Galliform epigenetic clock

Abstract

The present invention relates to an in vitro method for predicting the chronological age of a galliform subject, said method comprising the steps of: (a.) obtaining genomic DNA from biological sample material derived from a galliform subject to be tested or from a galliform population to be tested, (b.) determining the methylation levels of a specific set of CpG sites in the genomic galliform DNA obtained in step (a), and (c.) comparing the methylation levels of these CpG sites in the genomic galliform DNA from the sample to be tested with the methylation levels of the same CpG sites from age-related reference samples, thereby determining the epigenetic age and predicting the chronological age of the subject or of the population to be tested; wherein, for this specific set of CpG sites in step (b), the effect of genetic polymorphisms is eliminated by excluding CpG sites associated with single nucleotide polymorphisms, and the effect of gender-specific methylation differences on sex chromosomes is eliminated by excluding all CpG sites located on sex chromosomes.

Description

Technical Field

[0001] This invention relates to a method for determining the epigenetic age of Galliformes, and based on this method, to a method for assessing the inflammatory state of Galliformes. Background of the Invention Galliformes, such as chickens (Gallus gallus), are a significant source of meat and eggs for commercial production. Therefore, studying the factors influencing chicken growth, pathogen resistance, and meat quality is of great scientific and economic importance. Extensive genome-wide association studies have been conducted to elucidate the underlying genetic framework. Epigenetic modifications provide important supplementary and extended information on genetic variation, but remain relatively under-researched in chickens.

[0003] Animal methylomes can be highly diverse, ranging from some insect genomes with sparse methylation patterns and only tens of thousands of methylation markers to mammalian genomes with dense methylation patterns and tens of millions of methylation markers. To date, little is known about whole-genome DNA methylation patterns in non-mammalian vertebrates, and particularly birds.

[0004] DNA methylation is associated with the aging process and represents a highly specific epigenetic modification of the DNA region of CpG dinucleotides (5'-C-phosphate-G-3'), where a cytosine nucleotide is followed by a guanine nucleotide in the basic linear sequence of its 5'3' orientation. The collection of genomic methylation modifications constitutes the methylome of a particular cell.

[0005] Hypomethylated regions (LMRs) represent a key feature of the dynamic methylome. LMRs are localized reductions in the DNA methylation landscape and represent CpG-poor, distal regulatory regions that typically reflect the binding of transcription factors and other DNA-binding proteins. LMRs were initially described in mice (Stadler et al., Nature 480, 490–495 (2011)). The evolutionary conservation of LMRs outside of mammals remains unexplored.

[0006] Age-related DNA methylation changes on discrete sets of CpG in the human genome have been identified and used to predict age (Horvath, S. (2013). DNA methylation age of human tissues and cell types. Genome Biology 14:3156). These "epigenetic clocks" can independently assess tissue-specific or tissue-independent DNA methylation age and can predict mortality and time to death.

[0007] Epigenetic age is highly correlated with chronological age and also responds to environmental factors that accelerate or slow down the aging process, resulting in a significant deviation from chronological age.

[0008] Epigenetic age acceleration (epigenetic age > chronological age) indicates that the underlying tissues are aging faster than expected based on chronological age, while negative values ​​(epigenetic age < chronological age, age deceleration) indicate that the tissues are aging slower than expected. Epigenetic age acceleration is associated with many age-related conditions and diseases, such as inflammatory processes.

[0009] For animal husbandry, performance biomarkers are particularly useful tools because they help monitor large numbers of animals and provide objective quality assurance. The development of performance biomarkers for Galliformes, and especially for broilers, presents unique challenges due to their considerable economic importance and relatively short lifespan (up to 63 days).

[0010] Gut health is critical when it comes to Galliformes, particularly for the welfare and performance of poultry chickens. Gut diseases, which are often associated with inflammatory processes and affect the structural integrity of the gastrointestinal tract (GIT), lead to significant economic losses due to reduced body weight gain, poor feed conversion efficiency, increased mortality, and higher medication costs (M'Sadeq, SA, Wu, S., Swick, RA & Choct, M. (2015). Towards the control of necrotic enteritis in broiler chickens with in-feed antibiotics phasing-out worldwide. AnimalNutrition, 1, 1-11; Timbermont, L., Haesebrouck, F., Ducatelle, R. & VanImmerseel, F. (2011). Necrotic enteritis in broilers: an updated review on the pathogenesis. Avian Pathol, 40, 341-347).

[0011] Similar considerations apply to other bird species, particularly Galliformes species such as turkeys, quails, or pheasants.

[0012] Therefore, there is an urgent need for new descriptive and predictive markers for biological diseases (such as intestinal inflammation) to control evolving production processes and, where necessary, to intervene early.

