Method and device for predicting bull semen quality

Abstract

A method and device for predicting the bull semen quality. The method comprises: on the basis of the variation of methylation level values, using known methylation spectrum data of methylation sites of a bull sperm to select 83 methylation sites; on the basis of an elastic net regression algorithm, using the selected methylation sites to construct an epigenetic clock; and using the epigenetic clock to predict the age of a bull to be detected. The age of a bull can be predicted on the basis of an epigenetic clock, and the bull semen quality can be predicted on the basis of an epigenetic acceleration. The present invention can be applied to bull variety breeding and bull breeding.

Description

Method and device for predicting bull semen quality Technical Field

[0001] The invention belongs to the technical field of bioinformatics, and in particular relates to a method and a device for predicting bull semen quality. Background Art

[0002] An epigenetic clock is an age estimator constructed using the methylation levels of a set of age-related CpG sites combined with elastic net regression. This clock uses the methylation profile of genes to determine an organism's physiological age or epigenetic age. It can calculate an animal's age, indicate its health, and indicate aging. In recent years, with the advancement of research, epigenetic clocks have been developed for a variety of animals and tissues. However, due to the differences in methylation levels between sperm and other tissues, sperm epigenetic clocks are currently under research in the animal field.

[0003] Reduced representation bisulfite sequencing (RRBS) is a highly efficient, high-throughput sequencing technology used to analyze methylation levels at the single-nucleotide level. This technology combines restriction endonucleases with bisulfite sequencing to reveal enrichment information for high-CpG regions. Compared to whole-genome methylation sequencing, RRBS requires sequencing only approximately 1% of the genome, significantly reducing costs.

[0004] Semen quality is the most important reproductive trait for Holstein bulls. Reproductive performance determines whether a bull can generate significant economic returns for the stud farm and ranch, and plays a key role in improving pregnancy rates in Holstein cows. Artificial insemination is widely used in Holstein breeding, and semen from high-quality bulls significantly accelerates genetic progress. As a bull ages, its DNA methylation levels also change, and semen quality traits also change. Semen quality is the most important reproductive trait for Holstein bulls. SUMMARY OF THE INVENTION

[0005] This study has constructed the first epigenetic clock in Holstein bull sperm. This study aims to construct a bull sperm epigenetic clock, explore the relationship between age and semen quality traits, and investigate the pathways regulated by clock-related genes, providing a theoretical basis for analyzing epigenetic differences in Holstein bull sperm of different ages. The results are primarily divided into two parts: First, the construction of a Holstein bull sperm epigenetic clock and enrichment analysis of clock genes. Forty-nine Holstein bulls aged 12 months and older, unrelated within three generations, were selected. Frozen semen was obtained from each bull, and genomic DNA was extracted and subjected to reduced representation bisulfite sequencing (RRBS). Data analysis was then performed to identify shared CpG sites, which were then used to construct a bull sperm epigenetic clock using elastic network regression. Finally, the accuracy of the clock was verified using 22 sets of data from public databases and previous data from the research team. Clock CpG sites were used to identify clock genes, and GO and KEGG enrichment analyses were performed. Results showed that clock genes were enriched in terms and pathways related to reproduction and longevity. In addition, the results of the correlation analysis between chronological age, epigenetic acceleration, and semen quality traits showed that semen quality traits change with age, and the deformity rate, fresh sperm density, and post-frozen motility change accordingly. This provides a theoretical basis for clarifying the relationship between semen quality traits and aging, and is also new evidence for using epigenetic acceleration as a biomarker for reproduction and longevity. In summary, this study conducted a methylation analysis on the sperm genomic DNA of Holstein cattle and constructed the first epigenetic clock for Holstein cattle sperm. The results found that clock genes are enriched in pathways such as reproduction, disease, cancer, and longevity. It was also found that chronological age and epigenetic acceleration can affect semen quality traits. Technical issues

[0006] The technical problem to be solved by the present invention is how to predict the physiological age of an animal and / or how to use the epigenetic clock of sperm to predict the physiological age of a bull and / or how to use the epigenetic clock of sperm to predict the epigenetic age of a bull and / or how to predict the quality of bull semen and / or how to predict the quality of bull semen based on epigenetic age. Technical Solutions

[0007] In order to solve the above technical problems, the present invention first provides a method for predicting bull semen quality, which may include the following steps:

[0008] C1) Methylation site screening: obtaining known methylation profile data of bull sperm methylation sites, and screening the methylation sites in the known methylation profile data based on the variability of methylation level values ​​in the known methylation profile data to obtain screened methylation sites;

[0009] C2) Epigenetic clock construction: Using the methylation profile data of the methylation sites screened in the known methylation profile data, an epigenetic clock is constructed based on an elastic net regression algorithm; the formula of the epigenetic clock is as follows:

[0010]

[0011] In Formula I, represents physiological age or epigenetic age; a represents the coefficient corresponding to each CpG site; a1-ax represents the coefficient value corresponding to the first CpG site to the coefficient value corresponding to the xth CpG site; x represents the number of CpG sites, m represents the methylation level value of each CpG site; m1-mx represents the methylation level value from the first CpG site to the methylation level value of the xth CpG site; b represents the intercept value;

[0012] C3) Data acquisition: obtaining methylation profile data of the methylation sites in the bull sperm to be tested;

[0013] C4) Epigenetic age prediction: Based on the methylation profile data of the methylation sites of the sperm of the bull to be tested, the epigenetic age of the bull to be tested is predicted using the epigenetic clock;

[0014] C5) Semen quality prediction: calculating the difference between the epigenetic age of the bull to be tested and the actual physiological age of the bull to be tested to obtain the epigenetic acceleration of the bull to be tested; and predicting the semen quality of the bull to be tested based on the epigenetic acceleration.

[0015] In the above method, the semen quality may be sperm deformity rate, semen freshness and / or sperm motility after freezing.

[0016] In the above method, the methylation sites may include 83 methylation sites, the value of x may be 83, and the 83 methylation sites may be the 83 CpG sites in Table 3.

[0017] In order to solve the above technical problems, the present invention also provides a method for predicting the physiological age of an animal (specifically a bull), which may include the following steps:

[0018] B1) Methylation site screening: obtaining known methylation profile data of bull sperm methylation sites, and screening the methylation sites in the known methylation profile data based on the variability of methylation level values ​​in the known methylation profile data to obtain screened methylation sites;

[0019] B2) Epigenetic clock construction: Using the methylation profile data of the methylation sites screened in the known methylation profile data, an epigenetic clock is constructed based on an elastic net regression algorithm; the epigenetic clock formula can be as follows:

[0020]

[0021] In Formula I, represents physiological age or epigenetic age; a represents the coefficient corresponding to each CpG site; a1-ax represents the coefficient value corresponding to the first CpG site to the coefficient value corresponding to the x-th CpG site; x represents the number of CpG sites, m represents the methylation level value of each CpG site; m1-mx represents the methylation level value from the first CpG site to the methylation level value of the x-th CpG site; b represents the intercept value.

[0022] B3) Data acquisition: Acquire methylation profile data of the methylation sites in the bull sperm to be tested;

[0023] B4) Age prediction: Based on the methylation profile data of the methylation sites of the sperm of the bull to be tested, the age of the bull to be tested is predicted using the epigenetic clock.

[0024] In the above method, the methylation sites may include 83 methylation sites, the value of x may be 83, and the 83 methylation sites may be the 83 CpG sites in Table 3.

[0025] In the above method, the methylation profile data may be the methylation level value of the methylation site, and the methylation profile data may be derived from methylation sequencing. The age may be physiological age or epigenetic age. The bull may be a Holstein bull.

[0026] In order to solve the above technical problems, the present invention also provides a device for predicting the age of a bull, which may include the following modules:

[0027] A1) Methylation site screening module: used to obtain or receive known methylation profile data of bull sperm methylation sites, and screen the methylation sites in the known methylation profile data to obtain screened methylation sites based on the variability of methylation levels in the known methylation profile data;

[0028] A2) Epigenetic clock construction module: used to construct an epigenetic clock based on the elastic net regression algorithm using the methylation profile data of the methylation sites screened in the known methylation profile data; the formula of the epigenetic clock can be as follows:

[0029]

[0030] In Formula I, represents physiological age or epigenetic age; a represents the coefficient corresponding to each CpG site; a1-ax represents the coefficient value corresponding to the first CpG site to the coefficient value corresponding to the x-th CpG site; x represents the number of CpG sites, m represents the methylation level value of each CpG site; m1-mx represents the methylation level value from the first CpG site to the methylation level value of the x-th CpG site; b represents the intercept value.

[0031] A3) Data acquisition or receiving module: used to obtain or receive methylation profile data of methylation sites of bull sperm to be tested;

[0032] A4) Age prediction module: used to predict the age of the bull to be tested using the epigenetic clock based on the methylation profile data of the methylation sites of the sperm of the bull to be tested.

[0033] In the above device, the methylation sites may include 83 methylation sites, the value of x may be 83, and the 83 methylation sites may be the 83 CpG sites in Table 3. The mixing parameter α of the epigenetic clock may be 0.3, the penalty coefficient λ may be 0.7915253, and the intercept b may be -16.5241.

[0034] In the above device, the methylation profile data may be the methylation level value of the methylation site, and the methylation profile data may be derived from methylation sequencing. The age may be physiological age or epigenetic age. The bull may be a Holstein bull.

