Animal feed assessment based on DNA methylation

Abstract

The present invention relates to a method of evaluating the effect of at least one test component of an animal feed on at least the performance and / or the overall health of a test animal consuming an animal feed having the test component, the method comprising the steps of: (a) determining a test methylation profile of one or more preselected methylation sites within the DNA of the test animal; (b) comparing the test methylation profile from (a) to at least one control methylation profile from a control animal consuming animal feed free of the test component belonging to the same biologic classification unit as the test animal; and wherein the results of (a) are used to determine the performance and / or overall health of the test animal; and (c) comparing the test methylation profile from (a) to (i) a first reference methylation profile from a control animal having good performance and / or overall health belonging to the same biologic classification unit as the test animal; and / or (ii) a second reference methylation profile derived from a control animal having poor performance and / or overall health, belonging to the same bioclassification unit as the test animal; and wherein a significant similarity of the test methylation profile of (a) compared to the control methylation profile indicates that the test component has no effect on the performance and / or overall health of the test animal; and wherein a significant difference in the test methylation profile of (a) compared to the control methylation profile indicates that the test component has an effect on the performance and / or overall health of the test animal; and wherein a significant similarity of the test methylation profile of (a) to the first reference methylation profile and / or a difference of the test methylation profile of (a) to the second reference methylation profile indicates that the test animal has good performance and / or overall health as a control cell; and / or wherein the difference in the test methylation profile of (a) compared to the first reference methylation profile and / or the significant similarity of the test methylation profile of (a) to the second reference methylation profile indicates that the test animal has poor performance and / or overall health as a control animal; and wherein the test animal is selected from livestock or poultry.

Description

Invention Field

[0001] This invention relates to a method for determining the effect of a specific component of an animal feed on the overall health and / or performance of animals consuming that feed. Specifically, the method utilizes DNA methylation profiling analysis to determine whether the specific component improves the overall health and / or performance of the animal or actually has a harmful effect. The overall health and / or performance of the animal is characterized using a specific group of experimental subjects with CpG sites. Background of the Invention By 2050, the global population is estimated to reach 9 billion. To feed this growing population, agricultural practices must be as efficient as possible to sustainably utilize existing resources. For livestock production, feed costs are estimated to account for 60-70% of indirect costs; therefore, animal feed has become a clear focus for innovations aimed at improving productivity and sustainability.

[0003] Furthermore, animal feed consumption is directly related to their growth rate, feed efficiency (the ratio of weight gain to feed consumed), pathogenicity and resistance to metabolic diseases, and overall physiological development. The feed industry is constantly testing new nutritional concepts, ingredient compositions, nutrient sources, and feed additives. Generally, feeding trials measure animal variables directly related to return on investment, such as final weight, growth rate, and feed efficiency. However, these measurements may not necessarily identify subtle changes in a single trial, although these subtle changes may have significant cost-effectiveness for producers and / or health and welfare benefits for animals over time.

[0004] For example, the addition of probiotic feed additives to the diet has been shown to improve the development of the digestive and immune systems in animals. In cases where the diet is poorly digestible and / or where disease stress exists, the effects of these probiotic supplements can be readily observed phenotypically in terms of growth rate and disease resistance. However, in situations where high-quality feed ingredients are used and there is no pathogenic exposure, it is difficult to measure the benefits of feed additives such as probiotics using only growth indicators and overall physiological health. This makes testing and benchmarking feed additives difficult and expensive, as not all experiments can demonstrate significant effects of feed additives using phenotypic measurements. There is a need for more sensitive and reliable analyses to reduce the number of trials and, consequently, the costs required for feed additive studies under various conditions.

[0005] Evaluation of nutrient sources is another example of animal feed research that can be more sensitive and cost-effective. For instance, essential nutrients such as methionine, or vitamins and / or minerals, are often available in various forms from a wide range of commercially available products. When comparing two or more sources, very large dose-response trials with a large number of replicates in a large number of animals are required to mathematically model the optimal dose and source for each nutrient based on animal performance data such as feed intake, growth rate, milk or egg production, and feed efficiency. If the dose range chosen for such trials starts slightly too low or slightly too high, or does not contain sufficient levels, the expensive, large trials may not produce the data points needed for accurate modeling to define the optimal dose and source based on individual growth data. This unnecessarily wastes time, cost, effort, and animal lives. Therefore, analyses with increased sensitivity to determine optimal results with fewer animals and greater reliability would be a welcome addition to the evaluation of nutrient sources for animal feed.

[0006] Epigenetics is the study of hereditary traits caused by mechanisms other than changes in the fundamental DNA sequence. In other words, epigenetic markers "orchestrate" our genes. Epigenetic markers can be chemical (e.g., methylation), protein-based (e.g., histones), or a combination of both. DNA methylation is dynamic during development and cell differentiation, but some DNA methylation patterns may be retained as epigenetic memories, accumulating and / or inherited by the next generation. These changes may be responsible for heritable variations in gene activity, as DNA methylation events have been shown to be regulatory mechanisms associated with gene silencing, expression, chromatin remodeling, or imprinting. Epigenetics is attractive for animal breeding because it can identify causal relationships and heritability of complex traits and diseases. DNA methylation patterns are modified throughout an individual's life by environmental forces such as diet, stress, drugs, or pollution. Some environments are more likely to increase certain methylation patterns, and these patterns can contribute to epigenetic and / or phenotypic variation among individuals.

[0007] Recent studies have shown that DNA methylation patterns in animals contain important information about their husbandry conditions. For example, comparisons of genome-wide methylation and variation patterns at the DNA level have revealed that a significant portion of epigenetic variation may be associated with adaptation and environmental differences, such as captive salmon rearing (Le Luyer J et al., 2017, PNAS, Vol. 114, No. 49). Furthermore, Whelan, R. et al., 2023, Front. Vet. Sci., Sec. Veterinary Experimental and Diagnostic Pathology: 9, disclosed the use of epigenetic biomarkers for animal welfare monitoring. Koop et al. (Koop et al., 2021, International Journal of Legal Medicine, 135: 167–173) and Rhein et al. (Rhein et al., 2015, Frontiers in Genetics, 6: 182) also confirmed that methylation patterns are maintained postmortem and are consistent regardless of the level of decomposition in the sample. One of the few aspects that still affects the methylation pattern of a sample is the DNA integrity and quantity of DNA found in the sample.

[0008] In light of the above, there remains an urgent need to provide means that may be used to evaluate different nutrient sources, feed additives, ingredient compositions, nutritional concepts, and compound feeds.