[0013] Accordingly, the object of the present invention is to provide a robust method for determining the epigenetic age of Galliformes (e.g., chickens) with improved specificity, accuracy and precision; and to provide methods for determining inflammatory states.

[0014] Invention Summary This invention relates to an in vitro method for predicting the actual age of healthy Galliformes, the method comprising the following steps: (a.) Obtain genomic DNA from biological sample materials derived from the Galliformes species being tested or from the Galliformes population being tested. (b.) Determine the methylation levels of a specific set of CpG sites in the Galliformes DNA obtained in step (a.), and (c.) The methylation levels of these CpG sites in the genomic Galliformes DNA from the test samples were compared with the methylation levels of the same CpG sites from age-related reference samples. This determines the epigenetic age and predicts the actual age of the subject or the population to be tested; For this specific set of CpG sites in step (b) - Eliminating the effects of genetic polymorphisms by excluding CpG sites associated with single nucleotide polymorphisms, and - Eliminate the effects of sex-specific methylation differences on sex chromosomes by excluding all CpG sites located on sex chromosomes.

[0015] In the context of this invention, the term "healthy" specifically refers to Galliformes without inflammatory health problems. The inventors have discovered that the epigenetic age of uninflamed Galliformes subjects or populations of uninflamed Galliformes corresponds to their actual age, and the discrepancy between epigenetic age and actual age indicates an inflammatory process.

[0016] Furthermore, the present invention provides an in vitro method for determining the epigenetic age of Galliformes, the method comprising the following steps: (a.) Obtain genomic DNA from biological sample materials derived from the Galliformes species being tested or from the Galliformes population being tested. (b.) Determine the methylation levels of a specific set of CpG sites in the Galliformes DNA obtained in step (a.), and (c.) The methylation levels of these CpG sites in the genomic Galliformes DNA from the test samples were compared with the methylation levels of the same CpG sites from age-related reference samples. This determines the epigenetic age of the subject or the population to be tested; For this specific set of CpG sites in step (b) - Eliminating the effects of genetic polymorphisms by excluding CpG sites associated with single nucleotide polymorphisms, and - Eliminate the effects of sex-specific methylation differences on sex chromosomes by excluding all CpG sites located on sex chromosomes.

[0017] Finally, the present invention relates to an in vitro method for assessing inflammatory status in Galliformes, the method comprising the following steps: (a.) Obtain genomic DNA from biological sample materials derived from the Galliformes species being tested or from the Galliformes population being tested. (b.) Determine the methylation levels of a specific set of CpG sites in the Galliformes genomic DNA obtained in step (a.), and (c.) The methylation levels of these CpG sites in the genomic Galliformes DNA from the test samples were compared with the methylation levels of the same CpG sites from age-related reference samples. This determines the epigenetic age of the subject or the tested population, and (d.) Compare the epigenetic age of the subject or the population being tested obtained therefrom with their actual chronological age. Epigenetic age exceeding chronological age indicates inflammation, and For this specific set of CpG sites in step (b) - Eliminating the effects of genetic polymorphisms by excluding CpG sites associated with single nucleotide polymorphisms, and - Eliminate the effects of sex-specific methylation differences on sex chromosomes by excluding all CpG sites located on sex chromosomes. Invention Details This invention provides a novel epigenetic clock for Galliformes, in which CpG sites associated with previously unidentified confounding factors are removed. Therefore, the new clock provides significantly improved generalization ability and robustness.

[0019] More specifically, the inventors have identified several CpG (cytosine-phosphate-guanine) sites in the chicken (Gallus gallus) genome, whose DNA methylation levels are tissue-specific and tissue-independent at full age. From these CpG sites, the influence of genetic polymorphisms is eliminated by excluding CpG sites associated with single nucleotide polymorphisms (SNPs), and / or the influence of sex-specific methylation differences on sex chromosomes is eliminated by excluding all CpG sites located on sex chromosomes. SNPs of Galliformes can be found in specific databases, such as dbSNP databases. Similar considerations apply to the sex chromosomes of Galliformes.

[0020] In addition to the above, the methylation levels of a specific set of CpG sites can be tissue-specifically normalized. Normalization is achieved by calculating the average methylation value for each CpG from all samples of the same tissue and subtracting the resulting value from the value of that CpG (or by calculating the average methylation value for each LMR from all samples of the same tissue and subtracting the resulting value from the value of that LMR). This normalization is necessary for the different aging trajectories of individual tissues.

[0021] In other words, measuring DNA methylation at the CpG sites obtained in this way can determine the epigenetic age of chickens and accurately predict their actual age.

[0022] The methods described above, and especially the techniques for removing confounding factors from CpG sites, are easily transferred from chickens to other Galliformes.

[0023] Prediction of actual age Based on the above findings, a novel Galliformes multi-tissue age predictor (“epigenetic clock” / “methylation clock”) has been developed.