[0035] The value of x is less than or equal to the number of methylation sites in the known methylation profile data. The variability can be calculated based on the variance of the methylation level values ​​of the methylation sites in the known methylation profile data.

[0036] In order to solve the above technical problems, the present invention also provides a device for predicting bull semen quality, which may include the following modules:

[0037] D1) Methylation site screening module: used to obtain or receive known methylation profile data of bull sperm methylation sites, and screen the methylation sites in the known methylation profile data to obtain screened methylation sites based on the variability of methylation level values ​​in the known methylation profile data;

[0038] D2) Epigenetic clock construction module: used to construct an epigenetic clock based on the elastic network regression algorithm using the methylation profile data of the methylation sites screened in the known methylation profile data; the formula of the epigenetic clock is as follows:

[0039]

[0040] In Formula I, represents physiological age or epigenetic age; a represents the coefficient corresponding to each CpG site; a1-ax represents the coefficient value corresponding to the first CpG site to the coefficient value corresponding to the xth CpG site; x represents the number of CpG sites, m represents the methylation level value of each CpG site; m1-mx represents the methylation level value from the first CpG site to the methylation level value of the xth CpG site; b represents the intercept value;

[0041] D3) Data acquisition or receiving module: used to obtain or receive methylation profile data of methylation sites of the bull sperm to be tested;

[0042] D4) an epigenetic age prediction module: configured to predict the epigenetic age of the bull to be tested using the epigenetic clock based on the methylation profile data of the methylation sites of the sperm of the bull to be tested;

[0043] D5) Semen quality prediction module: used to calculate the difference between the epigenetic age of the bull to be tested and the actual physiological age of the bull to be tested to obtain the epigenetic acceleration of the bull to be tested; and predict the semen quality of the bull to be tested based on the epigenetic acceleration.

[0044] The semen quality may be sperm deformity rate, semen freshness and / or sperm motility after freezing.

[0045] The methylation sites may include 83 methylation sites, the value of x may be 83, and the 83 methylation sites may be the 83 CpG sites in Table 3.

[0046] In the above device, the methylation profile data may be the methylation level value of the methylation site, and the methylation profile data may be derived from methylation sequencing. The age may be physiological age or epigenetic age. The bull may be a Holstein bull.

[0047] The mixing parameter α of the epigenetic clock described above can be 0.3, the penalty coefficient λ can be 0.7915253, and the intercept b can be -16.5241.

[0048] In order to solve the above technical problem, the present invention further provides a computer-readable storage medium storing a computer program, wherein the computer program can enable a computer to execute the steps of the method described above.

[0049] In order to solve the above technical problems, the present invention also provides a computer device, including a memory, a processor and a computer program stored in the memory, wherein the processor executes the computer program to implement steps B1)-B4) of the method described above or steps C1)-C5) of the method described above.

[0050] In order to solve the above technical problems, the present invention also provides a computer program product, including a computer program, which can implement steps B1)-B4) of the above method or steps C1)-C5) of the above method when executed by a processor.

[0051] Any of the following applications of the method described above also fall within the scope of protection of the present invention:

[0052] D1) Application in bull breed selection;

[0053] D2) Application in animal breeding.

[0054] Any of the following applications of the device described above also fall within the scope of protection of the present invention:

[0055] D1) Application in bull breed selection;

[0056] D2) Application in animal breeding.

[0057] Any of the following applications of the computer-readable storage medium described above also falls within the scope of protection of the present invention:

[0058] D1) Application in bull breed selection;

[0059] D2) Application in animal breeding.

[0060] Any of the following applications of the computer device described above also falls within the scope of protection of the present invention:

[0061] D1) Application in bull breed selection;

[0062] D2) Application in animal breeding.

[0063] Any of the following applications of the computer program product described above also falls within the scope of protection of the present invention:

[0064] D1) Application in bull breed selection;

[0065] D2) Application in animal breeding.

[0066] The computer program product mentioned above may be a software product that implements its solution mainly through a computer program.

[0067] The computer-readable storage medium mentioned above refers to a carrier for storing data, which may be a magnetic tape, magnetic disk, floppy disk, optical disk, magneto-optical disk, ROM, PROM, VCD, DVD, hard disk, flash memory, USB flash disk, CF card, SD card, MMC card, SM card, memory stick (Memory Stick) or xD card, etc.

[0068] The bull mentioned above may be a Holstein bull.