[0009] Invention Description This invention attempts to address the aforementioned problems by providing a method for distinguishing the effects of different animal feeds on animals consuming those feeds using DNA methylation patterns. This method according to any aspect of the invention is not only accurate and reliable, but also saves the time, cost, and effort required to determine the effects of a particular animal feed or its components on the overall health or performance of animals over short or long periods. In particular, the methods according to any aspect of the invention can be used to determine and / or predict the effects of animal feed or its components on the growth rate and / or gut health of animals consuming those feeds over long periods without requiring long-term monitoring of the animal or a group of animals. The methods according to any aspect of the invention provide a method for predicting animal performance based on the animal's DNA methylation profile. Furthermore, since DNA methylation profiles can be altered, for example, by changing the animal's diet, the effects of animal feed components on animal performance can be determined using the methods according to any aspect of the invention. In particular, the methods according to any aspect of the invention also provide a method for monitoring the effects of feed or feed additive programs on the current or future performance of animals. The methods according to any aspect of the invention further provide a means of managing animal growth or processing operations by determining suitable animal feeds and / or additives for feeding animals to achieve optimal performance from the animals. Improved management can optimize animal performance and the animal products they produce.

[0010] This invention is based on the discovery that the components of animal feed can alter an animal's epigenome through epigenetics. Specifically, the ability to adapt to the environment and maintain adaptive biological patterns depends on epigenetic mechanisms, including DNA methylation. In particular, this invention is based on the discovery that animal feed can also lead to changes in an animal's epigenetic mechanisms, including changes in DNA methylation patterns, and that these patterns can be transferred to different products derived from that animal.

[0011] The inventors have unexpectedly discovered that this property can be used to identify an "epigmogenetic fingerprint" of the genome specific to a component of animal feed, which can not only improve the overall health and / or performance of an animal fed that feed, but may also improve the overall health and / or performance of all animals consuming the same animal feed or its components. Based on these findings, the present invention provides means for identifying the short-term and long-term specific effects of any component of an animal feed on the overall health and / or performance of animals consuming that feed. In particular, methods according to any aspect of the invention can be used to determine whether a specific component of any animal feed has a positive or negative effect on the overall health and / or performance of an animal. For example, component X in an animal feed can improve the overall health and / or performance of an animal consuming that feed in the short and / or long term, resulting in an animal with relatively good overall health and / or performance. In another example, component Y in an animal feed can impair the existing overall health and / or performance of an animal consuming that feed, resulting in an animal with relatively poor overall health and / or performance. More specifically, the methods according to any aspect of the invention can be used to determine whether a particular component of an animal feed, or the animal feed itself, has a positive or negative effect on the overall health and / or performance of an animal. In this way, the methods according to any aspect of the invention can then be used to accurately, reliably, and rapidly determine the specific effects of a component in an animal feed on an animal, and based on these results, it can be determined whether the component should be included in the animal's regular diet or should be removed from the animal's diet.

[0012] According to one aspect of the present invention, a method is provided for evaluating the effect of at least one test component of animal feed on the at least performance and / or overall health of test animals consuming animal feed containing the test component, the method comprising the steps of: (a) Determine the test methylation profile of one or more preselected methylation sites in the DNA of the test animal; (b) Compare the test methylation profile obtained from (a) with at least one control methylation profile from a control animal that consumed animal feed containing no test component and belonged to the same taxonomic unit as the test animal; and The results of (a) are used to determine the performance and / or overall health of the test animals; and The significant similarity between the test methylation profile and the control methylation profile in (a) indicates that the test component has no effect on the performance and / or overall health of the test animals; and The significant difference between the test methylation profile and the control methylation profile in (a) indicates that the test component has an effect on the performance and / or overall health of the test animals; and The test animals were selected from livestock or poultry.

[0013] According to another aspect of the invention, a method is provided for evaluating the effect of at least one test component of animal feed on the at least performance and / or overall health of test animals consuming animal feed containing the test component, the method comprising the following steps: (a) Determine the test methylation profile of one or more preselected methylation sites within the DNA of the test animal; and (c) Compare the test methylation profile obtained from (a) with the following (i) A first reference methylation profile obtained from a control animal of good performance and / or overall health that belongs to the same biological taxonomic unit as the test animal; and / or (ii) A second reference methylation profile obtained from control animals with poor performance and / or overall health that belong to the same biological taxonomic unit as the test animals; and The significant similarity between the test methylation profile in (a) and the first reference methylation profile, and / or the difference between the test methylation profile in (a) and the second reference methylation profile, indicate that the test animal has good performance and / or overall health, similar to control cells; and / or The difference between the test methylation profile in (a) and the first reference methylation profile, and / or the significant similarity between the test methylation profile in (a) and the second reference methylation profile, indicate that the test animal has poor performance and / or overall health as the control animal; and The test animals were selected from livestock or poultry.

[0014] The method according to this aspect of the invention further includes the following steps: (b) Compare the test methylation profile obtained from (a) with at least one control methylation profile from a control animal that consumed animal feed containing no test component and belonged to the same taxonomic unit as the test animal; and The results of (a) are used to determine the performance and / or overall health of the test animals; and The significant similarity between the test methylation profile and the control methylation profile in (a) indicates that the test component has no effect on the performance and / or overall health of the test animals; and The significant difference between the test methylation profile and the control methylation profile in (a) indicates that the test component has an effect on the performance and / or overall health of the test animals.

[0015] Specifically, the results of (a), namely the test methylation profile of one or more preselected methylation sites within the DNA of the test animal, are used to determine, in general (with the test component), the performance and / or overall health of the test animal. Specifically, the test methylation profile from (a) can be used to determine whether the test animal (fed the test component) has good performance and / or overall health or poor performance and / or overall health. The results of the comparisons made in (b) and (c) are then used to determine whether the test component has an effect on the overall health and / or performance of the animal, and whether that effect is positive or negative.

[0016] Specifically, the animal’s overall health and / or performance is a measurement of at least one of the animal’s indicators, and the indicator is selected from body weight or carcass weight, growth rate, animal feed intake, feed conversion ratio (FCR), digestive function, intestinal inflammatory status, medical costs, transition period, antibiotic use, mortality rate and combinations thereof.

[0017] Improvements in one or more of an animal’s metrics (e.g., but not limited to any of the following: improved feed conversion ratio (FCR); improved weight gain; improved feed efficiency; improved carcass quality; and / or improved milk production) can be measured using any method known in the art.