[0024] Therefore, the present invention provides an in vitro method for predicting the actual age of a healthy Galliformes, the method comprising the following steps: (a.) Obtain genomic DNA from biological sample materials derived from the Galliformes species being tested or from the Galliformes population being tested. (b.) Determine the methylation levels of a specific set of CpG sites in the Galliformes DNA genome obtained in step (a.), and (c.) The methylation levels of these CpG sites in the genomic Galliformes DNA from the test sample were compared with the methylation levels of the same CpG sites from an age-related reference sample. This determines the epigenetic age and predicts the actual age of the subject or the population to be tested; For this specific set of CpG sites in step (b) - Eliminating the effects of genetic polymorphisms by excluding CpG sites associated with single nucleotide polymorphisms, and - Eliminate the effects of sex-specific methylation differences on sex chromosomes by excluding all CpG sites located on sex chromosomes.

[0025] Galliformes is an order of birds that are bulky and forage on the ground. It includes turkeys, grouse, chickens, ptarmigans, quails, partridges, pheasants, francophores, the genus *Gallus*, and the family *Gallus*. This order comprises five families: Phasianidae (including chickens (*Gallus gallus*), quails, partridges, pheasants, turkeys, peacocks, and grouse), Quailidae, Guinea fowl, *Gallus*, and *Gallus*.

[0026] The method according to the invention is particularly applicable to chickens (Gallus gallus). Therefore, one specific embodiment of the invention is an in vitro method for predicting the true age of a healthy chicken (Gallus gallus), the method comprising the following steps: (a.) Obtain genomic DNA from biological sample materials derived from the chicken subject or the chicken population being tested. (b.) Determine the methylation levels of a specific set of CpG sites in the genomic chicken DNA obtained in step (a.), and (c.) The methylation levels of these CpG sites in the genomic chicken DNA from the test samples were compared with the methylation levels of the same CpG sites from age-related reference samples. This determines the epigenetic age and predicts the actual age of the subject or the population to be tested; For this specific set of CpG sites in step (b) - Eliminating the effects of genetic polymorphisms by excluding CpG sites associated with single nucleotide polymorphisms, and - Eliminate the effects of sex-specific methylation differences on sex chromosomes by excluding all CpG sites located on sex chromosomes.

[0027] An age-related reference sample was used as a control and represents the average methylation level at a predetermined and specific full age.

[0028] The term "actual age" refers to the calendar time that has elapsed since birth / hatching.

[0029] The epigenetic age depends on the biological state or condition of an individual or population and takes into account the living environment (such as stress, nutrition, etc.). The terms "epigmoid age," "methylation age," and "biological age" have the same meaning and are used interchangeably in the context of this application.

[0030] In the context of this invention, the terms "CpG site," "clock CpG," or "CpG location" refer to the location of a potentially methylated CpG. Methylation typically occurs in CpG-containing nucleic acids. These CpG-containing nucleic acids can be located, for example, in CpG islands, CpG duplexes, promoters, introns, or exons or intergenic regions of genes. For example, a potential methylation site may include a promoter / enhancer region indicating a gene.

[0031] "A specific set of CpG sites in the Galliformes / chicken DNA genome" refers to the CpG sites that show the best correlation with age.

[0032] Preferably, in addition to the above, the methylation level of the specific CpG sites in step (b) is tissue-specific normalized.

[0033] In a preferred embodiment of the present invention, the Galliformes object or group to be tested belongs to the species Galliformes, and the specific CpG sites in this group include or are composed of the CpG sites shown in Table 1.

[0034] In another embodiment, the Galliformes object or population to be tested belongs to the species *Gnaphalium affine*, and the specific set of CpG loci includes or consists of the CpG loci shown in Table 2. In another embodiment, the Galliformes object or population to be tested belongs to the species *Gnaphalium affine*, and the specific set of CpG loci includes or consists of the CpG loci shown in Table 3.

[0035] The methods used to predict the actual age of Galliformes can be used to test individual animals and to test complete groups of animals, such as chickens or broiler / laying chicken groups.

[0036] Biological sample materials derived from the test subject or the test population may be selected from, for example, body fluids, excretory materials, tissue materials (such as muscle tissue, intestinal tissue, organ tissue, skin tissue), feather materials (such as quill pens), or combinations thereof. Excretory materials include intestinal contents, feces and cecal excrement, waste samples and mixtures thereof, solutions, or suspensions. Examples of muscle tissue are the pectoralis major muscle, examples of intestinal tissue are the ileum and jejunum; and examples of organ tissue are spleen tissue or heart tissue. The term "waste sample" refers to mixed feces containing bedding residue.

[0037] The biological sample derived from the subject or population to be tested is preferably feces. Fecal sample material can be collected before death. DNA material isolated from feces contains a large amount of intestinal cell DNA (mucosa).

