Identifying breeding conditions for livestock using epigenetics

Abstract

The present invention relates to a method of authenticating a test animal-derived product sample, the method comprising the steps of: (a) determining a test methylation profile of one or more preselected methylation sites within a genomic material obtained from the test animal-derived product sample; and (b) comparing the test methylation profile obtained from (a) to a reference methylation profile obtained from a control animal having the same biological classification unit as the test animal from which the product sample is derived, wherein the control animal is cultivated under a known special type of animal husbandry; wherein a significant similarity of the test methylation profile of (a) to the reference methylation profile from the control animal indicates that the test animal has been cultivated under the same special type of animal husbandry as the control animal, and the test animal-derived product is so verified; wherein the difference in the test methylation profile of (a) compared to the reference methylation profile of the control animal indicates that the test animal has been cultivated in another special type of animal husbandry compared to the control animal; and wherein the preselected methylation site is a CpG site selected from gene or genomic DNA regions from control and test animals exhibiting the highest degree of methylation variation during method training; and wherein the test methylation profile has a significant similarity to the reference methylation profile when the test methylation profile overlaps the reference methylation profile defined by a plurality of training samples using principal component analysis and / or multi-dimensional scaling analysis; and wherein the methylation profile is determined using at least one method selected from the group consisting of PCR, methylation-specific PCR, real-time methylation-specific PCR, PCR assays using methylated DNA-specific binding proteins, quantitative PCR, DNA chip-based assays, pyrosequencing, bisulfate pyrosequencing, methylated DNA immunoprecipitation sequencing, and combinations thereof; and wherein the test animal is selected from livestock or poultry.

Description

Field of the Invention

[0001] The present invention relates to a method for determining conditions for culturing a product sample derived from an unknown animal based on a specific set of CpG sites, which provides a source for generating a DNA methylation profile specific to particular culturing conditions. In particular, DNA methylation profiling can be used to determine particular culturing conditions for animal-derived products. Particular culturing conditions can include organic, conventional culturing, etc., and can vary based on country and animal type. Background of the Invention There are significant variations in the environmental rearing conditions of agriculture-related animals. The conditions under which animals are reared can also affect the outcome of meat characteristics and the value of the meat when it is consistent with its value to the customer. For example, some consumers prefer organically reared chicken because they believe that the rearing standards used for organic production improve the welfare of the poultry. Another example is that consumers are concerned about whether the fish they purchase are farmed or wild-caught and also concerns related to farmed fish to ensure reduced escapees that can disrupt wild populations.

[0003] Currently, evaluating meat products to classify them based on the rearing environment is done via inspection, which may or may not be supported by artificial intelligence and / or blockchain technology. However, most evaluations still rely on data recording. In some cases, genetic testing can be utilized if the strains and thus the genetic backgrounds of the animals used in different rearing categories are different. For example, a slow-growing strain of broiler chickens is different from the ultra-high growth efficiency strains used in conventional production.

[0004] However, genetic testing of an animal will not be sufficient to evaluate whether a particular strain of animal was indeed reared in an environment classified as conventional, organic, farmed, or wild-caught. For example, if a slow-growing strain of broiler chickens is reared without reduced stocking density, access to outdoor activities, enrichment, or non-GMO feed, then genetic testing will not be able to determine this. Similarly, if a farmed fish strain escapes and is later caught by commercial fishing, then a genetic test alone will misclassify those fish. Therefore, there is still a need for a scientific evaluation of meat products that ensures proper classification based on environmental rearing conditions.

[0005] Epigenetics is the study of heritable traits caused by mechanisms other than changes in the underlying DNA sequence. In other words, epigenetic marks "orchestrate" our genes. Epigenetic marks can be chemical marks (such as methylation), protein-based marks (such as histones), or a combination of both. During development and cell differentiation, DNA methylation is dynamic, but some DNA methylation patterns can be maintained, accumulated, and / or inherited to the next generation as a form of epigenetic memory. Those changes may be responsible for heritable changes in gene activity, as DNA methylation events have been shown to be regulatory mechanisms related to gene silencing, expression, chromatin remodeling, or imprinting. Epigenetics is attractive for animal breeding because it can identify the causal relationships and heritability of complex traits and diseases. DNA methylation patterns are modified during an individual's life by environmental forces such as diet, stress, drugs, or pollution, among others. Some environments are more likely to increase certain methylation patterns, and these patterns can contribute to epigenetic and / or phenotypic variation among individuals.

[0006] Recent studies have shown that the DNA methylation patterns of animals contain important information about their rearing conditions. For example, comparison of the genome-wide methylation and variation patterns at the DNA level has revealed that a substantial proportion of epigenetic variation can be related to fitness and environmental differences, such as the captivity of salmon (Le Luyer J et al. 2017 PNAS vol114, no 49).

[0007] Koop et al. and Rhein et al. have also respectively confirmed that methylation patterns are maintained postmortem and are consistent regardless of the level of decomposition of the sample. One of the only aspects that still affects the methylation pattern of a sample will be the DNA integrity and the amount of DNA found in the sample (Koop et al., 2021 International Journal of Legal Medicine 135:167 - 173 and Rhein et al., 2015 Frontiers in Genetics 6:182).

[0008] In view of the above, there is still an urgent need to provide methods that may use epigenetics to identify and control markers of animal-derived products from animals reared under different conditions, particularly food and more particularly animal materials, which are derived to meet the living standards and dietary choices of consumers. Brief Description of the Drawings Figure 1 Principal component analysis (PCA) of CpG sites with a minimum coverage of 10 in all samples: 6458063 CpG sites Figure 2Principal component analysis (PCA) of the 201,246 identified differentially methylated positions (DMPs).

[0010] Description of the Invention The present invention attempts to solve the above problems by providing a method that uses DNA methylation patterns to distinguish one type of animal-derived product from another type of animal-derived product and is capable of determining the method and environment in which the animal from which the animal-derived product is derived was raised, and thereby accurately and reliably authenticate the product. The present invention is based on the discovery that the method and environment of raising can permanently alter the genome of an animal epigenetically. In particular, the ability to adapt to the environment and maintain an adapted biological pattern depends on epigenetic mechanisms, including DNA methylation. In particular, the present invention is based on the discovery that the method and environment of raising animals for food consumption can also lead to changes in the epigenetic mechanisms of the animals, including DNA methylation patterns, and that these patterns can be passed on to different products that can be derived from the animals.