[0069] The value of x is less than or equal to the number of methylation sites in the known methylation profile data. The variability can be calculated based on the variance of the methylation level values ​​of the methylation sites in the known methylation profile data. Beneficial effects

[0070] The present invention provides a theoretical basis for accurately predicting the epigenetic age and epigenetic acceleration of Holstein cattle, indicating the health status of Holstein cattle, and breeding high-quality breeding bulls. The present invention can be applied to the development or preparation of related products for predicting the epigenetic age and epigenetic acceleration of bulls, indicating the health status of Holstein cattle, and / or breeding high-quality breeding bulls. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 shows the quality control results of RRBS data from 49 Holstein cattle. Note: A. Q30 (%): indicates a base quality value of 30, an error rate of 0.1%, and an accuracy rate of 99.99%. After quality control of the 49 RRBS data, the Q30 values ​​were all greater than 81.25%, indicating that the data quality was acceptable. B. The number of clean reads obtained from the RRBS data in this study was between 22×10 6 -37×10 6 ; C. The RRBS data matching rate is between 53% and 81%, which is a high matching rate; D. The bisulfite conversion rate is greater than 99.62%, which is a qualified conversion rate.

[0072] Figure 2 shows the relationship between chronological age and semen quality traits of Holstein cattle. A shows the relationship between fresh sperm density and chronological age; the vertical axis is fresh sperm density (100 million sperm / mL), and the horizontal axis is chronological age (months). B shows the relationship between frozen semen motility and chronological age; the vertical axis is the percentage of frozen semen motility (%), and the horizontal axis is chronological age (months). C shows the relationship between semen collection volume and chronological age; the vertical axis is the semen collection volume (mL), and the horizontal axis is chronological age (months). D shows the relationship between sperm abnormality rate and chronological age; the vertical axis is the percentage of sperm abnormality (%), and the horizontal axis is chronological age (months). E shows the relationship between fresh semen motility and chronological age; the vertical axis is the percentage of fresh semen motility (%), and the horizontal axis is chronological age (months). F shows the relationship between semen effective sperm count and chronological age; the vertical axis is the number of effective sperm in semen (10 million sperm / straw), each straw containing 0.25 mL, and the horizontal axis is chronological age (months).

[0073] Figure 3 shows the relationship between post-freezing motility and fresh sperm density as a function of actual age. The left graph shows the relationship between post-freezing motility and actual age, with the vertical axis representing the percentage of post-freezing motility (%). The right graph shows the relationship between fresh sperm density and actual age, with the vertical axis representing fresh sperm density (100 million sperm / mL).

[0074] Figure 4 shows the epigenetic clock for Holstein bull sperm constructed using TOP variant loci. The vertical axis represents epigenetic age, and the horizontal axis represents chronological age.

[0075] Figure 5 shows the MAE, number of clock sites, and R values ​​at different degrees of variation. The left figure shows the MAE change analysis at different degrees of variation, with the MAE value on the vertical axis and the degree of variation on the horizontal axis. The middle figure shows the number of clock sites corresponding to different degrees of variation, with the number of clock CpG sites on the vertical axis and the degree of variation on the horizontal axis. The right figure shows the R value change analysis with the degree of variation, with the R value of the correlation between epigenetic age and chronological age on the vertical axis and the degree of variation on the horizontal axis.

[0076] FIG6 is a bubble chart showing GO enrichment analysis of clock genes in the epigenetic clock of Holstein bull sperm.

[0077] FIG7 is a bubble chart of the KEGG enrichment analysis of clock genes in the epigenetic clock of Holstein bull sperm.

[0078] Figure 8 shows the relationship between semen quality and epigenetic acceleration in Holstein bulls. A shows the relationship between sperm deformity rate and epigenetic acceleration, with the ordinate representing the percentage of sperm deformity rate (%); B shows the relationship between fresh sperm density and epigenetic acceleration, with the ordinate representing fresh sperm density (100 million sperm / mL); and C shows the relationship between post-freezing sperm motility and epigenetic acceleration, with the ordinate representing the percentage of post-freezing sperm motility (%). Modes for Carrying Out the Invention

[0079] The experimental methods in the following examples, unless otherwise specified, are conventional methods. The materials, reagents, and instruments used in the following examples, unless otherwise specified, are commercially available. The quantitative tests in the following examples were performed in triplicate, and the results were averaged.

[0080] Example 1: Construction and Verification of the Holstein Bull Sperm Epigenetic Clock

[0081] In this embodiment of the present invention, based on the methylation levels of multiple CpG dinucleotides in the epigenetic map of Holstein bull sperm and combined with elastic network regression, an age estimator, the Holstein bull sperm epigenetic clock, is constructed to accurately estimate actual age.

[0082] 1.RRBS Sequencing and Sequencing Data Quality Statistics

[0083] 1.1 Sequencing data acquisition

[0084] Frozen semen from 49 Holstein bulls was collected, of which 35 bulls had complete semen quality records (including data on six semen quality traits: effective sperm count, sperm deformity rate, fresh semen motility, fresh semen density, semen collection volume, and post-freezing motility, Table 1).

[0085]

[0086]

[0087] Note: Frozen semen is sealed in straw tubes, each straw tube contains 0.25 mL.