[0038] In a particularly preferred embodiment, the biological sample from the test subject or test group is a pooled fecal sample material from a Galliformes group. The pooled fecal sample material is obtained by combining and mixing individual fecal samples.

[0039] The sample size (i.e., the number of excrement samples to be collected; each sample collected at a specific location in the livestock shed) must be determined based on the actual stocking density, i.e., the actual number of animals belonging to the population to be tested.

[0040] Typically, at least 80 to 100 individual excrement samples are sufficient for most poultry flocks. For example, for a broiler flock of 20,000 animals, 96 individual samples are required for a 95% confidence level.

[0041] Several sampling methods can be used to obtain a combined excrement sample. In one embodiment, a combined excrement sample is obtained through systematic grid sampling (systematic random sampling). For this method, the housing or area of ​​the poultry population is divided into uniform grid-like chambers or sub-regions according to the required number of individual excrement samples (i.e., sample size). Random sample collection sites are then identified within the first grid chamber, and a first sample is collected at said site. Finally, using the same relative positions within each chamber, additional samples are sequentially obtained from adjacent chambers—for example, in a serpentine, angular, or zigzag pattern. Random starting points can be obtained using dice or a random number generator. For duplicate samples, the above process can optionally be repeated.

[0042] Step (b.) of the in vitro method for determining the epigenetic age of Galliformes, particularly chickens, may include a DNA methylation analysis process, preferably bisulfite sequencing. In this process, cytosine residues in the genomic DNA are converted to uracil, while 5-methylcytosine residues in the genomic DNA are not converted to uracil.

[0043] Whole-genome bisulfite sequencing is a genome-wide analysis of DNA methylation based on sodium bisulfite conversion of genomic DNA, followed by sequencing on a next-generation sequencing platform. These sequences are then re-aligned with a reference genome to determine the methylation status of CpG dinucleotides based on mismatches caused by the conversion of unmethylated cytosine to uracil.

[0044] For example, methylation levels can be achieved using commercial Illumina. TM The platform is used for measurement.

[0045] To quantify methylation levels, various established schemes can be used to calculate the β value of methylation, which is equal to the fraction of methylated cytosine at a specific position.

[0046] Step (c) can be performed using mathematical algorithms, particularly statistical prediction methods.

[0047] The selection of the specific CpG sites for the clock in step (b) can be accomplished using penalized regression. In this case, the newly sequenced test sample is evaluated by assessing the methylation values ​​using an existing regression function for the clock. Accordingly, a trained regression function is preferably applied in step (c).

[0048] Preferably, the Galliformes species or population to be tested are broiler chickens with a lifespan of up to 63 days.

[0049] Determination of epigenetic age The epigenetic age is usually dependent on the biological state or condition of an individual (or population).

[0050] Epigenetic age may match or not match chronological age. The deviation between epigenetic age and chronological age is referred to as aging acceleration or aging deceleration.

[0051] Therefore, epigenetic age can also be determined by comparing the methylation level of methylation markers (i.e., CpG sites) in the genomic Galliformes DNA of the test sample with the methylation status of the same markers (i.e., CpG sites) from an age-related reference sample. The term "age-related reference sample" should be understood as defined above.

[0052] More specifically, the present invention provides an in vitro method for determining the epigenetic age of Galliformes, the method comprising the following steps: (a.) Obtain genomic DNA from biological sample materials derived from the Galliformes species to be tested or from the Galliformes population to be tested. (b.) Determine the methylation levels of a specific set of CpG sites in the Galliformes DNA genome obtained in step (a.), and (c.) The methylation levels of these CpG sites in the genomic Galliformes DNA from the test sample were compared with the methylation levels of the same CpG sites from an age-related reference sample. This determines the epigenetic age of the subject or the population to be tested; For this specific set of CpG sites in step (b) - Eliminating the effects of genetic polymorphisms by excluding CpG sites associated with single nucleotide polymorphisms, and - Eliminate the effects of sex-specific methylation differences on sex chromosomes by excluding all CpG sites located on sex chromosomes.

[0053] The method implemented according to the present invention is particularly applicable to chickens (Gallus gallus). Therefore, one specific embodiment of the present invention is an in vitro method for determining the epigenetic age of Galliformes, the method comprising the following steps: (a.) Obtain genomic DNA from biological sample materials derived from the chicken subject or the chicken population being tested. (b.) Determine the methylation levels of a specific set of CpG sites in the chicken genomic DNA obtained in step (a.), and (c.) The methylation levels of these CpG sites in the genomic chicken DNA from the test samples were compared with the methylation levels of the same CpG sites from age-related reference samples. This determines the epigenetic age of the subject or the population to be tested; For this specific set of CpG sites in step (b) - Eliminating the effects of genetic polymorphisms by excluding CpG sites associated with single nucleotide polymorphisms, and - Eliminate the effects of sex-specific methylation differences on sex chromosomes by excluding all CpG sites located on sex chromosomes.

