Device, method, computer-readable storage medium, and application for predicting bull age using sperm epigenetic clock

Abstract

The present invention discloses an apparatus, method, computer-readable storage medium, and application for predicting bull age using a sperm epigenetic clock. The method uses known methylation profile data from bull sperm methylation sites to screen 83 selected methylation sites based on the variability of methylation levels. Using the elastic network regression algorithm, the selected methylation sites are used to construct an epigenetic clock. This epigenetic clock can be used to predict the age of the bull being tested. The epigenetic clock can be used to predict the age of the bull, and the epigenetic acceleration can be used to predict the quality of the bull's semen. This method can be applied to bull breed selection and breeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioinformatics, and specifically relates to a device, method, computer-readable storage medium, and application thereof for predicting the age of a bull using a sperm epigenetic clock. Background Art

[0002] An epigenetic clock is an age estimator constructed by combining the methylation levels of a set of age-related CpG sites 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 for analyzing 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, leading to changes in semen quality. Semen quality is the most important reproductive trait for Holstein bulls. Summary of the Invention

[0005] 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.

[0006] In order to solve the above technical problems, the present invention first provides a device for predicting the age of a bull, which may include the following modules:

[0007] A1) a methylation site screening module: used to obtain known methylation profile data of bull sperm methylation sites, and screen the methylation sites in the known methylation profile data based on the variability of methylation levels in the known methylation profile data to obtain screened methylation sites;

[0008] A2) Epigenetic clock construction module: for 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 may be as follows:

[0009] f(x)=a1m1+a2m2+a3m3+…+axmx+b Formula I

[0010] In formula I, f(x) 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; and b represents the intercept value.

[0011] A3) Data acquisition module: used to obtain methylation profile data of methylation sites of the bull sperm to be tested;

[0012] 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 using the epigenetic clock.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] In order to solve the above technical problems, the present invention also provides a method for predicting the physiological age of an animal, which may include the following steps:

[0017] 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 levels in the known methylation profile data to obtain screened methylation sites;

[0018] 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:

[0019] f(x)=a1m1+a2m2+a3m3+…+axmx+b Formula I

[0020] In formula I, f(x) 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; and b represents the intercept value.

[0021] B3) Data acquisition: obtaining methylation profile data of the methylation sites of the bull sperm to be tested;

[0022] 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.

[0023] 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.

[0024] In order to solve the above technical problems, the present invention also provides a method for predicting bull semen quality, characterized in that the method comprises the following steps:

[0025] 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;

[0026] 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:

[0027] f(x)=a1m1+a2m2+a3m3+…+axmx+b Formula I

[0028] In formula I, f(x) 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;

[0029] C3) Data acquisition: obtaining methylation profile data of the methylation sites of the bull sperm to be tested;

[0030] 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;

[0031] 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.

[0032] In the above method, the semen quality may be sperm deformity rate, semen freshness and / or sperm motility after freezing.

[0033] 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.

[0034] 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.

[0035] 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:

[0036] D1) a methylation site screening module: used to obtain 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;

[0037] D2) 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 is as follows:

[0038] f(x)=a1m1+a2m2+a3m3+…+axmx+b Formula I

[0039] In formula I, f(x) 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;

[0040] D3) Data acquisition module: used to obtain methylation profile data of methylation sites of the bull sperm to be tested;

[0041] 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;

[0042] 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.

[0043] The semen quality may be sperm deformity rate, semen freshness and / or sperm motility after freezing.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] Any of the following applications of the above-described apparatus and / or the above-described method and / or the above-described computer-readable storage medium also falls within the scope of protection of the present invention:

[0049] D1) Application in bull breed selection;

[0050] D2) Application in animal breeding.

[0051] 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.

[0052] This study has constructed the first epigenetic clock in Holstein bull sperm. This study aims to construct an epigenetic clock in bull sperm, 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 mainly include the following two parts: First, the construction of the epigenetic clock in Holstein bull sperm 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. Genomic DNA was extracted and subjected to reduced representational bisulfite sequencing (RRBS). Data analysis was then performed, and shared CpG sites were identified using elastic network regression to construct the bull sperm epigenetic clock. 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 perform GO and KEGG enrichment analysis. 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 actual 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 vitality 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 showed that clock genes are enriched in pathways such as reproduction, disease, cancer and longevity. It was also found that actual age and epigenetic acceleration will affect semen quality traits. 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1Quality 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 comparison rate is between 53% and 81%, which is a high comparison rate; D. The bisulfite conversion rate is greater than 99.62%, which is a qualified conversion rate.