[0011] The inventors have unexpectedly found that this property can be utilized to identify an "epigenetic fingerprint" on the genome that is specific to the method and environment of raising not just one animal but potentially all animals that have undergone the same method and environment of raising. Based on these findings, the present invention provides a method for identifying the specific environment or breeding conditions that an animal has experienced, particularly the raised animal from which an animal-derived product is derived, also known as livestock and poultry. In particular, the method according to any aspect of the present invention can be used to determine whether an animal-derived product is derived from an animal that has been raised under special conditions. More particularly, the method according to any aspect of the present invention can be used to determine whether an animal-derived product is derived from an animal that has been bred under special livestock farming conditions. In this way, the method according to any aspect of the present invention can then be used to accurately and reliably determine the specific livestock farming conditions under which the animal was raised, and then provide and / or confirm the authentication of any sample from the animal. Further, the method according to any aspect of the present invention can also be used to identify whether any animal-derived product has been accurately and reliably authenticated, particularly in cases where the authentication is based on the breeding conditions of the animal.

[0012] According to one aspect of the present invention, there is provided a method for authenticating a sample of an animal-derived product of a test animal, the method comprising the steps of: (a) determining a test methylation profile of one or more preselected methylation sites within genomic material obtained from the sample of the animal-derived product of the test animal; and (b) Compare the test methylation profile obtained in (a) with a reference methylation profile obtained from a control animal having the same taxonomic unit as the test animal from which the product sample is derived, wherein the control animal is bred under a known specific type of animal husbandry, and a significant similarity of the test methylation profile in (a) compared to the reference methylation profile from the control animal indicates that the test animal has been bred under the same specific type of animal husbandry as the control animal, and the product derived from the test animal is so certified; and wherein a difference of the test methylation profile in (a) compared to the reference methylation profile of the control animal indicates that the test animal has been bred under another specific type of animal husbandry compared to the control animal; wherein the preselected methylation sites are CpG sites selected from genes or genomic DNA regions of control and test animals that showed the highest degree of methylation variation during method training; and wherein the test methylation profile has a significant similarity to the reference methylation profile when the test methylation profile overlaps with the reference methylation profile defined by a plurality of training samples using principal component analysis and / or multi-dimensional scaling; and wherein the methylation profile is determined using at least one method selected from PCR, methylation-specific PCR, real-time methylation-specific PCR, PCR assay using methylation-DNA-specific binding proteins, quantitative PCR, DNA chip-based assay, pyrosequencing, bisulfite pyrosequencing, methylated DNA immunoprecipitation sequencing, and combinations thereof; and wherein the test animal is selected from livestock or poultry.

[0013] In particular, in step (b) of the method according to any aspect of the present invention, the test methylation profile determined in (a) is compared with a set of predefined reference methylation profiles of the same taxonomic unit as the test animal from which the product sample is derived, wherein each predefined reference methylation profile is specific for a control animal that has been bred under a known specific type of animal husbandry, and if the test methylation profile is significantly similar to one of the predefined reference methylation profiles, the product sample derived from the test animal has the same specific certification as the control animal from which the predefined reference methylation profile was obtained, and the product derived from the test animal is so certified.

[0014] According to another aspect of the present invention, there is provided a method for certifying a product sample derived from a test animal, the method comprising the following steps: (a) Determine a test methylation profile of one or more preselected methylation sites within genomic material obtained from a product sample derived from the test animal; and (b) Compare the test methylation profile determined in (a) with a set of predetermined reference methylation profiles of the same taxonomic unit of the test animal from which the product sample is derived, wherein each predetermined reference methylation profile is specific for a control animal that has been bred under a known particular type of animal husbandry, wherein if the test methylation profile is significantly similar to one of the predetermined reference methylation profiles, the product sample derived from the test animal has the same particular verification as the control animal from which the predetermined reference methylation profile was obtained, and the product derived from the test animal is so verified; and wherein the test animal is selected from livestock or poultry.

[0015] According to yet another aspect of the present invention, there is provided a method for verifying a product sample derived from a test animal, the method comprising the steps of: (a) Determine the methylation status of at least one CpG site within genomic material obtained from the product sample derived from the animal, (b) Compare the methylation status of the CpG site from (a) with that of a control animal of the same taxonomic unit as the test animal from which the product sample is derived, wherein the control animal has been bred under a known particular type of animal husbandry, wherein a significant similarity of the test methylation status in (a) of the test animal as compared to the CpG site in the control animal indicates that the test animal has been bred under the same particular type of animal husbandry as the control animal, and the product derived from the test animal is so verified; and wherein a difference of the test methylation status in (a) as compared to the CpG site in the control animal indicates that the test animal has been bred under a different particular type of animal husbandry as compared to the control animal; and wherein the test animal is selected from livestock or poultry.

[0016] As used herein, the term 'animal-derived product' refers to a product derived from an animal. In particular, the term 'test animal-derived product' refers to a sample or subject that is to be taken into consideration for introduction into an array in accordance with any aspect of the present invention. These products derived from animals can include meat and meat products, as well as fats, meats, bloods, processed meats and lesser-known products such as fish gelatin and crude rennet, poultry products (meat and eggs), dairy products (milk and cheese), and non-food products such as fibers (wool, mohair, cashmere, leather, etc.). Animal-derived products can also include products that can be prepared using animal products (e.g., fats), such as soaps, creams, etc. In one example, the animal-derived product is meat, eggs, blood, brain, sperm, milk and any other tissue or sample that provides genomic DNA. In particular, the animal-derived product is meat. In one example, the animal-derived product sample can be a single type of meat, different types of meat, a single part of one type of meat, different parts of a single type of meat or different parts of different types of meat. If the animal is an aquatic animal, then these products derived from the animal can include meat and meat products, as well as eggs, fats, meats, bloods, processed meats and lesser-known products, and non-food products such as fibers (shells, scales, etc.). Animal-derived products can also include products that are prepared using animal products (e.g., fish oil), such as tablets, powders, etc. In one example, the animal-derived product is meat, eggs, blood, brain, shell, scale, skin, tissue, abdominal muscle tissue or any other tissue or sample that provides genomic DNA. In particular, the animal-derived product is meat, skin, blood, trimmings or any organ from an aquatic animal. In particular, the trimmings can be used as a by-product for fish meal / fish oil, which ultimately goes into the animal feed industry or pets. The sample can be from any biological entity that has a DNA genome and DNA genome methylation. In particular, the methylation site is a CpG site.

[0017] The term 'certification' refers to a certificate or proof given by a designated certification body, which guarantees the quality of products derived from specific animals, including food for human consumption and / or use. The term 'quality certification' is interchangeable with the term 'certification'. These certifications usually appear on the packaging of animal-derived products (including food for consumption) and are printed by the product manufacturer. Examples of special food quality certifications can include 'breeding method (Haltungsform)', 'animal welfare (Tierwohl)', 'non-genetically engineered (Ohne Gentechnik)', 'halal', 'kosher', 'organic', 'free-range', 'pasture-raised', 'grass-fed', 'grain-fed','vegetarian', 'raised without hormones', etc. There are also different certifications based on the country. For example, like the breeding method in Germany, other certifications include Red Tractor (UK), Label Rouge (France), USDA grade (USA), etc., as well as other safety labels that confirm that the products sold have been prepared in accordance with specific religious or safety regulations. Specifically, the term 'certification' in this article refers to a certificate or proof given by a designated certification body, which guarantees the animal origin, quality, and / or the breeding method or animal husbandry technology experienced by the specific products derived from the animals for human consumption or use. According to any aspect of the present invention, the 'certification' of a'special type of animal husbandry' refers to a special and industrially acceptable method of breeding animals.