[0018] As used herein, the term "feed" is used interchangeably with "animal feed" and refers broadly to liquid or solid materials used to feed animals and to maintain the normal or accelerated growth of animals, including newborns or juvenile and developing animals. This term includes compounds, formulations, mixtures, or compositions suitable for ingestion by animals, particularly livestock. Animal feed typically comprises a variety of different components, which may be present in forms such as concentrates, premix byproducts, or pellets. Examples of feeds and feed components include, but are not limited to: total mixed ration (TMR), corn, soybean, forage, cereals, distillers' grains, germinated cereals, legumes, vitamins, amino acids, minerals, molasses, fiber, forage, hay, straw, silage, grains, leaves, cereal meal, solubles, and supplements. As used herein, the term "selected components of animal feed" refers to animal feed selected for analysis using methods according to any aspect of the invention. In one example, a feed or feed composition comprises a base food composition and one or more feed additives or feed additive compositions. As used herein, "feed additives" refers to components used to fortify a base feed containing additional ingredients to promote feed intake, treat or prevent disease, or modify metabolism for other purposes. Feed additives include premixes.

[0019] As used herein, the term "feed additive" refers to a substance added to feed. "Animal feed and its components" refers to the main components that constitute the largest percentage of animal feed and feed additives. Feed additives may be added to feed for a variety of reasons. For example, to enhance feed digestibility, supplement the nutritional value of feed, improve the recipient's immune defense, and / or improve the shelf life of feed. In some instances, feed additives supplement the nutritional value of feed and / or improve the recipient's immune defense. Representative feed additives include one or more components, such as medicated feed additives, enzymes, probiotic microorganisms, direct-feed microorganisms, antimicrobial agents, prebiotics, phytochemicals, immunomodulators, antibodies, plant extracts, essential oils, organic acids, antioxidants, amino acids, oligosaccharides, oleoresins, herbs, spices, saponins, and marine plants.

[0020] As used herein, a “premix” can be a composition consisting of trace components, such as, but not limited to, one or more of the following: vitamins, minerals, chemical preservatives, antibiotics, fermentation products, and other essential components. Premixes are typically compositions suitable for blending into commercial diets.

[0021] In particular, the components of animal feed according to any aspect of the invention may be selected from compound feeds, probiotics, vitamins, minerals, chemical preservatives, antibiotics and fermentation products.

[0022] As used herein, the term "performance" can be defined as follows: an animal's feed efficiency and / or weight gain, and / or feed conversion ratio, and / or the digestibility of nutrients in the feed (e.g., amino acid digestibility or phosphorus digestibility), and / or digestible or metabolizable energy in the feed, and / or nitrogen retention, and / or the animal's ability to avoid the negative effects of disease or an individual's immune response. Performance characteristics may include, but are not limited to: body weight; weight gain; mass; body fat percentage; height; body fat distribution; growth; growth rate; milk production; nutrient absorption; nutrient excretion; mineral absorption; mineral excretion, mineral retention; bone mineralization; bone strength; feed conversion ratio (FCR); average daily feed intake (ADFI); average daily gain (ADG); retention and / or the secretion of any one or more of copper, sodium, phosphorus, nitrogen, and calcium; amino acid retention or absorption; mineralization; bone mineralization; carcass yield and carcass mass. In particular, 'nutrient' can be fat, carbohydrate, protein, amino acids, etc.

[0023] "Improved animal performance" means the presence of increased feed efficiency, and / or increased weight gain and / or decreased feed conversion ratio, and / or improved digestibility of nutrients or energy in the feed, and / or by improved nitrogen retention, and / or by improved ability to avoid the negative effects of pathogenic intestinal diseases (such as necrotizing enterocolitis), and / or by improved immune responses in individuals due to the use of feed containing the feed additive composition described herein, compared to feeds that do not contain the feed additive composition described herein. In some embodiments, "improved animal performance" means the presence of increased feed efficiency and / or increased weight gain and / or decreased feed conversion ratio. Improvements in performance parameters can be relative to a control in which the feed used does not contain the component in question (i.e., the selected component in the feed to be tested).

[0024] Similarly, as used herein, the term 'general health' when referring to an animal means the animal's health condition and can be determined at least by measuring the presence of intestinal disease and / or inflammation and / or increased resistance to any disease in the animal. Measurements can be performed using any method known in the art. In one instance, general health can be measured by measuring the state of intestinal inflammation. 'Improved general health of an animal' means a reduced probability or condition of intestinal disease and / or inflammation and increased resistance to any disease. In one instance, 'improved general health of an animal' can be a reduced probability of intestinal inflammation (i.e., a positive state of intestinal inflammation) in the animal.

[0025] As used herein, the term "feed efficiency" refers to the amount of weight gain in an animal that occurs over a period of time when the animal is fed either randomly or in a prescribed amount of food. "Increased feed efficiency" means the increase in weight per unit of feed intake resulting from the use of a feed additive composition according to any aspect of the invention in the feed, compared to animals fed without the feed additive composition described herein.

[0026] As used herein, “feed conversion ratio” refers to a measure of an individual’s efficiency in converting feed quality into a desired increase in output, and is calculated by dividing the quality of food consumed over a specified period by the output. For example, if an animal is raised for meat (e.g., chicken), the output can be the quality obtained by the animal. If the animal is raised for another intended purpose (e.g., milk production or egg production), the output will be different. The term “feed conversion ratio” may be used interchangeably herein with the terms “feed conversion efficiency” or “feed conversion rate.” A “lower feed conversion ratio” or “improved feed conversion ratio” means that the amount of feed required to cause a specified weight gain in an animal is less than the amount of feed required to cause the same weight gain in an animal when the feed does not contain the specific feed additive composition.

[0027] The term “test” as used herein in conjunction with the terms individual and / or animal refers to an entity that has been subjected to the methods according to any aspect of the invention and is the basis for the analytical application of the invention. “(various) test individuals,” “(various) groups of test individuals,” or “test profile” or 'test animal derivatives’ are therefore profiles of (various) individuals or groups of individuals to be tested according to the invention, or profiles obtained or generated in this context. Conversely, the term “reference” should primarily refer to a predetermined entity used for comparison with the test entity. For example, the term 'reference animal' refers to an animal used for comparison or as a control when referring to 'test animal'. Similarly, the terms 'sample' and / or 'sample of test animal derivatives' as used according to any aspect of the invention refer to entities that may be subjected to the methods of the invention. Specifically, the sample can be any DNA sample obtained from a test animal that can be subjected to the method of the present invention to determine the effect of selected components of the animal feed on the overall health and / or performance of the animal by first determining the DNA methylation profile and then comparing the tested methylation profile with a control (a reference methylation profile from a control animal showing good or poor overall health and / or performance).

