotype of TT based on SNP loci, and total protein was extracted from the frozen semen samples using a protein extraction kit (Abmart).

[0131] 2. The total protein in the frozen sperm samples was detected by SDS-PAGE and Western blot, respectively. The primary antibody was either ATP5F1A antibody (Abcam, USA) or α-Tubulin antibody (proteintech product, catalog number 14555-1-AP) (as a control).

[0132] Test results are shown Figure 2(CC represents Chinese Holstein bulls with a genotype of CC homozygous based on SNP sites, and TT represents Chinese Holstein bulls with a genotype of TT homozygous based on SNP sites). The results showed that the ATP5F1A protein expression level of Chinese Holstein bulls with a genotype of CC homozygous based on SNP sites was significantly higher than that of Chinese Holstein bulls with a genotype of TT homozygous based on SNP sites.

[0133] This indicates that when the genotype at the SNP site changes from homozygous CC to homozygous TT, it leads to downregulation of ATP5F1A protein expression in sperm. The results suggest that the SNP site may be an important genetic mutation contributing to the reduced cryogenic tolerance of sperm from Chinese Holstein bulls.

[0134] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of a reagent kit in identifying the freeze resistance of bull sperm; The kit includes substances for detecting the SNP-based genotype of the bull to be tested; The SNP site is nucleotide Y at position 161 from the 5' end of SEQ ID NO:1 in the bull genome, and nucleotide Y is C or T; The sperm cryoprotection of bulls with a genotype of CC based on SNP loci is greater than that of bulls with a genotype of TT based on SNP loci. The bull in question is a Chinese Holstein bull.

2. The application according to claim 1, characterized in that: The substance used to detect the genotype of the bull under test based on the SNP site is a primer pair consisting of upstream primer F and downstream primer R; The upstream primer F is the single-stranded DNA molecule shown in SEQ ID NO:2; The downstream primer R is a single-stranded DNA molecule as shown in SEQ ID NO:

3.

3. Application of the molecular marker shown in SEQ ID NO: 1 in identifying the cryoprotective properties of bull sperm; the bull is a Chinese Holstein bull; The nucleotide Y at position 161 from the 5' end of SEQ ID NO:1 is C or T; The sperm cryoprotectiveness of bulls with a genotype of CC based on SNP loci is greater than that of bulls with a genotype of TT based on SNP loci.

4. Application of a reagent kit in screening bulls with different sperm cryoprotective properties; The kit includes substances for detecting the SNP-based genotype of the bull to be tested; The SNP site is nucleotide Y at position 161 from the 5' end of SEQ ID NO:1 in the bull genome, and nucleotide Y is C or T; The sperm cryoprotection of bulls with a genotype of CC based on SNP loci is greater than that of bulls with a genotype of TT based on SNP loci. The bull in question is a Chinese Holstein bull.

5. The application according to claim 4, characterized in that: The substance used to detect the genotype of the bull under test based on the SNP site is a primer pair consisting of upstream primer F and downstream primer R; The upstream primer F is the single-stranded DNA molecule shown in SEQ ID NO:2; The downstream primer R is a single-stranded DNA molecule as shown in SEQ ID NO:

3.

6. Application of the molecular marker shown in SEQ ID NO: 1 in screening bulls with different sperm cryoprotectiveness; The nucleotide Y at position 161 from the 5' end of SEQ ID NO:1 is C or T; The sperm cryoprotection of bulls with a genotype of CC based on SNP loci is greater than that of bulls with a genotype of TT based on SNP loci. The bull in question is a Chinese Holstein bull.

7. The application of a reagent kit in bull breeding; the goal of the bull breeding is to cultivate a bull breed with sperm that is resistant to freezing; The kit includes substances for detecting the SNP-based genotype of the bull to be tested; The SNP site is nucleotide Y at position 161 from the 5' end of SEQ ID NO:1 in the bull genome, and nucleotide Y is C or T; The sperm cryoprotection of bulls with a genotype of CC based on SNP loci is greater than that of bulls with a genotype of TT based on SNP loci. The bull in question is a Chinese Holstein bull.

8. The application according to claim 7, characterized in that: The substance used to detect the genotype of the bull under test based on the SNP site is a primer pair consisting of upstream primer F and downstream primer R; The upstream primer F is the single-stranded DNA molecule shown in SEQ ID NO:2; The downstream primer R is a single-stranded DNA molecule as shown in SEQ ID NO:

3.

9. Application of the molecular marker shown in SEQ ID NO: 1 in bull breeding; the goal of the bull breeding is to develop a bull breed with sperm that is resistant to freezing. The nucleotide Y at position 161 from the 5' end of SEQ ID NO:1 is C or T; The sperm cryoprotection of bulls with a genotype of CC based on SNP loci is greater than that of bulls with a genotype of TT based on SNP loci. The bull in question is a Chinese Holstein bull.

10. A method for identifying the cryogenic resistance of bull sperm, comprising the following steps: detecting whether the genotype of the bull to be tested is homozygous CC or homozygous TT based on the SNP locus, and then making the following judgment: the cryogenic resistance of the sperm of bulls with homozygous CC genotype based on the SNP locus is greater than that of bulls with homozygous TT genotype based on the SNP locus. The SNP site is nucleotide Y at position 161 from the 5' end of SEQ ID NO:1 in the bull genome, and nucleotide Y is C or T; The bull in question is a Chinese Holstein bull.

11. A method for identifying the freeze resistance of bull sperm, comprising the following steps: (1) Using the genomic DNA of the bull to be tested as a template, PCR amplification was performed using a primer pair consisting of upstream primer F and downstream primer R to obtain PCR amplification products; the PCR amplification products were sequenced, and then the following judgments were made: if the 161st position of the PCR amplification product is only C, then the genotype of the bull to be tested based on the SNP site is CC homozygous; if the 161st position of the PCR amplification product is only T, then the genotype of the bull to be tested based on the SNP site is TT homozygous. (2) Based on the results of (1), the following judgment is made: the sperm freezing resistance of bulls with the genotype CC based on the SNP site is greater than that of bulls with the genotype TT based on the SNP site. The SNP site is nucleotide Y at position 161 from the 5' end of SEQ ID NO:1 in the bull genome, and nucleotide Y is C or T; The upstream primer F is the single-stranded DNA molecule shown in SEQ ID NO:2; The downstream primer R is the single-stranded DNA molecule shown in SEQ ID NO:3; The bull in question is a Chinese Holstein bull.

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