[0088] Whole-genome DNA of semen was extracted and sequenced at Novogene using the Illumina platform, combined with a high-performance computing platform, to obtain raw sequencing data (Raw Data) of 49 samples by RRBS sequencing.

[0089] 1.2 Sequencing Data Quality Control

[0090] Trim_galore (related website: https: / / www.bioinformatics.babraham.ac.uk / projects / trim_galore / ) was used to perform data quality control on the raw RRBS sequencing data to obtain high-quality (quality control-compliant) clean reads. The quality control standard often uses Q30 to characterize the sequencing base quality, which refers to a base quality value of 30. This means that reads with a base quality value less than 30 are filtered out. For RRBS libraries, unmethylated Cs in the library can be converted to Ts after bisulfite conversion. The inclusion of a lambda sequence during library construction can assess conversion efficiency, with a bisulfite conversion rate of greater than 99%.

[0091] The quality of the data is shown in Figure 1. In summary, the data quality of this RRBS sequencing is acceptable and can be used for subsequent analysis.

[0092] 2. Correlation analysis between semen quality traits and chronological age of Holstein bulls

[0093] To investigate the changes in semen quality traits of Holstein bulls with chronological age, we used data from 35 groups of semen quality traits from 35 Holstein bulls to determine the correlation and significance between chronological age and semen quality traits (see the upper left and upper right panels in Figure 2).

[0094] A total of six semen quality traits were investigated, and two of them were found to be significantly correlated with chronological age (P < 0.05): fresh sperm density was significantly positively correlated with chronological age, while post-frozen motility was significantly negatively correlated with chronological age. Subsequently, the patterns of changes in fresh sperm density and post-frozen motility with chronological age were further discussed (Figure 3).

[0095] 3. Construction and Verification of the Epigenetic Clock in Holstein Bull Sperm

[0096] This study identified 531,394 CpG sites shared by 49 Holstein cattle sperm. Because the excessive number of CpG sites precluded elastic network regression training, CpG sites were screened. The methylation level variation of all CpG sites (obtained from variance calculations) was calculated, and all CpG sites were ranked from highest to lowest based on this variability. Here we select the CpG sites with the top 0.5% (top 0.5%), TOP 1.0% (top 1.0%), TOP 1.5% (top 1.5%), TOP 2.0% (top 2.0%), TOP 2.5% (top 2.5%), TOP 3.0% (top 3.0%) and TOP3.1% (top 3.1%) of the variation, and use the elastic net regression (elastic net regression) model to train these 7 groups of site data (CpG sites and their methylation levels). Then, we obtain the clock CpG sites of the epigenetic clock and the corresponding coefficients of the sites, and obtain 7 age estimators (7 epigenetic clocks).

[0097] To verify the accuracy of the seven age estimators (seven epigenetic clocks), we used public databases and the team's previous whole-genome methylation sequencing (WGBS) data from 22 Holstein cattle (data download link: https: / / www.ncbi.nlm.nih.gov / geo / ; data numbers are shown in Table 2 below) to validate the clocks (Figure 4). The main steps involved extracting the methylation levels of the seven clock CpG sites for each individual in the methylation data set and substituting them into the seven epigenetic clocks to calculate epigenetic age.

[0098]

[0099] Figure 4 shows the relationship between the actual age of Holstein cattle and the epigenetic age predicted by the clocks using seven different epigenetic clocks. A comparison revealed that, as the TOP rank of CpG site variation varied, the resulting clocks exhibited distinct characteristics in terms of MAE, number of end-point sites, and R values. It was observed that clocks constructed using CpG sites of different TOP ranks also influenced the accuracy of epigenetic age prediction. The MAE values ​​of the seven epigenetic clocks ranged from 8.2256 to 13.6913, the number of sites ranged from 52 to 110, and the R values ​​ranged from 0.84 to 0.93.

[0100] 4. Analysis of Clock Gene Enrichment in Holstein Cattle Sperm

[0101] Comparison of the seven DNA methylation clocks revealed that the elastic network regression training of the top 3% CpG sites yielded 83 clock sites. This clock showed the highest correlation between DNA methylation age and chronological age, and the lowest MAE (Figure 5). Therefore, the epigenetic clock constructed from the top 3% CpG sites was considered the most accurate of the seven epigenetic clocks and was used as the basis for subsequent analyses. This clock had a mixing parameter α of 0.3, a penalty coefficient λ of 0.7915253, an intercept of -16.5241, and 83 sites (Table 3). The MAE was 8.2256.