[0028] Livestock according to any aspect of the invention includes terrestrial and aquatic livestock. In particular, livestock can be domesticated animals selected from terrestrial and aquatic livestock or poultry. Specifically, terrestrial livestock can include cattle, sheep, pigs, goats, horses, camels, donkeys, mules, rabbits, etc., and poultry can include chickens, turkeys and other gallinaceous birds, ducks, geese, quails, etc. As used herein, the term 'livestock' can also include poultry and refers to any farm animal or animal that may be used for agriculture.

[0029] As used herein, the term "aquatic livestock" refers specifically to any organism that is entirely raised in water or primarily lives in water, in particular compared to terrestrial animals. These aquatic livestock may live in various bodies of water, such as seas, oceans, rivers, lakes, ponds, etc. More specifically, aquatic livestock according to any aspect of the invention can be any fish, cephalopod, aquatic mollusc, or aquatic crustacean at all life stages (including eggs, sperm, and gametes). Even more specifically, 'aquatic animal' means animals of the following species: (i) belonging to the superclass Aranea (Gnaphaloidea). Agnatha ) and cartilaginous fish ( Chondrichthyes ), lobe-finned fish ( Sarcopterygii ) and Actinopterygii ( Actinopterygii (ii) Fish belonging to the phylum Mollusca ( Mollusca (iii) aquatic mollusks belonging to the subphylum Crustacea ( CrustaceaAquatic crustaceans. More specifically, aquatic livestock according to any aspect of the invention can be aquatic livestock used for aquaculture. Some non-limiting examples of aquatic animals according to any aspect of the invention include Australian lungfish, carp, catfish, halibut, marbled crayfish, marine and brackish water fish, shrimp, Chinese mitten crab, mussels, oysters, pangasius, rainbow trout, salmon trout, scallops, sea bass, sea bream, soft-shell crab, soft-shell turtle, tiger prawn, tilapia, turbot, whiteleg shrimp, shrimp, octopus, squid, and other decapod crustaceans, bivalves, and gastropods.

[0030] As used herein, the term “comprising” should be interpreted to encompass both “including” and “consisting of”, both meanings being clearly defined and thus individually disclosing aspects of the invention. When used herein, “and / or” should be understood to mean that each of the two specified features or components is accompanied by or not accompanied by a specific disclosure of the other. For example, “A and / or B” should be considered as (i) A, (ii) B, and (iii) A and B, respectively, as if each were individually set forth herein. In the context of this invention, the terms “about” and “approximately” indicate, as those skilled in the art will understand, a range of accuracy that still ensures the technical effect of the features under discussion. This term typically indicates a deviation from the indicated numerical value of ±20%, ±15%, ±10%, and, for example, ±5%. As those skilled in the art will understand, the specific deviation of a numerical value regarding a given technical effect depends on the nature of that technical effect. For example, natural or biotechnological effects can generally have greater such deviations than artificial or modified technical effects. When referring to a singular noun, the use of the indefinite or definite article, such as "a," "a kind," or "the / that," includes the plural form of the noun unless otherwise explicitly stated.

[0031] In the context of this invention, the terms “methylation profile,” “methylation pattern,” “methylation status,” or “methylation state” are used herein to describe the status, condition, or state of methylation of a genomic sequence, and such terms refer to the methylation-related characteristics of a DNA segment at a particular genomic locus. These characteristics include, but are not limited to, whether any cytosine (C) residues within the DNA sequence are methylated, the location of methylated C residues, the percentage of methylated C residues at any particular residue segment, and allelic differences in methylation due to, for example, differences in allelic origin.

[0032] The term "methylation state" refers to the state of a specific methylation site (i.e., methylation relative to unmethylation), meaning that the residue or methylation site is methylated or unmethylated. Based on the methylation state of one or more methylation sites, a methylation profile can then be determined. Accordingly, the term "methylation profile" or "methylation pattern" refers to the relative or absolute concentration of methylated or unmethylated C residues at any specific segment of genomic material in a biological sample. For example, if cytosine (C) residues within a DNA sequence that are not normally methylated are methylated, it can be described as "hypermethylated"; while if cytosine (C) residues within a DNA sequence that are normally methylated are not methylated, it can be described as "hypomethylated." Similarly, if cytosine (C) residues within a DNA sequence (e.g., DNA from a sample nucleic acid from a test subject) are methylated compared to another sequence from a different region or different individual (e.g., a standard nucleic acid relative to a normal nucleic acid or a reference sequence), that sequence is considered hypermethylated compared to the other sequence. Alternatively, a sequence is considered hypomethylated compared to another sequence if the cytosine (C) residues within the DNA sequence are unmethylated compared to that of the other sequence from a different region or individual. These sequences are referred to as “differentially methylated.” The level of differential methylation can be measured in various ways known to those skilled in the art. As a non-limiting example, one approach is to measure the methylation level of individual queried CpG sites determined by bisulfite sequencing.

[0033] The term "hypermethylation" refers to an increase in the average methylation level corresponding to the presence of 5-mCyt at one or more CpG dinucleotides within the DNA sequence of the test DNA sample, relative to the amount of 5-mCyt found at the corresponding CpG dinucleotide in a normal control DNA sample. Specifically, a control refers to an animal known to have good performance and / or overall health, or an animal known to have poor performance and / or overall health.

[0034] The term "hypomethylation" refers to the average methylation level corresponding to a reduction in the presence of 5-mCyt at one or more CpG dinucleotides within the DNA sequence of the test DNA sample, relative to the amount of 5-mCyt found at the corresponding CpG dinucleotide in a normal control DNA sample. Specifically, a control refers to an animal known to have good performance and / or overall health, or an animal known to have poor performance and / or overall health.

[0035] As used herein, “methylated nucleotide” or “methylated nucleotide base” refers to the presence of a methyl moiety on a nucleotide base, which is typically not present in recognized typical nucleotide bases. For example, cytosine in its usual form does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at position 5 on its pyrimidine ring. Therefore, cytosine in its usual form may not be considered a methylated nucleotide, while 5-methylcytosine may be. In another instance, thymine may contain a methyl moiety at position 5 on its pyrimidine ring; however, for the purposes of this document, thymine may not be considered a methylated nucleotide when present in DNA. Typical nucleotide bases for DNA are thymine, adenine, cytosine, and guanine. Typical bases for RNA are uracil, adenine, cytosine, and guanine. Accordingly, a “methylation site” is a location in a target gene nucleic acid region where methylation is likely to occur. For example, a location containing CpG is a methylation site where cytosine may or may not be methylated. Specifically, the term "methylated nucleotide" refers to a nucleotide carrying a methyl group attached to a methylated nucleotide position. These methylated nucleotides are commonly found in nature, and to date, methylated cytosines, primarily occurring in the context of dinucleotide CpG and in the context of CpNpG- and CpNpN- sequences, are considered the most common. In principle, other naturally occurring nucleotides may also be methylated, but they will not be considered for any aspect of this invention.