[0054] Figure 2 Figure 1 shows the relationship between chronological age and semen quality traits in Holstein cattle. A represents the relationship between fresh sperm density and chronological age; the vertical axis represents fresh sperm density (100 million sperm / mL), and the horizontal axis represents chronological age (months). B represents the relationship between frozen semen motility and chronological age; the vertical axis represents the percentage of frozen semen motility (%), and the horizontal axis represents chronological age (months). C represents the relationship between semen collection volume and chronological age; the vertical axis represents semen collection volume (mL), and the horizontal axis represents chronological age (months). D represents the relationship between sperm abnormality rate and chronological age; the vertical axis represents the percentage of sperm abnormality (%), and the horizontal axis represents chronological age (months). E represents the relationship between fresh semen motility and chronological age; the vertical axis represents the percentage of fresh semen motility (%), and the horizontal axis represents chronological age (months). F represents the relationship between effective sperm count and chronological age; the vertical axis represents effective sperm count (10 million sperm / vial, each 0.25 mL straw), and the horizontal axis represents chronological age (months).

[0055] Figure 3 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 post-freezing motility and actual age, with the vertical axis representing the fresh sperm density (100 million sperm / mL).

[0056] Figure 4 This is 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.

[0057] Figure 5 The left panel shows the MAE, number of clock sites, and R value at different degrees of variation. The vertical axis shows the MAE value and the horizontal axis shows the degree of variation. The middle panel shows the number of clock sites corresponding to different degrees of variation. The vertical axis shows the number of clock CpG sites and the horizontal axis shows the degree of variation. The right panel shows the R value as the degree of variation changes. The vertical axis shows the correlation between epigenetic age and chronological age and the horizontal axis shows the degree of variation.

[0058] Figure 6Bubble plot showing GO enrichment analysis of clock genes in the epigenetic clock of Holstein bull sperm.

[0059] Figure 7 Bubble chart showing KEGG enrichment analysis of clock genes in the epigenetic clock of Holstein bull sperm.

[0060] Figure 8 Figure 2 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); C shows the relationship between post-freezing sperm motility and epigenetic acceleration, with the ordinate representing the percentage of post-freezing sperm motility (%). DETAILED DESCRIPTION

[0061] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0062] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0063] Example 1: Construction and Verification of the Holstein Bull Sperm Epigenetic Clock

[0064] The present invention constructs an age estimator, the Holstein bovine sperm epigenetic clock, based on the methylation levels of multiple CpG dinucleotides in the Holstein bovine sperm epigenetic map and combined with elastic network regression to accurately estimate the actual age.

[0065] 1.RRBS sequencing and sequencing data quality statistics

[0066] 1.1 Sequencing data acquisition

[0067] 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).

[0068] Table 1. Semen quality record information

[0069]

[0070]

[0071] Note: Frozen semen is sealed in straw tubes, each straw tube contains 0.25 mL.

[0072] 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 RRBS sequencing of 49 samples.

[0073] 1.2 Sequencing Data Quality Control

[0074] 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 sequencing reads with a base quality value less than 30 are filtered out. For RRBS libraries, bisulfite conversion allows unmethylated Cs to be converted to Ts. The inclusion of a lambda sequence during library construction allows for assessment of conversion efficiency, which is required to be greater than 99%.

[0075] The quality of the data obtained can be seen in Figure 1 In summary, the data quality of this RRBS sequencing is qualified and can be used for subsequent analysis.

[0076] 2. Correlation analysis between semen quality traits and chronological age of Holstein bulls

[0077] In order to explore the changes of semen quality traits of Holstein bulls with chronological age, the data of 35 groups of semen quality traits of 35 Holstein bulls were used to obtain the correlation and significance between chronological age and semen quality traits (see Figure 2 (center left upper picture and right upper picture)

[0078] A total of 6 semen quality traits were investigated, and two of them were found to be significantly correlated with actual age (P < 0.05): fresh sperm density was significantly positively correlated with actual age, and frozen motility was significantly negatively correlated with actual age. Subsequently, the patterns of fresh sperm density and frozen motility changing with actual age were further discussed ( Figure 3 ).