[0102] Epigenetic clock formula: ;

[0103] CpG sites and coefficients of the epigenetic clock in Holstein bull sperm. In formula I, Represents physiological age or epigenetic age; a represents the coefficient corresponding to each CpG site; a1-ax represents the coefficient corresponding to the first CpG site to the coefficient corresponding to the x-th CpG site; x represents the ranking of the CpG sites, m represents the methylation level value of each CpG site; m1-mx represents the methylation level value from the first CpG site to the methylation level value of the x-th CpG site; b represents the intercept value (the intercept b in the top 3% epigenetic clock formula is -16.5241).

[0104]

[0105]

[0106] Note: In the CpG site column, the number before the “:” is the NCBI Reference Sequence number, and the number after the “:” is the sequence position within that NCBI Reference Sequence number. For example, NC_037328.1:118215697 indicates the CpG site at position 118215697 in the NCBI Reference Sequence: NC_037328.1 (May 11, 2018).

[0107] Further exploration of the epigenetic clock derived from training on the top 3.0% CpG sites revealed 83 genes containing these clock CpG sites, termed "clock genes." GO enrichment analysis and KEGG enrichment analysis were performed on these clock genes (see Figure 6). GO enrichment analysis revealed 23 significant GO terms, including histone amination (P-value = 5.67E-04), folate entry into cells (P-value = 1.10E-03), intrauterine embryonic development (P-value = 2.53E-02), negative regulation of the canonical Wnt signaling pathway (P-value = 4.26E-02), sperm-egg recognition (P-value = 4.61E-02), endocytosis (P-value = 4.72E-02), and gene expression regulation (P-value = 4.99E-02). Folic acid is a key methyl donor and an essential raw material for DNA methylation. Sperm-egg recognition and embryonic development are both crucial initial stages of animal life. Notably, the paternal DNA methylation pattern is retained during early embryonic development, while the maternal methylation pattern disappears during the blastocyst stage. Following this disappearance, the maternal methylation pattern acquires a new paternal-like pattern. These GO terms are primarily involved in processes such as reproduction and embryonic development. This confirms the practical significance of the epigenetic clock, as previously reported in the literature.

[0108] KEGG enrichment analysis of clock genes (see Figure 7) revealed 11 significant pathways, including the longevity regulating pathway (multiple species), pathways in cancer, and proteoglycans in cancer. Genes enriched in the longevity regulating pathway include CLPB, HDAC1, RPS6KB2, AKT1S1, ADCY2, IRS2, and PRKAG3. The Hippo signaling pathway is involved in regulating cell death and growth and plays a crucial role in tumor development and prevention. Some studies suggest that manipulating the Hippo signaling pathway could potentially regulate glucose metabolism, providing new insights into the development of cancer therapies.

[0109] 5. Correlation analysis between semen quality traits and epigenetic acceleration in Holstein cattle

[0110] Semen quality is one of the key reproductive traits of Holstein cattle. Semen quality significantly impacts the probability of successful pregnancy and the economic benefits of animal husbandry. Epigenetic acceleration (Δage) is the difference between clock-estimated epigenetic age and actual (physiological) age. Previous studies have found correlations between epigenetic acceleration and various traits of experimental tissues and individuals. The sign and magnitude of acceleration can reflect the aging and health of the tissues and even individuals involved. To examine the relationship between semen quality (including fresh sperm density, post-freezing motility, semen collection volume, deformity rate, fresh sperm motility, and viable sperm count) and epigenetic acceleration, a Pearson correlation analysis was performed (see Figure 8). The results showed significant correlations between epigenetic acceleration and deformity rate, fresh sperm density, and epigenetic acceleration (P < 0.05), and a significant positive correlation between post-freezing motility and epigenetic acceleration.

[0111] The present invention further provides a computer device comprising a memory, a processor, and a computer program stored in the memory, wherein the processor executes the computer program to implement the following steps:

[0112] B1) Methylation site screening: receiving known methylation profile data of bull sperm methylation sites, and screening the methylation sites in the known methylation profile data to obtain screened methylation sites based on the variability of methylation level values ​​in the known methylation profile data;

[0113] B2) Epigenetic clock construction: Using the methylation profile data of the methylation sites selected from the known methylation profile data, an epigenetic clock is constructed based on an elastic net regression algorithm; the epigenetic clock formula is as follows:

[0114]

[0115] In Formula I, represents physiological age or epigenetic age; a represents the coefficient corresponding to each CpG site; a1-ax represents the coefficient value corresponding to the first CpG site to the coefficient value corresponding to the xth CpG site; x represents the number of CpG sites, m represents the methylation level value of each CpG site; m1-mx represents the methylation level value from the first CpG site to the methylation level value of the xth CpG site; b represents the intercept value;

[0116] B3) Data acquisition: Acquire methylation profile data of the methylation sites in the bull sperm to be tested;

[0117] B4) Age prediction: Based on the methylation profile data of the methylation sites of the sperm of the bull to be tested, the age of the bull to be tested is predicted using the epigenetic clock.