[0018] In one example, the special certification or certification of sample X can be based on the type of animal husbandry in which the test animals are raised. In Germany, this is called 'breeding method'. There are at least four types / conditions in which animals can be raised. These four levels of animal husbandry include stable housing (Stallhaltung), stable housing plus (StallhaltungPlus), outside climate (Auβenklima), and premium, which are also known as breeding methods 1, 2, 3, and 4 respectively. Animal products derived from animals raised under different animal husbandry conditions can result in different DNA methylation profiles. The special type of animal husbandry can vary depending on the country in which the method according to any aspect of the present invention is implemented. Regardless of the different terms used in different countries to describe different special animal husbandry practices, the general concept of the method according to any aspect of the present invention is the same and applicable in any of these countries.

[0019] For example, in Germany, different special types of livestock husbandry techniques for domestic and poultry animals can be referred to as 'keeping methods', and as mentioned above, are officially and industrially accepted as being divided into at least four types / conditions of animals that can be kept. These four levels of livestock husbandry include cage / barn keeping (Stallhaltung), upgraded cage / barn keeping (StallhaltungPlus), outdoor climate (Auβenklima), and premium. Similarly, in France, domestic and poultry animals may be labeled with 'French Red Label', 'organic', or other pictograms that show the keeping methods the animals have undergone before obtaining products derived from the animals. In the UK for domestic and poultry animals, there is the Red Tractor Food Assurance accreditation scheme, which includes at least three levels of livestock husbandry, including Certified Standards, Enhanced Welfare, and Free Range. Other existing labels in the UK include RSPCA Assured, which accredits specific animal welfare standards, and several organic meat accreditation schemes, such as the Organic Farmers and Growers Certification and the Soil Association Organic Standard. Examples of meat accreditation in the United States (USA) include those provided by the United States Department of Agriculture (USDA), which as examples include Carcass Quality and organic accreditation. The USDA has also approved some third-party accreditation schemes, such as those provided by the non-profit A Greener World, which includes Certified Animal Welfare Approved that defines the accreditation of livestock husbandry related to animal welfare and Certified Grassfed that defines the accreditation of specific feed types in livestock husbandry. In particular, the special type of livestock husbandry can be selected from: - Certified Standards, cage / barn keeping (Stallhaltung), or its equivalent; - Enhanced Welfare, upgraded cage / barn keeping (StallhaltungPlus), or its equivalent; - Free Range, outdoor climate (Auβenklima), premium, or its equivalent; and - organic.

[0020] The term 'correspondence' as used herein refers to different terms used in different countries for the same or similar special type of animal husbandry. For example, the conditions under which animals are raised in the UK under a special type of animal husbandry according to 'certified standards' may be the same or at least quite similar to the conditions under which animals are raised in Germany according to cage / shed confinement (Stallhaltung). The same conditions of the special type of animal husbandry may exist in another country under different terms. 'Correspondence' thus refers to the same or quite similar breeding conditions practiced in different countries under different names or certifications.

[0021] The term "test" used herein with the term subject and / or animal refers to an entity subjected to the method according to any aspect of the present invention and is the basis for the analytical application of the present invention. Thus, a "(individual) test subject", "(individual) group of test subjects" or "test profile" or "test animal-derived product" is a profile obtained or generated in the context of a (individual) subject or group of subjects tested according to the present invention. In contrast, the term "reference" shall mean an entity, mostly predetermined, for comparison with a test entity. For example, the term 'reference animal' refers to an animal used for comparison or as a control for a 'test animal'. Similarly, the term 'sample' and / or 'test animal-derived product sample' used according to any aspect of the present invention refers to an entity that can be subjected to the method of the present invention. In particular, a sample can be any (test) animal-derived product that can be subjected to the method of the present invention to determine a specific assay by first determining a DNA methylation profile, and then comparing the test methylation profile to a control. Blockchain can also be used to make information more accessible to consumers.

[0022] The method according to any aspect of the invention can be used to identify unknown samples (i.e. animal-derived product samples) based on DNA methylation patterns. These DNA methylation patterns can then be compared with reference DNA methylation patterns to trace the animal-derived product samples back to the slaughterhouse or farmhouse from which they were produced, and then determine whether the unknown sample corresponds to an animal that has been bred according to any of the animal husbandry techniques under consideration. In this way, a purchaser or consumer of meat can verify that meat that is sold or marketed as organic or non-organic, for example, is indeed what it claims to be.

[0023] Livestock according to any aspect of the present invention include terrestrial livestock and aquatic livestock. In particular, livestock can be a farmed animal selected from terrestrial and aquatic livestock or poultry. In particular, terrestrial livestock can include cattle, sheep, pigs, goats, horses, camels, donkeys, mules, rabbits, etc., and poultry can include chickens, turkeys and other quail-like birds, ducks, geese, quails, etc. As used herein, the term 'livestock' can also include poultry and refers to any farm animal or animal that can be used for agriculture.

[0024] As used herein, the term "aquatic livestock" refers to any organism that is raised entirely in water or lives primarily in water, especially as compared to terrestrial animals. These aquatic livestock may live in different water forms, such as the sea, ocean, river, lake, pond, etc. More particularly, the aquatic livestock according to any aspect of the present invention can be any fish, cephalopod, aquatic mollusk or aquatic crustacean at all life stages, including eggs, sperm and gametes. Even more particularly, 'aquatic animals' means animals of the following species: (i) fish belonging to the superclass Agnatha and the classes Chondrichthyes, Sarcopterygii and Actinopterygii; (ii) aquatic mollusks belonging to the phylum Mollusca; and (iii) aquatic crustaceans belonging to the subphylum Crustacea. Even more particularly, the aquatic livestock according to any aspect of the present invention can be aquatic livestock for aquaculture. Some non-limiting examples of aquatic animals according to any aspect of the present invention include lungfish, carp, catfish, halibut, marbled crayfish, marine and brackish fishes, sea shrimp, mitten crabs, shellfish, oysters, pangasius, rainbow trout, salmonids, scallops, black bass, sea bream, soft-shell crabs, turtles, tiger prawns, tilapia, turbot, white-leg prawn, shrimp, octopus, squid and other decapod crustaceans, bivalves and gastropods.