[0036] As used herein, a “CpG site” or “methylation site” is a nucleotide within a nucleic acid (DNA or RNA) that is susceptible to methylation, either through a naturally occurring event in vivo or through an event in vitro that chemically methylates the nucleotide.

[0037] As used herein, the terms "methylation marker" or "CpG site" refer to CpG sites that are potentially methylated. Methylation typically occurs in CpG-containing nucleic acids. CpG-containing nucleic acids can be located, for example, in CpG islands, CpG duplexes, promoters, introns, or exons of genes. For example, a potential methylation site may encompass the promoter / enhancer region of the gene being indicated. "A collection of specific CpG sites in DNA" refers to CpG sites that demonstrate the best association with an animal's performance and / or growth rate.

[0038] As used in this article, "methylated nucleic acid molecule" refers to a nucleic acid molecule containing one or more methylated nucleotides.

[0039] As used herein, the term 'epigenetic change' refers to chemical (e.g., methylation) or protein (e.g., histone) changes occurring on the genomic body or its promoter. Through epigenetic changes, environmental factors such as diet, stress, and prenatal nutrition can leave an imprint on genes passed from one generation to the next.

[0040] As used herein, the term "bisulfite" encompasses 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 the methylated cytosine, and thus can be used for differential modification of DNA sequences based on the methylation state of DNA, as illustrated in U.S. Patent Publication US 2010 / 0112595 (Menchen et al.). As used herein, reagents for "differentially modifying" methylated or unmethylated DNA encompass any reagent that modifies methylated and / or unmethylated DNA in a process by which products originating from methylated and unmethylated DNA can be distinguished, thereby allowing identification of the DNA methylation state. Such processes may include, but are not limited to, chemical reactions (e.g., C-to-U conversion via bisulfite) and enzymatic treatments (e.g., cleavage by methylation-dependent endonucleases). Therefore, enzymes that preferentially cleave or digest methylated DNA are those capable of cleaving or digesting DNA molecules with much higher efficiency when DNA is methylated, while enzymes that preferentially cleave or digest unmethylated DNA exhibit significantly higher efficiency when DNA is not methylated.

[0041] In the context of this invention, the terms also include any “non-bisulfite-based method” and “non-bisulfite-based quantitative method” for testing the methylation state at any given methylation site to be tested. Such terms refer to any method that does not require the use of bisulfite for quantifying methylated or unmethylated nucleic acids. These terms also refer to methods that do not require bisulfite treatment for preparing the nucleic acid to be quantified. Examples of non-bisulfite-based methods include, but are not limited to, methods using one or more methylation-sensitive enzymes for digesting nucleic acids, and methods using reagents that bind nucleic acids based on methylation state for isolating nucleic acids. The terms “methyl-sensitive enzyme” and “methylation-sensitive restriction enzyme” refer to DNA restriction endonucleases whose activity depends on the methylation state of their DNA recognition site. For example, there are methyl-sensitive enzymes that cleave or digest only when their DNA recognition sequence is unmethylated. Therefore, unmethylated DNA samples will be cleaved into smaller fragments than methylated DNA samples. Similarly, highly methylated DNA samples will not be cleaved. In contrast, there are methyl-sensitive enzymes that cleave only when their DNA recognition sequence is methylated. As used in this article, the terms “cutting,” “slicing,” and “digesting” are used interchangeably.

[0042] A “biological sample” according to any aspect of the invention may comprise any biological material containing genomic material obtained from a subject or group of subjects, and may be liquid, solid, or both, and may be tissue or bone, or bodily fluids such as blood, lymph, saliva, semen, etc. In particular, biological samples used in the invention may comprise biological cells or fragments thereof.

[0043] As used herein, the term "preselected methylation site" refers to a methylation site selected from genes or regions exhibiting the highest degree of methylation variation during method training and meeting certain quality criteria, such as considering a minimum sequencing coverage of ≥5x and ≥5 eligible CpG sites. Additionally, genes with an average methylation level <0.1 or >0.9 can be excluded due to its limited dynamic range.

[0044] The “reference methylation profile” can be defined based on multiple training samples using multivariate statistical methods, such as principal component analysis or multidimensional scaling transformation.

[0045] As used herein, the term "predetermined reference profile" refers to a typical or standard methylated profile of the genomic material of a class of reference animals that are recognized or shown in the industry to have good performance and / or good health. In one instance, a predetermined reference profile may be used in the context of control animals that have demonstrated good performance traits (i.e., control animals exhibiting values ​​considered good among at least one of the following: body weight or carcass weight, growth rate, feed intake, feed conversion ratio (FCR), digestive function, intestinal inflammation status, medical costs, transition period, antibiotic use, mortality, and combinations thereof). Specifically, the term "predetermined reference profile" as used herein may be used in the context of control animals that have good performance and / or overall health, wherein, compared to baseline values ​​of animals belonging to the same taxonomic unit, the control animals exhibit improved body weight or carcass weight, growth rate, feed intake, feed conversion ratio (FCR), digestive function, intestinal inflammation status, reduced medical costs, transition period, antibiotic use, mortality, reproductive measurements, and combinations thereof. Examples of reproductive measurements can include the percentage of live births, the number of offspring, and so on.

[0046] As used herein, the term "baseline" in relation to performance or performance traits refers to various aspects of an animal when it is fed an animal diet free of one or more optional feed supplements. Examples of baseline performance include an animal's meat / milk production and / or meat / milk production efficiency. The experimental subject group with respect to a predetermined reference profile of control animals may also include profiles from different samples obtained from different parts of control animals (animals with varying levels of performance and / or overall health). For example, the experimental subject group with predetermined reference profiles may include at least one profile regarding eggs, at least one profile regarding meat (muscle, tissue, organs, etc.), at least one profile regarding milk, and so on. Each of these samples may have its own unique predetermined methylated reference profile, which also constitutes part of the experimental subject group with respect to the predetermined reference profiles. The experimental subject group may also include predetermined reference profiles regarding these animal-derived products and their respective animals, which are specific to each characteristic of the animal in relation to its overall health and / or performance.