[0118] The methylation sites include 83 methylation sites, the value of x is 83, and the 83 methylation sites are the 83 CpG sites in Table 3.

[0119] The present invention further provides a computer device comprising a memory, a processor, and a computer program stored in the memory, wherein the processor executes the computer program to implement the following steps:

[0120] C1) Methylation site screening: obtaining known methylation profile data of bull sperm methylation sites, and screening the methylation sites in the known methylation profile data based on the variability of methylation level values ​​in the known methylation profile data to obtain screened methylation sites;

[0121] C2) Epigenetic clock construction: using the methylation profile data of the methylation sites screened in the known methylation profile data to construct an epigenetic clock based on an elastic net regression algorithm; the epigenetic clock formula is as follows:

[0122]

[0123] In Formula I, represents physiological age or epigenetic age; a represents the coefficient corresponding to each CpG site; a1-ax represents the coefficient value corresponding to the first CpG site to the coefficient value corresponding to the xth CpG site; x represents the number of CpG sites, m represents the methylation level value of each CpG site; m1-mx represents the methylation level value from the first CpG site to the methylation level value of the xth CpG site; b represents the intercept value;

[0124] C3) Data acquisition: obtaining methylation profile data of the methylation sites in the bull sperm to be tested;

[0125] C4) Epigenetic age prediction: Based on the methylation profile data of the methylation sites of the sperm of the bull to be tested, the epigenetic age of the bull to be tested is predicted using the epigenetic clock;

[0126] C5) Semen quality prediction: calculating the difference between the epigenetic age of the bull to be tested and the actual physiological age of the bull to be tested to obtain the epigenetic acceleration of the bull to be tested; and predicting the semen quality of the bull to be tested based on the epigenetic acceleration.

[0127] The semen quality includes sperm deformity rate, semen freshness and sperm motility after freezing.

[0128] The methylation sites include 83 methylation sites, the value of x is 83, and the 83 methylation sites are the 83 CpG sites in Table 3.

[0129] CROSS-REFERENCE TO RELATED APPLICATIONS

[0130] This application claims priority to the Chinese patent application (application number 202310803474.9) filed on July 3, 2023, the entire contents of which are incorporated herein by reference. Industrial Applicability

[0131] This study analyzed methylation of Holstein cattle sperm genomic DNA and constructed the first epigenetic clock for Holstein cattle sperm. The results revealed that sperm epigenetic clock genes are enriched in pathways related to reproduction, disease, cancer, and longevity. They also found that chronological age and epigenetic acceleration influence semen quality traits. The methods and devices of this invention can be used to develop or manufacture products that accurately predict Holstein cattle semen quality and / or epigenetic age and / or epigenetic acceleration. They can also be used to assess the health of Holstein cattle and to select high-quality sires.

Claims

1. A method for predicting bull semen quality, characterized in that: The method comprises the following steps: C1) Methylation site screening: used to obtain known methylation spectrum data of bull sperm methylation sites, and screen the methylation sites in the known methylation spectrum data according to the variability of methylation level values ​​in the known methylation spectrum data to obtain screened methylation sites; C2) Epigenetic clock construction: for using the methylation spectrum data for screening methylation sites in the known methylation spectrum data to construct an epigenetic clock based on an elastic network regression algorithm; the formula of the epigenetic clock is as follows: ； In Formula I, represents physiological age or epigenetic age; a represents the coefficient corresponding to each CpG site; a1-ax represents the coefficient value corresponding to the first CpG site to the coefficient value corresponding to the xth CpG site; x represents the number of CpG sites, and m represents the methylation level value of each CpG site; m1-mx represents the methylation level value from the first CpG site to the methylation level value of the xth CpG site; b represents the intercept value; C3) Data acquisition: obtaining the methylation profile data of the methylation sites of the bull sperm to be tested; C4) Epigenetic age prediction: based on the methylation spectrum data of the methylation sites of the sperm of the bull to be tested, the epigenetic age of the bull to be tested is predicted using the epigenetic clock; C5) Semen quality prediction: calculating the difference between the epigenetic age of the bull to be tested and the actual physiological age of the bull to be tested, and obtaining the epigenetic acceleration of the bull to be tested; The semen quality of the bull to be tested is predicted based on the epigenetic acceleration.

2. The method according to claim 1, characterized in that: The semen quality includes sperm deformity rate, semen freshness and / or sperm motility after freezing.

3. The method according to claim 1, characterized in that: The methylation sites include 83 methylation sites, the value of x is 83, and the 83 methylation sites are the 83 CpG sites in Table 3.

4. The method according to claim 2, characterized in that: The methylation sites include 83 methylation sites, the value of x is 83, and the 83 methylation sites are the 83 CpG sites in Table 3.