[0025] As used herein, the term "comprising" shall be interpreted to cover both "including" and "consisting of", both of which meanings are expressly intended, and thus the individual disclosed aspects of the present invention. When used herein, "and / or" shall be regarded as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" shall be regarded as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually recited herein. In the context of the present invention, the terms "about" and "approximately" denote the range of accuracy that a person skilled in the art will understand still ensures the technical effect of the feature under consideration. The terms generally denote a deviation of ±20%, ±15%, ±10%, and for example ±5% from the indicated value. As will be understood by a person of ordinary skill in the art, the specific deviation of a value for a given technical effect will depend on the nature of the technical effect. For example, natural or biological technical effects generally have a greater such deviation than artificial or engineering technical effects. In instances where an indefinite or definite article is used when referring to a singular noun, e.g., "a", "an", or "the", this includes the plural form of the said noun unless expressly stated otherwise.

[0026] In the context of the present invention, the terms "methylation profile", "methylation pattern", "methylation status", or "methylation state" are used herein to describe the condition, situation, or circumstances of genomic sequence methylation, and such terms refer to the characteristics of a DNA segment at a specific genomic locus related to methylation. Such characteristics include, but are not limited to, whether any cytosine (C) residue in the DNA sequence is methylated, the position of one or more methylated C residues, the percentage of methylated C at any specific residue segment, and allelic differences in methylation attributable to, for example, differences in allelic origin.

[0027] The term "methylation state" refers to the state of a specific methylation site (i.e., methylated versus non-methylated), which means that the residue or methylation site is methylated or not methylated. Then, based on the methylation state of one or more methylation sites, a methylation profile can be determined. Thus, the term "methylation profile" or also "methylation pattern" refers to the relative or absolute concentration of methylated C residues or non-methylated C residues at any specific residue sequence segment in the genomic material of a biological sample. For example, if one or more cytosine (C) residues that are generally not methylated in a DNA sequence are methylated, it can be called "hypermethylated"; while if one or more cytosine (C) residues that are generally methylated in a DNA sequence are not methylated, it can be called "hypomethylated". Similarly, if one or more cytosine (C) residues in a DNA sequence (e.g., the DNA of a sample nucleic acid from a test subject) are methylated compared to another sequence from a different region or different individual (e.g., relative to a normal nucleic acid or a standard nucleic acid of a reference sequence), the said sequence is considered hypermethylated compared to the other sequence. On the other hand, if one or more cytosine (C) residues in a DNA sequence are not methylated compared to another sequence from a different region or different individual, the said sequence is considered hypomethylated compared to the other sequence. These sequences are called "differentially methylated". The measurement of the differential methylation level can be carried out in a variety of ways known to those skilled in the art. As a non-limiting example, one method is to measure the methylation level of individual queried CpG sites determined by bisulfite sequencing method.

[0028] The term "hypermethylation" refers to the average methylation state corresponding to the presence of an increase in 5-mCyt at one or more CpG dinucleotides in the DNA sequence of a test DNA sample, relative to the amount of 5-mCyt found at the corresponding CpG dinucleotides in a normal control DNA sample. In particular, the control refers to an animal-derived product obtained from a control animal that has been bred according to a known specific type of animal husbandry or breeding method. The known specific type of animal husbandry refers to a breeding method implemented on the control animal, where the breeding method is known and confirmed before obtaining the animal-derived product from the control animal.

[0029] The term "hypomethylation" refers to the average methylation state corresponding to the presence of a decrease in 5-mCyt at one or more CpG dinucleotides in the DNA sequence of a test DNA sample, relative to the amount of 5-mCyt found at the corresponding CpG dinucleotides in a normal control DNA sample. In particular, the control refers to an animal-derived product obtained from a control animal that has been bred according to a known specific type of animal husbandry or breeding method. The known specific type of animal husbandry refers to a breeding method implemented on the control animal, where the breeding method is known and confirmed before obtaining the animal-derived product from the control animal.

[0030] As used herein, "methylated nucleotide" or "methylated nucleobase" refers to the presence of a methyl moiety on a nucleobase, where the methyl moiety is not typically present in the recognized canonical nucleobases. For example, cytosine in its normal form does not contain a methyl moiety on the pyrimidine ring, but 5-methylcytosine contains a methyl moiety at position 5 of its pyrimidine ring. Thus, cytosine in its normal form may not be considered a methylated nucleotide, while 5-methylcytosine may be considered a methylated nucleotide. In another example, thymine may contain a methyl moiety at position 5 of its pyrimidine ring; however, for the purposes of this disclosure, when thymine is present in DNA, it may not be considered a methylated nucleotide. The canonical nucleobases of DNA are thymine, adenine, cytosine, and guanine. The canonical bases of RNA are uracil, adenine, cytosine, and guanine. Accordingly, a "methylation site" is a position within a nucleic acid region of a target gene where methylation has the potential to occur. For example, a position containing CpG is a methylation site, where the cytosine may or may not be methylated. In particular, the term "methylated nucleotide" refers to a nucleotide carrying a methyl group that is attached to a position of the nucleotide that is susceptible to methylation. These methylated nucleotides typically occur in nature and, to date, have predominantly occurred in the context of the dinucleotide CpG, but methylated cytosines in the context of CpNpG- and CpNpN-sequences may also be considered the most common. In principle, other naturally occurring nucleotides may also be methylated, but they will not be considered for any aspect of the present invention.

[0031] As used herein, a "CpG site" or "methylation site" is a nucleotide within a nucleic acid (DNA or RNA) that is susceptible to methylation by an event occurring naturally in vivo or an event performed in vitro to chemically methylate a nucleotide.

[0032] As used herein, a "methylated nucleic acid molecule" refers to a nucleic acid molecule that contains one or more methylated nucleotides.

[0033] As used herein, the term 'epigenetic change' refers to chemical (e.g., methylation) or protein (e.g., histone) changes that occur to a gene body or its promoter. Through epigenetic changes, environmental factors such as diet, stress, and prenatal nutrition can imprint genes that are passed from one generation to the next.

[0034] As used herein, the term "bisulfite" includes any suitable type of bisulfite, such as sodium bisulfite, or another chemical reagent capable of chemically converting cytosine (C) to uracil (U) without chemically modifying methylated cytosine and thus can be used to differentially modify DNA sequences based on the methylation status of DNA, e.g., U.S. Patent Publication US2010 / 0112595 (Menchen et al.). As used herein, a reagent that "differentially modifies" methylated or unmethylated DNA includes any reagent that modifies methylated and / or unmethylated DNA in a process that produces distinguishable products from methylated and non-methylated DNA, thereby allowing identification of the DNA methylation status. Such processes can include, but are not limited to, chemical reactions (e.g., C to U conversion by bisulfite) and enzymatic treatments (e.g., cleavage by methylation-dependent endonucleases). Thus, an enzyme that preferentially cleaves or digests methylated DNA is an enzyme that can cleave or digest a DNA molecule with much higher efficiency when the DNA is methylated, while an enzyme that preferentially cleaves or digests unmethylated DNA exhibits significantly higher efficiency when the DNA is unmethylated.