[0047] Predetermined reference profiles for experimental subjects can be prepared for different samples, said different samples being animals belonging to the same taxonomic unit as the test animal, which have been industry-recognized as exhibiting good performance and / or good health. Again, for each product derived from an animal, there may be a predetermined reference profile for experimental subjects, said animals exhibiting at least one characteristic indicating good performance and / or good health. For example, a predetermined reference profile for cattle may include at least one profile regarding meat (muscle, tissue, organs, etc.), at least one profile regarding milk, and a second experimental subject group with at least predetermined reference profiles, one of which is regarding sperm, tissue, blood, etc., from live cattle, and each profile relates to one characteristic indicating good performance and / or good health. For example, one of the predetermined reference profiles in this experimental subject group is a tissue sample of a cow with improved body weight or carcass weight; another is a tissue sample of a cow with high growth rate; another is a tissue sample of a cow with good feed intake; another has high feed conversion ratio (FCR); another has good digestive function; another profile is a tissue sample of a cow with mild intestinal inflammation; another is a tissue sample of a cow without intestinal inflammation; and yet another predetermined reference profile is a tissue sample of a cow exhibiting a combination of characteristics of a cow with good performance / good overall health. Each of these samples may have its own unique predetermined methylated reference profile, which also constitutes part of the experimental subject group of the predetermined reference profiles. A third experimental subject group regarding the predetermined reference profiles of cattle may include at least one profile regarding meat (muscle, tissue, organs, etc.), at least one profile regarding milk, and a fourth experimental subject group of at least predetermined reference profiles, one of which is regarding sperm, tissue, blood, etc., of live cattle, and each profile relates to a characteristic indicating poor performance and / or poor health. For example, one of the predetermined reference profiles in this experimental group might be a tissue sample from cattle with low body weight or carcass weight; another might be a tissue sample from cattle with low growth rate; another might be a tissue sample from cattle with poor feed intake; another might be a tissue sample from cattle with low feed conversion ratio (FCR); another might be a tissue sample from cattle with poor digestive function; another might be a tissue sample from cattle with severe intestinal inflammation; and yet another might be a tissue sample from cattle exhibiting a combination of characteristics of cattle with poor performance / poor overall health. Each of these samples may have its own unique predetermined methylated reference profile, which also constitutes part of the experimental group of predetermined reference profiles. In another instance, the experimental group could be based on animals from different taxa.In a further example, the experimental group can be based on a timeline, meaning there are different DNA methylation profiles from control animals at different time points, each specific to control animals with good performance and / or overall health or poor performance and / or overall health.

[0048] Several predefined reference profiles may exist, and comparing the methylation profile of a test sample with the predefined reference profiles in the compilation allows for the identification of specific predefined reference profiles that are (significantly) similar to the methylation profile of the test sample. Test animals from which the test sample is derived can then be confirmed to currently have good performance and / or good overall health, or are expected to have good performance and / or good overall health in the future. In one example, the predefined reference profiles may include methylation profiles of meat (i.e., breast, thigh, kidney, liver, shoulder, rib, intestine, etc.) from different parts of an animal (chicken, goat, cow, lamb, sheep, etc.) that has good performance and / or good overall health.

[0049] In particular, the experimental subject group of a predetermined reference methylation profile according to any aspect of the invention is unique to the DNA sample from which different test animal derivatives or animal parts are derived. That is, each predetermined reference methylation profile is unique to a single animal derivative or animal part. The experimental subject group of a predetermined reference methylation profile can therefore include many different predetermined reference methylation profiles from different parts of one or more animals belonging to the same biological taxonomic unit as the test animal. There will also be different experimental subject groups with respect to predetermined reference methylation profiles for different animal taxonomic units, and the relevant experimental subject group for a predetermined reference methylation profile unique to an animal taxonomic unit will depend on the animal taxonomic unit of the test animal.

[0050] In the context of this disclosure, and particularly in the context of comparisons of methylation profiles (e.g., comparisons between test profiles (i.e., between test subjects (i.e., sample X) and reference profiles)), the term "significantly similar" should mean similarity observed by statistical means (i.e., by using bioinformatics) and / or by visual observation. For example, significant similarity is observed if a test profile overlaps with a reference profile defined by multiple training samples using multivariate statistical methods such as principal component analysis or multidimensional scaling transformation. In particular, a test profile is significantly similar to a predetermined reference profile if more than 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% of methylation patterns / profiles overlap with a reference profile. The similarity of a test profile with more than one, such as 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 experimental errors that occur in all methods.

[0051] Animal-derived product samples may be of a single type of meat, different types of meat, a single cut of a type of meat, different cuts of a single type of meat, or different cuts of different types of meat. Samples can come from any biological entity with a DNA genome and DNA genome methylation. Specifically, the methylation site is a CpG site. The biological entity can be any animal excluding pigs. Specifically, the animal can be selected from chickens, lambs, camels, cattle, goats, sheep, horses, donkeys, turkeys, ducks, geese, quails, rabbits, and mules. More specifically, the animal can be selected from cattle, sheep, goats, camels, chickens, geese, ducks, and turkeys. The term 'meat' as used herein can therefore be understood to include chicken meat, lamb meat, beef, adult lamb, goat meat, camel meat, chicken, goose meat, duck meat, turkey meat, and mixtures thereof.

[0052] One or more of the preselected methylation sites in (a) are methylation sites associated with tissue-specific gene expression, preferably wherein the preselected methylation sites are associated with gene expression in a unique tissue.

[0053] The organization may be selected from: (i) Metabolic tissue, such as intestinal tissue, preferably the ileum or jejunum. (ii) Muscle tissue, (iii) Skin tissue, and (iv) Organ tissues, preferably liver and / or pancreatic tissues.

[0054] In particular, any difference according to any aspect of the invention refers to a difference in methylation, and is either hypomethylation or hypermethylation.

[0055] According to another aspect of the invention, an in vitro method is provided for establishing the effect of at least one test component of an animal feed on the performance and / or overall health of a test animal consuming the animal feed, the method comprising the steps of: (a) Determine the test methylation level of the set of specific CpG sites in the test DNA of the test animal prior to consumption of the animal's feed (T0). (b) Determine the methylation level of the same set of specific CpG sites in the test DNA obtained from the test animal at a specific time point T1 after the animal has consumed its feed, and (c) Compare the methylation levels of the same set of specific CpG sites from (a) and (b) with the methylation levels of the same CpG sites from a reference that correlates DNA methylation patterns with animal performance and / or overall health. This establishes the role of the test components in animal feed on the performance and / or overall health of the test animals. The test animals were selected from livestock or poultry.

[0056] In particular, the method according to this aspect of the invention can be used to predict the effect of at least one test component of an animal feed on the performance and / or overall health of a test animal consuming that feed at a next time point T2.