5. A method for predicting the physiological age of an animal, characterized in that: The method comprises the following steps: B1) Methylation site screening: obtaining known methylation spectrum data of bull sperm methylation sites, and screening the methylation sites in the known methylation spectrum data according to the variability of methylation level values ​​in the known methylation spectrum data to obtain screened methylation sites; B2) Epigenetic clock construction: using the methylation spectrum data of the methylation sites screened in the known methylation spectrum data, an epigenetic clock is constructed based on an elastic network regression algorithm; the formula of the epigenetic clock is as follows: ； In Formula I, represents physiological age or epigenetic age; a represents the coefficient corresponding to each CpG site; a1-ax represents the coefficient value corresponding to the first CpG site to the coefficient value corresponding to the xth CpG site; x represents the number of CpG sites, and m represents the methylation level value of each CpG site; m1-mx represents the methylation level value from the first CpG site to the methylation level value of the xth CpG site; b represents the intercept value; B3) Data acquisition: obtaining the methylation profile data of the methylation sites of the bull sperm to be tested; B4) Age prediction: Based on the methylation spectrum data of the methylation sites of the sperm of the bull to be tested, the age of the bull to be tested is predicted using the epigenetic clock.

6. The method according to claim 5, characterized in that: The methylation sites include 83 methylation sites, the value of x is 83, and the 83 methylation sites are the 83 CpG sites in Table 3.

7. A device for predicting the age of a bull, characterized in that: The device comprises the following modules: A1) Methylation site screening module: used to obtain known methylation spectrum data of bull sperm methylation sites, and screen the methylation sites in the known methylation spectrum data according to the variability of methylation level values ​​in the known methylation spectrum data to obtain screened methylation sites; A2) Epigenetic clock construction module: used to use the methylation spectrum data of the methylation sites screened in the known methylation spectrum data to construct an epigenetic clock based on the elastic network regression algorithm; the formula of the epigenetic clock is as follows: ； In Formula I, represents physiological age or epigenetic age; a represents the coefficient corresponding to each CpG site; a1-ax represents the coefficient value corresponding to the first CpG site to the coefficient value corresponding to the xth CpG site; x represents the number of CpG sites, and m represents the methylation level value of each CpG site; m1-mx represents the methylation level value from the first CpG site to the methylation level value of the xth CpG site; b represents the intercept value; A3) Data receiving module: used to receive the methylation spectrum data of the methylation sites of the bull sperm to be tested; A4) Age prediction module: used to predict the age of the bull to be tested based on the methylation spectrum data of the methylation sites of the sperm of the bull to be tested and using the epigenetic clock.

8. The device according to claim 7, characterized in that: The methylation sites include 83 methylation sites, the value of x is 83, and the 83 methylation sites are the 83 CpG sites in Table 3.

9. A device for predicting the quality of bull semen, characterized in that: The device comprises the following modules: D1) Methylation site screening module: used to obtain known methylation spectrum data of bull sperm methylation sites, and screen the methylation sites in the known methylation spectrum data according to the variability of methylation level values ​​in the known methylation spectrum data to obtain screened methylation sites; D2) Epigenetic clock construction module: used to use the methylation spectrum data of the methylation sites screened in the known methylation spectrum data to construct an epigenetic clock based on the elastic network regression algorithm; the formula of the epigenetic clock is as follows: ； In Formula I, represents physiological age or epigenetic age; a represents the coefficient corresponding to each CpG site; a1-ax represents the coefficient value corresponding to the first CpG site to the coefficient value corresponding to the xth CpG site; x represents the number of CpG sites, and m represents the methylation level value of each CpG site; m1-mx represents the methylation level value from the first CpG site to the methylation level value of the xth CpG site; b represents the intercept value; D3) Data acquisition module: used to obtain the methylation spectrum data of the methylation sites of the bull sperm to be tested; D4) an epigenetic age prediction module: used to predict the epigenetic age of the bull to be tested based on the methylation spectrum data of the methylation sites of the sperm of the bull to be tested and using the epigenetic clock; D5) a semen quality prediction module: used to calculate the difference between the epigenetic age of the bull to be tested and the actual physiological age of the bull to be tested, and obtain the epigenetic acceleration of the bull to be tested; The semen quality of the bull to be tested is predicted based on the epigenetic acceleration.

10. A computer-readable storage medium storing a computer program, characterized in that: The computer program enables a computer to execute the steps of the method according to any one of claims 1 to 4 and / or the steps of the method according to claim 5 or 6.

11. Any of the following uses of the method according to any one of claims 1 to 6: D1) Application in bull breed selection; D2) Application in animal breeding.

12. Any of the following uses of the device according to any of claims 7 to 9: D1) Application in bull breed selection; D2) Application in animal breeding.

13. Any of the following applications of the computer-readable storage medium of claim 10: D1) Application in bull breed selection; D2) Application in animal breeding.

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