[0035] In the context of the present invention, also included are any "bisulfite-free methods" and "bisulfite-free quantification methods" for testing the methylation status at any given methylation site to be tested. Such terms refer to any method for quantifying methylated or non-methylated nucleic acids that does not require the use of bisulfite. The terms also refer to methods for preparing nucleic acids to be quantified that do not require bisulfite treatment. Examples of bisulfite-free methods include, but are not limited to, methods for digesting nucleic acids using one or more methylation-sensitive enzymes and methods for separating nucleic acids using reagents that bind nucleic acids based on the methylation status. The terms "methylation-sensitive enzyme" and "methylation-sensitive restriction enzyme" are DNA restriction endonucleases that rely on the methylation status of their DNA recognition sites to exert their activity. For example, there are methylation-sensitive enzymes that only cleave or digest at a sequence when its DNA recognition sequence is not methylated. Thus, an unmethylated DNA sample will be cut into smaller fragments than a methylated DNA sample. Similarly, a hypermethylated DNA sample will not be cut. In contrast, there are methylation-sensitive enzymes that only cleave at a sequence when its DNA recognition sequence is methylated. As used herein, the terms "cut", "cleave", and "digest" are used interchangeably.

[0036] A "biological sample" in the context of the present invention can include any biological material obtained from a subject or group of subjects that contains genomic material and can be liquid, solid, or both, can be tissue or bone, or a body fluid such as blood, lymph, etc. In particular, a biological sample for use in the present invention can include biological cells or fragments thereof.

[0037] As used herein, the term "preselected methylation site" refers to a methylation site selected from a gene or region that showed the highest degree of methylation variation during method training and that meets certain quality criteria, such as considering a minimum sequencing coverage of ≥5x and ≥5 qualified CpG sites. In addition, genes with an average methylation level of <0.1 or >0.9 can be excluded due to their limited dynamic range. Multivariate statistical methods, such as principal component analysis or multidimensional scaling analysis, can be used to define a "reference methylation profile" based on multiple training samples.

[0038] The term "predetermined reference profile" as used herein refers to the typical or standard methylation profile of genomic material of a class of reference animal-derived products that have been proven to be correctly labeled or assayed. In one example, the predetermined reference profile can be used in the context of a control animal that has been correctly assayed (i.e., the control animal has been bred according to known specific types of animal husbandry or rearing methods). In particular, the term "predetermined reference profile" herein can be used in the context of a control animal that has been correctly assayed (i.e., based on the animal husbandry technique for breeding or rearing the control animal. The control animal may thus have been reared under Stallhaltung, StallhaltungPlus, Außenklima, and Premium conditions. The set of predetermined reference profiles for the control animal can also include profiles of different samples that have been obtained from different parts of the control animal (animals reared under at least one, two, three, or four animal husbandry conditions). For example, the set of predetermined reference profiles can include the profile of at least one egg, the profile of at least one meat (muscle, tissue, organ, etc.), the profile of at least one milk, etc. Each of these samples may have its own unique predetermined methylation reference profile, which also forms part of the set of predetermined reference profiles. The set can also include the predetermined reference profile for each of these animal-derived products that is specific for each of the four animal husbandry techniques.

[0039] Predetermined reference spectral groups can be prepared for different samples from animals that have been proven to be raised according to at least one of two, three, four or more livestock husbandry techniques. Here again, there may be a predetermined reference spectral group for each product, which is derived from animals that have been bred according to at least one of these four livestock husbandry techniques. For example, the predetermined reference spectral group can include the spectrum of at least one egg, the spectrum of at least one meat (muscle, tissue, organ, etc.), the spectrum of at least one milk, etc. from animals that have been raised according to livestock husbandry belonging to German husbandry category 1, cage / shed keeping (Stallhaltung). Each of these samples may have its own unique predetermined methylation reference spectrum, which also forms part of the predetermined reference spectral group. The second predetermined reference spectral group can include the spectrum of at least one egg, the spectrum of at least one meat (muscle, tissue, organ, etc.), the spectrum of at least one milk, etc. from animals that have been raised according to livestock husbandry belonging to German husbandry category 2, upgraded cage / shed keeping (StallhaltungPlus). The third predetermined reference spectral group can include the spectrum of at least one egg, the spectrum of at least one meat (muscle, tissue, organ, etc.), the spectrum of at least one milk, etc. from animals that have been raised according to livestock husbandry belonging to German husbandry category 3, outdoor climate (Auβenklima). The fourth predetermined reference spectral group can include the spectrum of at least one egg, the spectrum of at least one meat (muscle, tissue, organ, etc.), the spectrum of at least one milk, etc. from animals that have been raised according to livestock husbandry belonging to German husbandry category 4, premium conditions. In one example, the predetermined reference spectral group can include all four different groups. In yet another example, the groups can be based on different livestock husbandry techniques found in a particular location, land, country or geographical location. The number of predetermined reference spectral groups can vary depending on the location where the method is implemented and what the animal husbandry and / or livestock husbandry techniques implemented in the country or region may be.

[0040] The methylation spectra of different types of meat from animals grown under a particular livestock husbandry technique can be the same. The methylation spectra of different types of meat from different species of animals raised under a particular livestock husbandry technique can also be the same.

[0041] There may be several compilations of predetermined reference spectra, and comparing the methylation spectrum of a test sample with the predetermined reference spectra in the compilation can enable the identification of a particular predetermined reference spectrum that is (significantly) similar to the methylation spectrum of the test sample, and then it can be confirmed whether the test sample has been raised under a particular livestock husbandry technique. In one example, the predetermined reference spectrum can include the methylation spectra of different parts of the meat (i.e., chest, thigh, kidney, liver, shoulder, rib, intestine, etc.) from animals (chickens, goats, cows, lambs, sheep, etc.) that have been raised under a particular livestock husbandry technique.

[0042] In particular, a predetermined reference methylation profile according to any aspect of the present invention is specific for different test animal-derived products. That is, each predetermined reference methylation profile is specific for a single animal-derived product. Thus, the predetermined reference methylation profile set may include many different predetermined reference methylation profiles from different parts of animals or several animals having the same taxonomic unit as the test animal. For different animal taxonomic units, there will also be different predetermined reference methylation profile sets, and the relevant predetermined reference methylation profile set unique to the animal taxonomic unit will depend on the animal taxonomic unit of the test animal.

[0043] In the context of the present disclosure, and particularly in the context of a comparison of methylation profiles (e.g., a comparison between a test profile (from one or more test subjects, i.e., sample X) and a reference profile), the term "significantly similar" shall mean similarity observed by statistical methods (i.e., by using bioinformatics) and / or also by using eye observation. For example, significant similarity is observed if the test profile overlaps with a reference profile defined by multiple training samples through multivariate statistical methods such as principal component analysis or multidimensional scaling analysis. In particular, if more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the methylation patterns / profiles overlap with the reference profile, the test profile is significantly similar to the predetermined reference profile. The similarity of the test profile to more than one, e.g., two, three, or even all reference profiles reduces the significance of the similarity. Of course, the similarity between the test methylation profile and the reference methylation profile takes into account the experimental errors that occur in all methods.