[0054] Preferably, in addition to the above, the methylation level of the specific CpG sites in step (b) is tissue-specifically normalized.

[0055] In a preferred embodiment of the present invention, the Galliformes object or group to be tested belongs to the species Galliformes, and the specific CpG sites in this group include or are composed of the CpG sites shown in Table 1.

[0056] In another embodiment, the Galliformes object or population to be tested belongs to the species *Gnaphalium affine*, and a specific set of CpG loci includes or consists of the CpG loci shown in Table 2. In another embodiment, the Galliformes object or population to be tested belongs to the species *Gnaphalium affine*, and a specific set of CpG loci includes or consists of the CpG loci shown in Table 3.

[0057] The methods used to predict the actual age of Galliformes can be used to test individual animals and to test complete groups of animals, such as chickens or broiler / laying chicken groups.

[0058] The sample materials and sampling conditions are as described above. Preferably, the biological sample materials derived from the test subject or the test group are selected from body fluids, excrement materials, tissue materials such as muscle tissue, organ tissue such as intestinal tissue, skin tissue, feather materials, or combinations thereof.

[0059] Step (b.) of the in vitro method for determining the epigenetic age of Galliformes, particularly chickens, may include a DNA methylation analysis process, preferably bisulfite sequencing. Step (c) can be performed using mathematical algorithms, particularly statistical prediction methods. Defining the CpG of the clock, i.e., the selection of a specific set of CpG sites in step (b), can be accomplished using penalized regression. In this case, the newly sequenced test sample is evaluated by assessing the methylation value by applying an existing regression function of the clock. Accordingly, a trained regression function is preferably applied in step (c).

[0060] Preferably, the Galliformes species or population to be tested are broiler chickens with a lifespan of up to 63 days.

[0061] Assessment of inflammatory status The inventors have discovered that in Galliformes, particularly in chickens (Gallus gallus), the mismatch between epigenetic age and chronological age, especially the acceleration of epigenetic age (i.e., epigenetic age > chronological age), is an early indicator of inflammatory processes.

[0062] Therefore, the present invention also relates to an in vitro method for assessing the inflammatory state of Galliformes, the method comprising the following steps: (a.) Obtain genomic DNA from biological sample materials derived from the Galliformes species to be tested or from the Galliformes population to be tested. (b.) Determine the methylation levels of a specific set of CpG sites in the Galliformes DNA obtained in step (a.), and (c.) The methylation levels of these CpG sites in the genomic Galliformes DNA from the test samples were compared with the methylation levels of the same CpG sites from age-related reference samples. This determines the epigenetic age of the subject or the tested population, and (d.) Compare the epigenetic age of the subject or the population being tested obtained therefrom with their actual chronological age. An epigenetic age higher than the chronological age indicates inflammation. For this specific set of CpG sites in step (b) - The effects of genetic polymorphisms are eliminated by excluding CpG sites associated with single nucleotide polymorphisms, and - Eliminate the effects of sex-specific methylation differences on sex chromosomes by excluding all CpG sites located on sex chromosomes.

[0063] In one specific embodiment, the present invention relates to an in vitro method for assessing the inflammatory status of poultry (Gallus gallus), the method comprising the following steps: (a.) Obtain genomic DNA from biological sample materials derived from the chicken subject or the chicken population being tested. (b.) Determine the methylation levels of a specific set of CpG sites in the genomic chicken DNA obtained in step (a.), and (c.) The methylation levels of these CpG sites in the genomic chicken DNA from the test samples were compared with the methylation levels of the same CpG sites from age-related reference samples. This determines the epigenetic age of the subject or the tested population, and (d.) Compare the epigenetic age of the subject or the population being tested obtained therefrom with their actual chronological age. An epigenetic age higher than the chronological age indicates inflammation. For this specific set of CpG sites in step (b) - Eliminating the effects of genetic polymorphisms by excluding CpG sites associated with single nucleotide polymorphisms, and - Eliminate the effects of sex-specific methylation differences on sex chromosomes by excluding all CpG sites located on sex chromosomes.

[0064] Preferably, in addition to the above, the methylation level of the specific CpG sites in step (b) is tissue-specific normalized.

[0065] In a preferred embodiment of the present invention, the Galliformes object or group to be tested belongs to the species Galliformes, and the specific CpG sites in this group include or are composed of the CpG sites shown in Table 1.

[0066] In another embodiment, the Galliformes object or population to be tested belongs to the species *Gnaphalium affine*, and the specific set of CpG loci includes or consists of the CpG loci shown in Table 2. In another embodiment, the Galliformes object or population to be tested belongs to the species *Gnaphalium affine*, and the specific set of CpG loci includes or consists of the CpG loci shown in Table 3.