[0057] According to one aspect of the invention, an in vitro method is provided for establishing the effect of at least one test component of an animal feed on the performance and / or overall health of a test animal consuming the animal feed, the method comprising the steps of: (a) Determine the test methylation level of the set of specific CpG sites in test DNA obtained from test animals that consumed the animal's feed. (b) Determine the methylation levels of the same set of specific CpG sites in DNA obtained from control animals that did not consume the animal feed, and (c) Compare the methylation levels of the same set of specific CpG sites from (a) and (b) with the methylation levels of the same CpG sites from a reference that correlates DNA methylation patterns with animal performance and / or overall health. This establishes the role of the test components in animal feed on the performance and / or overall health of the test animals. The test animals were selected from livestock or poultry.

[0058] According to another aspect of the present invention, a method is provided for evaluating the effect of at least one component of an animal feed on the intestinal inflammatory status of meat-producing and / or milk-producing test animals consuming the animal feed, the method comprising the following steps: (a) Determine the test methylation profile of one or more preselected methylation sites within the genomic material obtained from the test animal; and (b) Compare the test methylation profile obtained from (a) with at least one first reference methylation profile obtained from a control animal with a positive intestinal inflammation state belonging to the same taxonomic unit as the test animal and / or at least one second reference methylation profile obtained from a control animal with a negative intestinal inflammation state belonging to the same taxonomic unit as the test animal; and Significant similarity between the test methylation profile (a) and the first or second reference methylation profile of the control animal indicates that the test animal has a positive or negative intestinal inflammatory state, as in the control animal. and The difference between the test methylation profile in (a) and the first or second reference methylation profile in the control animal indicates whether the test animal has a negative or positive intestinal inflammatory state, similar to that in the control animal.

[0059] The term "negative intestinal inflammatory state" refers to intestinal material showing no signs of inflammatory process. As used herein, the terms "intestinal inflammation" and "enteritis" are used interchangeably and have the same meaning. The expression "classifying the intestinal inflammatory state" refers both to the classification of the presence or absence of an ongoing inflammatory process (yes / no) and to classification into different degrees of inflammation (e.g., severe, moderate, or mild inflammation). Specifically, a positive intestinal inflammatory state is further classified into categories of severe, moderate, or mild inflammation based on average methylation levels. The genomic material used according to any aspect of the invention may be obtained from an intestinal test sample, and the intestinal test sample is intestinal tissue, preferably the ileum or jejunum.

[0060] According to a further aspect of the present invention, a method is provided for evaluating the effect of at least one test component of animal feed on the growth rate and / or feed conversion ratio (FCR) of meat-producing and / or milk-producing test animals consuming animal feed containing the test component, the method comprising the following steps: (a) Determine the test methylation profile of one or more preselected methylation sites in the DNA of the test animal; and (b) Compare the test methylation profile obtained from (a) with at least one control methylation profile from a control animal that consumed animal feed containing no test component and belonged to the same taxonomic unit as the test animal; and The results of (a) were used to determine the growth rate and / or feed conversion ratio (FCR) of the test animals; and The significant similarity between the test methylation profile and the control methylation profile in (a) indicates that the test component has no effect on the growth rate and / or FCR of the test animals; and The significant difference between the test methylation profile and the control methylation profile in (a) indicates that the test component has an effect on the growth rate and / or FCR of the test animals.

[0061] According to this aspect of the invention, the following further steps are provided: (c) Compare the test methylation profile obtained from (a) with the following (i) First reference methylation profile obtained from control animals with rapid growth rate and / or FCR belonging to the same taxonomic unit as the test animals; and / or (ii) A second reference methylation profile obtained from control animals with slow growth rate and / or FCR, belonging to the same taxonomic unit as the test animals; and Significant similarity between the test methylation profile in (a) and the first reference methylation profile, and / or difference between the test methylation profile in (a) and the second reference methylation profile, indicate that the test animal has a rapid growth rate and / or FCR; and / or The difference between the test methylation profile in (a) and the first reference methylation profile and / or the significant similarity between the test methylation profile in (a) and the second reference methylation profile indicates that the test animal has a slow growth rate and / or FCR.

[0062] According to a further aspect of the present invention, a method is provided for evaluating the effect of at least one component of an animal feed on the growth rate of a meat-producing, egg-producing, and / or milk-producing test animal consuming the animal feed, the method comprising the following steps: (a) Determine the test methylation profile of one or more preselected methylation sites within the genomic material obtained from the test animal; and (b) Compare the test methylation profile obtained from (a) with at least one first reference methylation profile obtained from a control animal with a slow growth rate belonging to the same taxonomic unit as the test animal and / or at least one second reference methylation profile obtained from a control animal with a fast growth rate belonging to the same taxonomic unit as the test animal; and Significant similarity between the tested methylation profile (a) and the first or second reference methylation profile of the control animal indicates that the tested animal has a slow or fast growth rate, similar to the control animal; and The difference between the test methylation profile in (a) and the first or second reference methylation profile of the control animal indicates whether the test animal has a fast or slow growth rate, similar to that of the control animal.

[0063] As used herein, the term 'animal-derived product' refers to a product derived from an animal. Specifically, the term 'testing animal-derived product' refers to the discussed sample or object to be introduced into the array according to any aspect of the invention. These animal-derived products can include meat and meat products, as well as fat, fresh meat, blood, processed meat, and lesser-known products such as gelatin and 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., fat), such as soaps, creams, etc. In one instance, animal-derived products are meat, eggs, blood, brain, sperm / sperm, milk, and any other tissue or sample providing genomic DNA. In particular, animal-derived products are meat. In one instance, an animal-derived product sample can be a single type of meat, different types of meat, a single part of a type of meat, different parts of a single type of meat, or different parts of different types of meat. Where the animal is aquatic, these products derived from the animal can include meat and meat products, as well as eggs, fat, fresh meat, blood, rumen, processed meat, and lesser-known products, and non-food products such as fibers (shells, scales, etc.). Animal-derived products can also include products that can be prepared using animal products (such as fish oil), such as tablets, powders, etc. In one instance, animal-derived products are meat, eggs, blood, brain, shells, scales, skin, tissue, abdominal muscle tissue, or any other tissue or sample that provides genomic DNA.

[0064] Specifically, animal-derived products are meat, skin, blood, scraps, or any organs from aquatic animals. Scraps are particularly used as a byproduct of fishmeal / fish oil production, ultimately for use in the animal feed industry or for pets. Samples can come from any biological entity with a DNA genome and DNA genome methylation. Specifically, the methylation site is a CpG site.