[0044] An animal-derived product sample can be a single type of meat, different types of meat, a single part of one type of meat, different parts of a single type of meat, or different parts of different types of meat. The sample can be from any biological entity having a DNA genome and DNA genome methylation. In particular, the methylation site is a CpG site. The biological entity can be any animal excluding pigs. In particular, the animal can be selected from chickens, lambs, camels, cows, goats, sheep, horses, donkeys, turkeys, ducks, geese, quails, rabbits, and mules. More particularly, the animal can be selected from cows, sheep, goats, camels, chickens, geese, ducks, and turkeys. The term'meat' herein can thus be understood to include chicken meat, lamb meat, beef, mutton, goat meat, camel meat, chicken meat, goose meat, duck meat, turkey meat, and mixtures thereof.

[0045] One or more of the preselected methylation sites in (a) are methylation sites related to tissue-specific gene expression, preferably where the preselected methylation site is related to the gene expression of a particular tissue.

[0046] The tissue may be selected from (i) metabolic tissues, such as intestinal tissue, preferably ileum or jejunum, (ii) muscle tissue, (iii) skin tissue, and (iv) organ tissue, preferably liver and / or pancreatic tissue.

[0047] According to a further aspect of the invention, there is provided an assay of a product sample derived from a test animal, which demonstrates that the test animal has been reared under a special type of animal husbandry, wherein the method for determining that the test animal has been reared under a unique type of animal husbandry comprises the steps of: (a) determining a test methylation profile of one or more preselected methylation sites within genomic material obtained from the product sample derived from the test animal; and (b) comparing the test methylation profile obtained from (a) with a set of predetermined reference methylation profiles of the same taxonomic unit of the test animal from which the product sample is derived, wherein each predetermined reference methylation profile from the set is a reference methylation profile of a control animal that has been reared under a known special type of animal husbandry, wherein if the test methylation profile of (a) is significantly similar to one of the predetermined reference methylation profiles, it indicates that the test animal has been reared under the same special type of animal husbandry as the control animal from which the reference methylation profile is derived, and the product derived from the test animal is so assayed; and wherein the test animal is selected from livestock or poultry.

[0048] According to yet a further aspect of the invention, there is provided a method of verifying the assay of a product sample derived from a test animal, the product sample derived from the test animal having been assayed as derived from a test animal that has been reared under a special type of animal husbandry, the method comprising the steps of: (a) determining a test methylation profile of one or more preselected methylation sites within genomic material obtained from the product sample derived from the test animal; and (b) comparing the test methylation profile obtained from (a) with a reference methylation profile obtained from a control animal having the same taxonomic unit as the test animal from which the product sample is derived, wherein the control animal is reared under the special type of animal husbandry under which the test animal has been assayed as having been reared, A significant similarity of the test methylation profile of (a) compared to the reference methylation profile from the control animal indicates that the test animal has been reared under the same specific type of animal husbandry as the control animal, and that the product derived from the test animal has been correctly authenticated, and a difference of the test methylation profile of (a) compared to the reference methylation profile of the control animal indicates that the test animal has not been reared under the same specific type of animal husbandry as the control animal, and that the product derived from the test animal has been falsely authenticated; wherein the preselected methylation sites are CpG sites selected from genes or genomic DNA regions of control and test animals that showed the highest degree of methylation variation during method training; and wherein the test methylation profile has a significant similarity to the reference methylation profile when the test methylation profile overlaps with the reference methylation profile defined by a plurality of training samples using principal component analysis and / or multidimensional scaling analysis; and wherein the methylation profile is determined using at least one method selected from PCR, methylation-specific PCR, real-time methylation-specific PCR, PCR assays using methylation-DNA-specific binding proteins, quantitative PCR, DNA chip-based assays, pyrosequencing, bisulfite pyrosequencing, methylated DNA immunoprecipitation sequencing, and combinations thereof; and wherein the test animal is selected from livestock or poultry.

[0049] According to a further aspect of the invention, there is provided a method of determining whether a product sample derived from a test animal has been obtained from a test animal that has been reared under a specific type of animal husbandry, the method comprising: - comparing a test methylation profile obtained from genomic material of a product sample derived from the test animal with a reference methylation profile obtained from a control animal of the same taxonomic unit as the test animal from which the product sample is derived, wherein the control animal has been reared under a known specific type of animal husbandry; and wherein the test animal is selected from livestock or poultry.

[0050] The method according to the aspect of the invention comprises the following steps: a) determining a test methylation profile of one or more preselected methylation sites within genomic material obtained from a product sample derived from the test animal; and b) comparing the test methylation profile determined in (a) with a set of predefined reference methylation profiles of the same taxonomic unit as the test animal from which the product sample is derived, wherein each predefined reference methylation profile from the set is a reference methylation of a control animal that has been reared under a known specific type of animal husbandry, A significant similarity of the test methylation profile of (a) compared to any of the reference methylation profiles of the group from the control animals indicates that the test animal has been reared under the same specific type of animal husbandry as the control animal from which the reference methylation profile is derived; and a difference of the test methylation profile of (a) compared to the reference methylation profile of the control animal indicates that the test animal has been reared under a different specific type of animal husbandry compared to the control animal; and wherein the preselected methylation sites are CpG sites selected from genes or genomic DNA regions of control and test animals that showed the highest degree of methylation variation during method training; and wherein the test methylation profile has a significant similarity to the reference methylation profile when the test methylation profile overlaps with the reference methylation profile defined by multiple training samples using principal component analysis and / or multidimensional scaling analysis; and wherein the methylation profile is determined using at least one method selected from PCR, methylation-specific PCR, real-time methylation-specific PCR, PCR assays using methylation-DNA-specific binding proteins, quantitative PCR, DNA chip-based assays, pyrosequencing, bisulfite pyrosequencing, methylated DNA immunoprecipitation sequencing, and combinations thereof; and wherein the test animal is selected from livestock or poultry.

[0051] The method DNA chip-based assay refers to a DNA methylation-based assay that can be performed on a chip. For example, a bead-based chip.

[0052] In particular, the difference according to any aspect of the present invention refers to a difference in methylation and is hypomethylation or hypermethylation.

[0053] In particular, one or more preselected methylation sites according to any aspect of the present invention are selected from the list of CpG sites in Tables 2a and 2b.