[0067] Biological sample materials derived from the test subject or the test population may be selected from, for example, body fluids, excretory materials, tissue materials (such as muscle tissue, intestinal tissue, organ tissue, skin tissue), feather materials (such as quill pens), or combinations thereof. Excretory materials include intestinal contents, feces and cecal excrement, waste samples and mixtures thereof, solutions, or suspensions. Examples of muscle tissue are the pectoralis major muscle, examples of intestinal tissue are the ileum and jejunum; and examples of organ tissue are spleen tissue or heart tissue. The term "waste sample" refers to mixed feces containing bedding residue.

[0068] The biological sample derived from the subject or population to be tested is preferably feces. Fecal sample material can be collected before death. DNA material isolated from feces contains a large amount of intestinal cell DNA (mucosa).

[0069] In a particularly preferred embodiment, the biological sample derived from the test subject or the test group is a pooled fecal sample material derived from a Galliformes group, such as a chicken population. The pooled fecal sample material is obtained by combining and mixing individual fecal samples.

[0070] The sample size (i.e., the number of excrement samples to be collected; each sample collected at a specific location in the livestock shed) must be determined based on the actual stocking density, i.e., the actual number of animals belonging to the population to be tested.

[0071] Typically, at least 80 to 100 individual excrement samples are sufficient for most poultry flocks. For example, for a broiler flock of 20,000 animals, 96 individual samples are required for a 95% confidence level.

[0072] Several sampling methods can be used to obtain a combined excrement sample. In one embodiment, a combined excrement sample is obtained through systematic grid sampling (systematic random sampling). For this method, the housing or area of ​​the poultry population is divided into uniform grid-like rooms or sub-areas based on the desired number of individual excrement samples (i.e., sample size). Random sample collection sites are then identified within the first grid room, and a first sample is collected at said site. Finally, using the same relative positions within each room, additional samples are sequentially obtained from adjacent rooms—for example, in a serpentine, angular, or zigzag pattern. Random starting points can be obtained using dice or a random number generator. For duplicate samples, the above process can optionally be repeated.

[0073] As an example of a broiler flock, excrement samples can be collected and analyzed daily during the initial growth phase (starting phase, days 5 to 10), and / or during the enhanced growth phase (days 11 to 18), and optionally also daily in the later stages. Alternatively, excrement sample material, particularly fecal sample material, from the broiler flock can be collected and analyzed daily starting from day 10.

[0074] Preferably, the target Galliformes species or population is a broiler chicken with a lifespan of up to 63 days.

[0075] The chicken's life cycle begins with the egg being taken from the hen in the hatchery, and then incubated at a constant temperature for 21 days until the hen hatches. Although precocious chickens may be as big as 72 hours at this stage, they are called day-old chickens. These chickens are separated by sex, with hens being raised for about a year to lay eggs.

[0076] Broiler chickens have a significantly shorter lifespan, ranging from 21 to 170 days. In the United States, broiler chickens are slaughtered at an average age of 47 days, with a slaughter weight of 2.6 kg, while in Europe, the average slaughter age is 42 days (with a weight of 2.5 kg).

[0077] Broilers are typically housed in flocks in a coop that can hold more than 20,000 chickens, fed up to three different feed types (chick feed, mid-season feed, and finishing feed) throughout the production cycle. These feed types are tailored to specific production stages: the initial growth stage (starting stage, days 5 to 10), the growth enhancement stage (starting around day 11), and the finishing stage. Feeding protocols also influence methylation levels. Therefore, unoptimized feed can also lead to accelerated aging (epigenetic age > chronological age).

[0078] In addition, birds are typically exposed to a variety of external environmental factors, such as bacteria, viruses, parasites, diet, or climate. These factors affect the outcome of the production cycle in terms of group performance or group coherence, and are manifested in different methylation patterns in individuals or groups, which can lead to detectable aging acceleration.

[0079] Step b), determining the methylation level of a specific set of CpG (cytosine-phosphate-guanine) sites (“clock CpGs”) in the Galliformes genome or chicken DNA, may include a DNA methylation analysis process, preferably bisulfite sequencing. Step (c) can be performed using mathematical algorithms, particularly statistical prediction methods.

[0080] The CpGs of the clock, i.e., the selection of a specific set of CpG sites in step (b), can be accomplished using penalized regression. In this case, the methylation values ​​of the newly sequenced test samples are evaluated by applying an existing regression function of the clock. Accordingly, a trained regression function is preferably applied in step (c).

[0081] As shown above, epigenetic age is associated with the health status of Galliformes poultry, and particularly with inflammatory conditions. Therefore, based on the method described according to the invention, the necessity of treatment or nutritional intervention can be assessed.