[0079] 3. Construction and Verification of the Epigenetic Clock in Holstein Bull Sperm

[0080] This study identified 531,394 common CpG sites in sperm from 49 Holstein cattle. Because the large 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 ranked from highest to lowest based on this variation. Here, we selected 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 top 3.1% (top 3.1%) of the variation, and used the elastic net regression model to train these seven groups of site data (CpG sites and their methylation levels). Subsequently, the clock CpG sites of the epigenetic clock and the corresponding coefficients of the sites were obtained, and seven age estimators (seven epigenetic clocks) were obtained.

[0081] In order to verify the accuracy of the above 7 age estimators (7 sets of epigenetic clocks), the online public database and the whole genome methylation sequencing (WGBS) data of 22 Holstein cattle individuals previously collected by the research team (data download website: https: / / www.ncbi.nlm.nih.gov / geo / , data numbers are shown in Table 2 below) were used for clock verification ( Figure 4 The main steps are to extract the methylation levels of 7 groups of clock CpG sites of the individual in the methylation data to be tested, and substitute them into 7 groups of epigenetic clocks to obtain the epigenetic age.

[0082] Table 2. Verification data information

[0083]

[0084]

[0085] Figure 4 The study examined the relationship between the actual age of Holstein cattle and the epigenetic age predicted by the clocks using seven different epigenetic clocks. Comparison revealed that the clocks exhibited distinct characteristics in terms of MAE, number of end-stage sites, and R values, depending on the TOP rank of CpG site variation. It was also observed that clocks constructed using different TOP CpG site rank influence the accuracy of epigenetic age prediction. MAE values ​​for the seven epigenetic clocks ranged from 8.2256 to 13.6913, the number of sites ranged from 52 to 110, and R values ​​ranged from 0.84 to 0.93.

[0086] 4. Analysis of Clock Gene Enrichment in Holstein Cattle Sperm

[0087] By comparing the 7 DNA methylation clocks obtained, it was found that there were 83 clock sites obtained after elastic network regression training of the CpG sites with the TOP 3% variation, and the DNA methylation age of this clock had the highest correlation with the actual age and the lowest MAE ( Figure 5 ), the epigenetic clock constructed from the top 3% of CpG sites was considered the most accurate of the seven epigenetic clocks, and subsequent analyses were based on this clock. 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), resulting in a MAE of 8.2256.

[0088] Epigenetic clock formula: f(x)=a1m1+a2m2+a3m3+…+axmx+b Formula I

[0089] CpG sites and coefficients of the Holstein bovine sperm epigenetic clock. In formula I, f(x) represents physiological age or epigenetic age; a represents the coefficient corresponding to each CpG site; a1-ax represents the coefficients corresponding to the first CpG site to the xth CpG site; x represents the rank of the CpG sites; m represents the methylation level of each CpG site; m1-mx represents the methylation level from the first CpG site to the xth CpG site; and b represents the intercept value (the intercept b in the top 3% epigenetic clock formula is -16.5241).

[0090] Table 3. Apparent clock coefficient values ​​constructed from the top 3% of CpG sites

[0091]

[0092] 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 NCBI Reference Sequence: NC_037328.1 (11-MAY-2018).

[0093] Further exploration of the epigenetic clock obtained by training the TOP 3.0% CpG sites of variation yielded 83 genes with clock CpG sites, which were named "clock genes". GO enrichment analysis and KEGG enrichment analysis were performed on these clock genes. GO enrichment analysis of clock genes (see Figure 6 ), enriched for 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). Folate is an important 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, and a new methylation pattern similar to the paternal pattern is subsequently acquired. These GO terms are primarily involved in processes such as reproduction and embryonic development. This also confirms the practical significance of the epigenetic clock mentioned in published literature.

[0094] KEGG enrichment analysis of clock genes (see Figure 7 ), with 11 pathways significantly enriched, including the longevity regulating pathway (multiple species), the cancer pathway (pathways incancer), and the proteoglycans in cancer pathway. 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 key role in tumor development and prevention. Some studies suggest that manipulating the Hippo signaling pathway might be able to regulate glucose metabolism, providing new clues for the development of cancer therapies.