[0054] According to yet another aspect of the present invention, there is provided the use of DNA methylation profiling for the authentication of animal-derived product samples, wherein the authentication of the animal-derived product samples is based on animals that have been reared under a specific type of animal husbandry, and wherein the DNA methylation profiling comprises the following steps: (a) determining a test methylation profile of one or more preselected methylation sites within genomic material obtained from an animal-derived product sample from the test animal; and (b) comparing the test methylation profile obtained from (a) with a reference methylation profile obtained from a control animal of the same taxonomic unit as the test animal from which the product sample is derived, wherein the control animal has been reared under a known specific type of animal husbandry, and wherein the test animal is selected from livestock or poultry.

[0055] More particularly, the special type of animal husbandry is selected from: - certified standard, stall housing or its equivalent; - enhanced welfare, upgraded stall housing (StallhaltungPlus) or its equivalent; - free-range pasture, outdoor climate, premium or its equivalent; and - organic. Examples The foregoing describes preferred embodiments which, as will be understood by those skilled in the art, may be subject to variations or modifications in design, construction or operation without departing from the scope of the claims. Such variations are, for example, intended to be covered by the scope of the claims.

[0057] Example 1 Wet-Lab methods Broiler chicken breast meat was obtained from three different German supermarkets to obtain as many replicate samples as possible of the German certified standard called rearing method. There are four special rearing method categories for broiler chicken meat in Germany, which include rearing method 1 (stall housing), 2 (upgraded stall housing), 3 (outdoor climate) and 4 (premium). From rearing methods 1 - 4, the rearing conditions are increasingly improved in line with the animal welfare requirements to be evaluated. For example, the space requirement increases from a maximum of 39 kg of chicken / m in stall housing 2 to 21 kg / m in the premium category 2 . Additional requirements defining each rearing method category include the genetic lineage of the broiler chickens, the duration of rearing (the last 81 days in rearing methods 3 and 4), the amount of enrichment and outdoor activity access available, and the type of ingredients fed to the animals.

[0058] Based on the sample collection, 2 replicate samples of breast meat of rearing method 2 from each of the 3 grocery stores were obtained, for a total of 6 replicate samples, 3 replicate samples of breast meat of rearing method 2 from 1 grocery store were obtained, and 3 replicate samples of breast meat of rearing method 4 from 1 grocery store were obtained. Unfortunately, it was not possible to obtain clearly labeled samples of rearing method 1, so that category was excluded. In total, there were 12 samples, including 3 out of the 4 available rearing method categories (Table 1).

[0059] Table 1. Sample identification of 12 breast meat samples obtained from 3 out of 4 special rearing method conditions. DNA sample id Sequencing sample name Type Supermarket Feeding conditions Replicate sample CHKN-0015 SR_15 Frozen Aldi Feeding method 2 Sample 1 CHKN-0016 SR_16 Frozen Aldi Feeding method 2 Sample 2 CHKN-0018 SR_18 Frozen Lidl Feeding method 2 Sample 1 CHKN-0019 SR_19 Frozen Lidl Feeding method 2 Sample 2 CHKN-0021 SR_21 Frozen Rewe Feeding method 2 Sample 1 CHKN-0022 SR_22 Frozen Rewe Feeding method 2 Sample 2 CHKN-0024 SR_24 Frozen Aldi Feeding method 3 Sample 1 CHKN-0025 SR_25 Frozen Aldi Feeding method 3 Sample 2 CHKN-0026 SR_26 Frozen Aldi Feeding method 3 Sample 3 CHKN-0027 SR_27 Frozen Lidl Feeding method 4 Sample 1 CHKN-0028 SR_28 Frozen Lidl Feeding method 4 Sample 2 CHKN-0029 SR_29 Frozen Lidl Feeding method 4 Sample 3 Genomic DNA was purified from chest tissue samples using the DNeasy Blood & Tissue Kit (Qiagen), and quantified using PicroGreen or NanoDrop TM 2000.

[0061] Genomic DNA (500 ng) from chest tissue samples was used to prepare libraries for whole-genome bisulfite sequencing (WGBS). Sequencing of the libraries was performed by a third party on the NovaSeq platform, which generated 125 GB of data per sample with 20X coverage.

[0062] Calculation methods Treatment: The assembled version 5.0 of the chicken (Gallus gallus) genome was used as the reference sequence, and sequencing reads were trimmed and mapped using BSMAP 1 version 2.5. After deduplication using picard 2 the methylation ratio was determined using a Python script (methratio.py) distributed with the BSMAP package. For all further analyses, only CpGs covered by at least ten reads were considered, which resulted in 6,458,063 CpG sites.

[0063] Differential methylation analysis Differential methylation analysis was performed between different rearing method groups using MethylKit 3 (version 1.12.0). MethylKit uses logistic regression to calculate p-values and the sliding linear model method 4 to adjust the p-values to q-values.

[0064] CpG sites with an FDR below 0.05 and a methylation change greater than 25% between groups were considered significantly differentially methylated positions (DMPs), resulting in 201,246 CpG sites. Examples of CpG sites are provided in Table 2.

[0065] methylKit generates a 'prcomp' object, which can be used to extract and plot the principal components. Principal component analysis (PCA) is a dimensionality reduction method that can transform a large dataset into a few principal components. The first few principal components usually retain most of the variation present in the dataset and are useful for highlighting the grouping structure in the data.

[0066] Results PCA of all samples using CpG sites with a minimum coverage of 10 for all 12 samples showed a distinct clustering based on the rearing regime, also called the rearing condition ( Figure 1 ). When running a second PCA using the differentially methylated positions, the clustering based on the rearing regime, also called the rearing condition, became even clearer ( Figure 2 ).

[0067] Overall, the plot of the first two principal components of CpGs before and after differential methylation analysis revealed a meaningful clustering of the samples. Differentially methylated positions (DMPs), i.e., CpG sites, were able to effectively cluster different groups in a meaningful way. Specific CpG sites related to the rearing condition classification were identified and allowed for the generation of a reference profile for future evaluation of chicken samples to determine the rearing category.

Claims

1. A method for assaying a product sample derived from a test animal, the method comprising the steps of: (a) determining a test methylation profile of one or more preselected methylation sites within genomic material obtained from the product sample derived from the test animal; and (b) comparing the test methylation profile obtained in (a) with a reference methylation profile obtained from a control animal of the same taxonomic unit as the test animal from which the product sample is derived, wherein the control animal is bred under a known specific type of animal husbandry, wherein a significant similarity of the test methylation profile in (a) compared to the reference methylation profile from the control animal indicates that the test animal has been bred under the same specific type of animal husbandry as the control animal, and the product derived from the test animal is assayed as such; and wherein a difference of the test methylation profile in (a) compared to the reference methylation profile of the control animal indicates that the test animal has been bred under a different specific type of animal husbandry compared to the control animal; and wherein the preselected methylation sites are CpG sites selected from genes or genomic DNA regions of control and test animals that showed the highest degree of methylation variation during method training; and wherein the test methylation profile has a significant similarity to the reference methylation profile when the test methylation profile overlaps with the reference methylation profile defined by multiple training samples using principal component analysis and / or multidimensional scaling analysis; and wherein at least one method selected from PCR, methylation-specific PCR, real-time methylation-specific PCR, PCR assay using methylation-DNA-specific binding proteins, quantitative PCR, DNA chip-based assay, pyrosequencing, bisulfite pyrosequencing, methylated DNA immunoprecipitation sequencing, and combinations thereof is used to determine the methylation profile; and wherein the test animal is selected from livestock or poultry.