[0082] Such interventions may include providing individualized (customized) treatments to the individuals or groups being tested to make the predicted epigenetic age closer to the actual age of the individuals or groups.

[0083] Furthermore, such treatment or intervention may include feeding or administering health-promoting substances, such as animal feed additives or therapeutic agents. The term "administration" or related terms include oral administration. Oral administration can be performed via drinking water, oral gavage, aerosol spraying, or animal feed. The term "animal feed additive" refers to any additive used to beneficially influence the good health of animals or to beneficially influence the environment. Examples of animal feed additives are digestibility enhancers, i.e., substances that improve dietary digestibility when fed to animals by acting on target feed materials; gut microbiota stabilizers; microorganisms or other chemically defined substances that, when fed to animals, have a positive effect on the gut microbiota; or substances that have a beneficial effect on the environment. Preferably, the health-promoting substance is selected from probiotics, prebiotic agents, phytothenic agents, organic / fatty acids, bacteriophages, and lysozymes, or any combination thereof.

[0084] In addition to the foregoing, this invention also relates to the use of the methods disclosed herein in the development of conventional analytical tools, such as real-time PCR, targeted sequencing / panel sequencing, immunoprecipitation of methylated DNA as input to both, chip / array technology, or methylated DNA sequencing.

[0085] The application of the method according to the invention is, for example, (i) to help assess the health status of Galliformes (e.g., chickens); (ii) to monitor the progression or recurrence of clinical and subclinical diseases; or (iii) to study the effects of drugs, feed compounds and / or special diets on biological age - and thus on the health status of Galliformes (e.g., chickens).

[0086] The application of the method described in this invention is particularly helpful in avoiding losses in animal performance such as weight gain and feed conversion ratio.

[0087] Example method sample Animals were stratified into four tissues (pectoralis major, ileum, spleen, and jejunum) and three age groups (3d, 15d, and 34d), and 14d, 16d, and 35d in the case of the jejunum. From each of these 12 groups, DNA was prepared from three independent animals, resulting in 36 genomic DNA samples.

[0088] Whole-genome bisulfite sequencing Whole-genome bisulfite sequencing libraries were prepared using the Swift Biosciences Accel-NGS Methyl-Seq DNA Library Kit. Two sequencing libraries were encoded into a single sequencing lane. Sequencing was performed on the Illumina HiSeq X platform using a standard paired-end sequencing protocol with 105 nucleotide reads.

[0089] Segment Mapping Reads were trimmed and mapped using BSMAP 2.5 (Xi Y, Li W. 2009. BSMAP: wholegenome bisulfite sequence MAPping program. BMC Bioinformatics 10:232. doi:10.1186 / 1471-2105-10-232.), using the Gallus gallus genome assembly version 5.0 as the reference sequence. Picard was used to remove duplicates. The methylation ratio was determined using the Python script (methratio.py) distributed with the BSMAP package, by dividing the number of reads with methylated CpG at a given genomic location by the number of reads covering that location.

[0090] Standardization and SNP filtering of methylation data All CpGs listed as Gallus gallus genomic SNPs in the dbSNP database were screened. All CpGs and LMRs mapped to Galliformes sex chromosomes W and Z were screened and removed from the dataset. For the whole-genome clock, analysis was limited to CpGs showing chain-specific coverage greater than 10 in each sequencing sample, resulting in a set of 257,913 CpGs. The data were then normalized by calculating the average methylation value of each CpG across all samples from the same tissue and subtracting that value from the CpG's methylation value. For the LMR clock, the analysis was limited to CpGs in hypomethylated regions that showed chain-specific coverage greater than 5 in each sequencing sample, resulting in a set of 67,651 LMRs. The average methylation value of these LMRs was calculated and normalized by calculating the average of each LMR across all samples from the same tissue and subtracting that value from the LMR's value.

[0091] Determination of the methylation clock in chicken DNA Then, a penalized regression model (implemented in the R package) is applied to regress the animal's chronological age to the normalized methylation value of the CpG probe. In the case of the LMR clock, a penalized regression model is applied to regress the animal's chronological age to the normalized mean methylation value of the LMR.

[0092] result Whole genome clock The α parameter of glmnet was varied between 0 and 1, and 0.7 was chosen (for elastic network regression) because this value resulted in a fit close to the best fit and a controllable number of CPGs. A λ value of 0.4016 was chosen for cross-validation on the training data. This determined a set of 45 CpGs and corresponding β values, which defined the weights of these CpGs used in the chicken methylation clock. The mean squared error of 6-fold cross-validation with an α value of 0.7 and a λ value of 0.4016 was 11.538. This indicates that the clock can be predicted for new samples with an error of approximately 3.4 days. To apply the clock to new samples, the methylation ratio of that sample at the 45 clock CpGs must be provided, and the `predict.cv` command of the glmnet package must be executed with the trained clock.