[0095] 5. Correlation Analysis between Holstein Cattle Semen Quality Traits and Epigenetic Acceleration

[0096] Semen quality is one of the main representative traits of Holstein cattle reproduction. The quality of semen greatly affects the probability of successful pregnancy and the economic benefits of animal husbandry. Epigenetic acceleration (Δage) is the difference between the epigenetic age estimated by the clock and the actual (physiological) age. Previous studies have found that there is a correlation between epigenetic acceleration and many traits of test tissues and test individuals. The positive and negative as well as the size of the acceleration can reflect the aging and health of the test tissues and even individuals. In order to examine the relationship between semen quality (including fresh semen density, post-freezing vitality, semen collection volume, deformity rate, fresh semen vitality, and effective sperm count) and epigenetic acceleration, a Pearson correlation analysis was performed for the two (see Figure 8 The results showed that there was a significant correlation between the sperm deformity rate and epigenetic acceleration, and between fresh sperm density and epigenetic acceleration (P < 0.05). There was also a significant positive correlation between post-freezing motility and epigenetic acceleration.

[0097] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A device for predicting the age of a bull, characterized by: The device includes the following modules: A1) Methylation site screening module: for obtaining known methylation profile data of bull sperm methylation sites, screening the methylation sites in the known methylation profile data based on the variability of methylation levels in the known methylation profile data, and selecting methylation sites with a variability in the top 3.0% to obtain screening methylation sites; A2) Epigenetic clock construction module: for 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: f(x)=a1m1+a2m2+a3m3+…+axmx+b Formula I In formula I, f(x) 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 of the first CpG site to the methylation level value of the xth CpG site; b represents the intercept value; A3) Data acquisition module: used to obtain methylation profile data of methylation sites of the bull sperm to be tested; A4) an age prediction module: configured 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; The methylation sites include 83 methylation sites, the value of x is 83, and the 83 methylation sites are shown in Table 3: Table 3, 2. A method for predicting the physiological age of an animal, characterized in that: The method comprises the following steps: B1) Methylation site screening: Obtain known methylation profile data of bull sperm methylation sites, and screen the methylation sites in the known methylation profile data based on the variability of methylation levels in the known methylation profile data. Select methylation sites with a variability in the top 3.0% to obtain screening methylation sites. 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 formula of the epigenetic clock is as follows: f(x)=a1m1+a2m2+a3m3+…+axmx+b Formula I In formula I, f(x) 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 of the first CpG site to the methylation level value of the xth CpG site; b represents the intercept value; B3) Data acquisition: obtaining methylation profile data of the methylation sites of the bull sperm to be tested; 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; The methylation sites include 83 methylation sites, the value of x is 83, and the 83 methylation sites are shown in Table 3 of claim 1.

3. A method for predicting bull semen quality, characterized in that: The method comprises the following steps: C1) Methylation site screening: obtaining known methylation profile data of bull sperm methylation sites, screening the methylation sites in the known methylation profile data based on the variability of methylation levels in the known methylation profile data, and selecting methylation sites with a variability in the top 3.0% to obtain screening methylation sites; C2) Epigenetic clock construction: using the methylation profile data for screening methylation sites 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: f(x)=a1m1+a2m2+a3m3+…+axmx+b Formula I In formula I, f(x) 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 of the first CpG site to the methylation level value of the xth CpG site; b represents the intercept value; C3) Data acquisition: obtaining methylation profile data of the methylation sites of the bull sperm to be tested; 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; 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; Predicting the semen quality of the bull to be tested based on the epigenetic acceleration; The methylation sites include 83 methylation sites, the value of x is 83, and the 83 methylation sites are shown in Table 3 of claim 1.

4. The method according to claim 3, wherein: The semen quality includes sperm deformity rate, semen freshness and / or sperm motility after freezing.

5. A device for predicting bull semen quality, characterized in that: The device includes the following modules: D1) Methylation site screening module: for obtaining known methylation profile data of bull sperm methylation sites, screening the methylation sites in the known methylation profile data based on the variability of methylation levels in the known methylation profile data, and selecting methylation sites with a variability in the top 3.0% to obtain screening methylation sites; D2) 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 is as follows: f(x)=a1m1+a2m2+a3m3+…+axmx+b Formula I In formula I, f(x) 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 of 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 methylation profile data of methylation sites of the bull sperm to be tested; 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; 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; Predicting the semen quality of the bull to be tested based on the epigenetic acceleration; The methylation sites include 83 methylation sites, the value of x is 83, and the 83 methylation sites are shown in Table 3 of claim 1.

6. A computer-readable storage medium storing a computer program, characterized in that: The computer program causes a computer to execute the steps of the method according to claim 2 and / or the steps of the method according to claim 3 or 4.

7. Any of the following applications of the apparatus of claim 1 and / or the method of any one of claims 2 to 4 and / or the apparatus of claim 5 and / or the computer-readable storage medium of claim 6: D1) Application in bull breed selection; D2) Application in animal breeding.

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