2. The method according to claim 1, wherein in step (b), the test methylation profile determined in (a) is compared with a set of predetermined reference methylation profiles of the same taxonomic unit as the test animal from which the product sample is derived, wherein each predetermined reference methylation profile is specific for a control animal that has been bred under a known specific type of animal husbandry, and wherein if the test methylation profile is significantly similar to one of the predetermined reference methylation profiles, the product sample derived from the test animal has the same specific assay as the control animal from which the predetermined reference methylation profile was obtained, and the product derived from the test animal is assayed as such.

3. A method for verifying the assay of a product sample derived from a test animal, the product sample derived from the test animal having been assayed as derived from a test animal that has been bred under a specific type of animal husbandry, the method comprising the steps of: (a) determining a test methylation profile of one or more preselected methylation sites within genomic material obtained from the product sample derived from the test animal; and (b) Compare the test methylation profile obtained in (a) with a reference methylation profile obtained from a control animal of the same taxonomic unit as the test animal from which the product sample is derived, wherein the control animal is raised under a particular type of animal husbandry under which the test animal has been certified to be raised, wherein a significant similarity of the test methylation profile in (a) compared to the reference methylation profile from the control animal indicates that the test animal has been raised under the same particular type of animal husbandry as the control animal, and the product derived from the test animal is correctly certified, and wherein a difference of the test methylation profile in (a) compared to the reference methylation profile of the control animal indicates that the test animal has not been raised under the same particular type of animal husbandry as the control animal, and the product derived from the test animal is incorrectly certified; wherein the preselected methylation sites are CpG sites selected from genes or genomic DNA regions of control and test animals that showed the highest degree of methylation variation during method training; and wherein the test methylation profile has a significant similarity to the reference methylation profile when the test methylation profile overlaps with the reference methylation profile defined by multiple training samples using principal component analysis and / or multidimensional scaling analysis; and wherein the methylation profile is determined using at least one method selected from PCR, methylation-specific PCR, real-time methylation-specific PCR, PCR assay using methylation-DNA-specific binding proteins, quantitative PCR, DNA chip-based assay, pyrosequencing, bisulfite pyrosequencing, methylated DNA immunoprecipitation sequencing, and combinations thereof; and wherein the test animal is selected from livestock or poultry.

4. A method for determining whether a product sample derived from a test animal is obtained from a test animal that has been raised under a particular type of animal husbandry, the method comprising the steps of: a) determining a test methylation profile of one or more preselected methylation sites within genomic material obtained from the product sample derived from the test animal; and b) comparing the test methylation profile determined in (a) with a set of predefined reference methylation profiles of the same taxonomic unit as the test animal from which the product sample is derived, wherein each predefined reference methylation profile from the set is a reference methylation of a control animal that has been raised under a known particular type of animal husbandry, wherein a significant similarity of the test methylation profile in (a) compared to any of the reference methylation profiles from the control animals of the set indicates that the test animal has been raised under the same particular type of animal husbandry as the control animal from which the reference methylation profile is derived; wherein a difference of the test methylation profile in (a) compared to the reference methylation profile of the control animal indicates that the test animal has been raised under another particular type of animal husbandry compared to the control animal; and wherein the preselected methylation sites are CpG sites selected from genes or genomic DNA regions of control and test animals that showed the highest degree of methylation variation during method training; and wherein the test methylation profile has a significant similarity to the reference methylation profile when the test methylation profile overlaps with the reference methylation profile defined by multiple training samples using principal component analysis and / or multidimensional scaling analysis; and wherein the methylation profile is determined using at least one method selected from PCR, methylation-specific PCR, real-time methylation-specific PCR, PCR assay using methylation DNA-specific binding proteins, quantitative PCR, DNA chip-based assay, pyrosequencing, bisulfite pyrosequencing, methylated DNA immunoprecipitation sequencing, and combinations thereof; and wherein the test animal is selected from livestock or poultry.

5. The method according to any one of the preceding claims, wherein the special type of animal husbandry for breeding the control and / or test animals is selected from at least: - certified standards, stall / cage rearing (Stallhaltung) or its equivalent; - enhanced welfare, upgraded stall / cage rearing (StallhaltungPlus) or its equivalent; - free-range pasture, outdoor climate (Auβenklima), premium or its equivalent; and - organic.

6. The method according to any one of the preceding claims, wherein the livestock includes terrestrial livestock and aquatic livestock.

7. The method according to claim 6, wherein the aquatic livestock is selected from carp, salmon, tilapia, catfish, marine and brackish water fish, soft-shelled turtles, Australian lungfish, sea shrimp, Chinese mitten crabs, marbled crayfish and other decapod crustaceans, bivalves, gastropods, and the terrestrial livestock is selected from cattle, goats, sheep, pigs, horses, donkeys, rabbits, and mules and / or poultry selected from chickens, turkeys, ducks, geese, and quails.

8. The method according to any one of the preceding claims, wherein the animal-derived product is meat, muscle, at least one organ, milk, collagen, feathers, blood, and / or bone.

9. The method according to any one of the preceding claims, wherein one or more of the preselected methylation sites in (a) are methylation sites related to tissue-specific gene expression, preferably wherein the preselected methylation site is related to the gene expression of a special tissue.

10. The method according to any one of the preceding claims, wherein the one or more preselected methylation sites are selected from the list of CpG sites in Tables 2a and 2b.

11. Use of DNA methylation profiling for assaying animal-derived product samples, wherein the assaying of the animal-derived product samples is based on animals bred under a special type of animal husbandry, and wherein the DNA methylation profiling comprises the following steps: (a) determining a test methylation profile of one or more preselected methylation sites within genomic material obtained from a product sample derived from the test animal; and (b) comparing the test methylation profile obtained from (a) with a reference methylation profile obtained from a control animal having the same taxonomic unit as the test animal from which the product sample is derived, wherein the control animal is bred under a known special type of animal husbandry, and Wherein the test animals are selected from domestic livestock or poultry.

12. Use according to claim 11, wherein the special type of animal husbandry for breeding the control and / or test animals is selected from at least: - Certified standard, stall housing or the like; - Enhanced welfare, upgraded stall housing (StallhaltungPlus) or the like; - Free-range pasture, outdoor climate, premium or the like; and - Organic.

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