[0093] Figure 1 The mean square error of the training clock that results in the minimum error for a given α value at the λ value is shown.

[0094] Figure 2 The figure shows the number of CpG values ​​that result in the minimum error at a given α value and λ value.

[0095] Table 1: Clock CpGs (genome-wide methylation, α=0.7, λ=0.4016, #CpGs: 45).

[0096] 1 Correction coefficients for different organizations. The corresponding values ​​must be subtracted.

[0097] LMR clock Example 1: The α parameter of glmnet was varied between 0 and 1 and 0.84 was chosen (for elastic network regression) because this value leads to a fit close to the best fit and a controllable amount of LMR. A λ value of 0.3194 was chosen for cross-validation on the training data. This determined a set of 39 LMRs and corresponding β values, which define the weights of these LMRs used in the chicken methylation clock. The mean squared error of 6-fold cross-validation with an α value of 0.84 and a λ value of 0.3194 was 13.4831. This indicates that the clock can be predicted for new samples with an error of approximately 3.7 days. To apply the clock to new samples, the methylation ratio of that sample at the 39 clock LMRs must be provided, and the `predict.cv` command of the glmnet package must be executed with the trained clock.

[0098] Figure 3 The diagram shows the mean squared error of the training clock that results in the minimum error for a given α value at the λ value. Figure 4 This shows the number of LMRs that result in the minimum error for a given α value at the λ value.

[0099] Table 2: Clock CpG (LMR methylation, α=0.84, λ=0.3194, #LMR's: 39).

[0100] 1 Correction coefficients for different organizations. The corresponding values ​​must be subtracted.

[0101] Example 2: The α value varied between 0 and 1 and was chosen to be 0.9 (elastic network regression). This determined a set of 32 LMRs and their corresponding β values, which defined the weights of these LMRs used in the chicken methylation clock (Table 3).

[0102] Table 3. Clock LMR (α=0.9, λ=0.3147).

[0103] Correction factors are specified for different organizations. To make corrections, the corresponding values ​​must be subtracted.

[0104] Figure 5 The diagram shows the root mean square error of the training clock that results in the minimum error for a given α value at the λ value.

[0105] Figure 6 This shows the number of LMRs that result in the minimum error for a given α value at the λ value.

[0106] Age prediction in pectoralis major tissue from completely independent validation datasets: To validate the LMR clock, whole-genome bisulfite sequencing was performed on six samples (pectoralis major muscle) from two age groups (day 14 and day 28) in completely independent animal experiments. The root mean square errors of age prediction were 2.7 days and 3.8 days, respectively, consistent with the prediction errors obtained after cross-validation. Results are as follows... Figure 7 As shown.

[0107] Accelerated aging as a biomarker of inflammatory processes Chickens were injected with CpG or control GpC on the second day after hatching and on days 13-16, 27-30, and 34-35. Jejunal tissue was collected, and genomic DNA was isolated from samples taken on days 14, 16, and 35, and whole-genome bisulfite sequencing was performed using standard protocols.

[0108] Analysis of jejunal samples showed a clear and highly consistent acceleration of age, particularly on days 14 and 16. Figure 8 The control group received an injection of the non-inflammatory agent GpC and showed no response.

Claims

1. Used to predict the health of Galliformes ( Galliformes An in vitro method for determining age, the method comprising the following steps: (a.) Obtain genomic DNA from pectoral muscle material derived from either the Galliformes species being tested or from the Galliformes population being tested. (b.) Determine the methylation levels of a specific set of CpG sites in the Galliformes DNA obtained in step (a.), and (c.) The methylation levels of these CpG sites in the genomic Galliformes DNA from the test samples were compared with the methylation levels of the same CpG sites from age-related reference samples. This determines the epigenetic age and predicts the actual age of the subject or the population to be tested; The method is characterized in that, for the specific set of CpG sites in step (b) - Eliminating the effects of genetic polymorphisms by excluding CpG sites associated with single nucleotide polymorphisms, and - Eliminate the effect of sex-specific methylation differences on sex chromosomes by excluding all CpG sites located on sex chromosomes. The Galliformes objects or groups to be tested belong to the species Galliformes, and the specific CpG sites in this group consist of the CpG sites shown in Table 2.

2. The method according to claim 1, characterized in that... The methylation levels of this set of specific CpG sites in step (b) are tissue-specific normalized.

3. The method according to claim 1 or claim 2, characterized in that... Step (b.) includes a DNA methylation analysis procedure.

4. The method according to claim 3, wherein the DNA methylation analysis process is bisulfite sequencing.

5. The method according to claim 1 or claim 2, characterized in that... The target species or population of Galliformes is a broiler chicken with a lifespan of up to 63 days.

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