[0065] The test methylation profile of step (a) according to any aspect of the invention can be determined by contacting the genomic material of the test animal with a DNA methylation-based array.

[0066] As used herein, the term "array" refers to an intentionally created collection of probe molecules, which may be prepared synthetically or biosynthetically. The probe molecules in an array may be the same as or different from each other. Arrays can take various forms, such as libraries of soluble molecules; libraries of compounds tethered to resin beads, silica chips, or other solid supports.

[0067] In particular, DNA methylation-based arrays provide a convenient platform for the simultaneous analysis of a large number of CpG sites, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1000, 5000, 10,000, 100,000 or more sites or loci. Specifically, the array contains a variety of different probe molecules that can be attached to a substrate or otherwise spatially differentiated within the array. Examples of arrays that can be used according to any aspect of the invention include slide arrays, silicon wafer arrays, liquid arrays, bead-based arrays, and the like. In one example, the array technology used according to any aspect of the invention combines a miniaturized array platform, a high level of assay multiplexing, and scalable automation for sample handling and data processing.

[0068] In particular, an array according to any aspect of the invention can be an array of arrays, also known as a composite array, having multiple individual arrays configured to allow simultaneous processing of multiple samples. Examples of composite arrays and the techniques underlying them are disclosed in at least US 6,429,027 and US 2002 / 0102578. The substrate of a composite array may include multiple individual array locations, each having multiple probes, and each physically separated from other measurement locations on the same substrate, such that fluid contact with one array location is prevented from contacting another array location. Each array location may have multiple different probe molecules, which are either directly attached to the substrate or attached to the substrate via rigid particles in pores (also referred to herein as beads in pores).

[0069] In one example, the array substrate can be a bundle or array of optical fibers as described in US6,023,540, US6,200,737, and / or US6,327,410. The bundle or array of optical fibers may have probes attached directly to the fibers or via beads. Those skilled in the art can readily determine which substrate is best suited for an array according to any aspect of the invention. WO2004110246 further discloses other substrates that may be used in an array according to any aspect of the invention, and methods for attaching beads to the substrate.

[0070] In one instance, the surface of the substrate may be physically modified to allow for probe attachment or array localization. For example, the surface of the substrate may be modified to contain chemically modified sites that can be used for covalent or non-covalent attachment of probe molecules or particles already attached to probe molecules. Probes may be attached using any of a variety of methods known in the art, including inkjet printing, spotting techniques, photolithography, or mask-based printing. These techniques are disclosed in more detail in WO2004110246.

[0071] In one example, a DNA methylation-based array according to any aspect of the invention can be a bead-based array, wherein the beads are associated with a solid support, such as those commercially available from Illumina, Inc. (San Diego, Calif.). Bead arrays available according to any aspect of the invention can also be in fluid form, such as a fluid flow from a flow cytometer or similar device. Commercially available fluid forms for distinguishing beads include, for example, those used in Luminex's XMAP™ technology or Lynx Therapeutics' MPSS™ method.

[0072] As used herein, the terms “solid support,” “support,” and “substrate” are used interchangeably and refer to a material or group of materials having one or more rigid or semi-rigid surfaces. In many instances, at least one surface of the solid support will be substantially flat, although in some instances it may be desirable to physically separate regions of synthesis of different compounds with, for example, holes, raised areas, pins, etched trenches, etc.

[0073] DNA methylation arrays according to any aspect of the invention can be extremely high-density arrays, for example, having approximately 10,000,000 probes / cm². 2 Approximately 2,000,000,000 probes / cm 2 Or approximately 100,000,000 probes / cm 2 Up to approximately 1,000,000,000 probes / cm 2 Those arrays. High-density arrays according to any aspect of the invention are particularly useful for including a large number of CpG sites from different species on the array.

[0074] DNA methylation arrays according to any aspect of the invention can be used to simultaneously or sequentially analyze or evaluate multiple such loci as needed. In one instance, multiple different probe molecules can be attached to a substrate or otherwise spatially distinguished within the array. Each probe is typically specific to a particular locus and can be used to differentiate the methylation status of the locus.

[0075] As used herein, the term "probe molecule" refers to a surface-fixed molecule that can be recognized by a specific target. Probes used in arrays can be specific to methylated alleles at CpG sites, unmethylated alleles at CpG sites, or both.

[0076] As used herein, the term "target" refers to a molecule that has an affinity for a given probe molecule. Targets can be naturally occurring or artificial molecules. They can also be employed in their unchanged state or as aggregates of other kinds. Targets can be covalently or non-covalently attached to binding members, directly or via specific binding substances. Examples of targets that can be employed according to any aspect of the invention are methylated and unmethylated CpG sites. Targets are sometimes referred to in the art as anti-probes. No difference in meaning is contemplated when the term "target" is used herein.

[0077] Specifically, the probe molecule according to any aspect of the invention comprises a nucleic acid sequence complementary to a unique CpG site. The array according to any aspect of the invention thus comprises several unique or exclusive locations, each containing a specific probe molecule complementary to a unique CpG site of the animal. The array thus comprises multiple locations, each having a specific probe molecule complementary to a unique CpG site of the animal.

[0078] As used herein, the term "complementary" refers to hybridization or base pairing between nucleotides or nucleic acids, such as between the two strands of a double-stranded DNA molecule, or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are generally A and T (or A and U), or C and G. Two single-stranded RNA or DNA molecules are said to be complementary when the nucleotides of one strand, which are optimally aligned and compared and have appropriate nucleotide insertions or deletions, pair with at least about 80%, typically at least about 90% to 95%, and more preferably about 98% to 100%, of the nucleotides of the other strand. Perfect complementarity refers to 100% complementarity in sequence length. For example, a 25-base probe is perfectly complementary to a target when all 25 bases of the probe are complementary to the adjacent 25-base sequence of the target, without any mismatch in probe length.

[0079] According to one aspect of the invention, a method is provided for establishing a reference for associating DNA methylation patterns with the performance and / or overall health of at least one animal, the method comprising: (a) Determine the performance and / or overall health of a specific animal tissue - methylation ratio and readout coverage of genomic CpG sites in associated training samples; (b) Using the reading coverage cutoff value, define the set of CpG sites with methylation ratios in all training samples from step (a); and (c) By applying a penalized regression model, the methylation rate of step (b) is used as input and the performance and / or overall health associated with the training samples are used as dependent variables; This yields a set of CpG sites, with corresponding weighting factors and the intercept of a penalized regression model as parameters for defining a reference that correlates DNA methylation patterns with animal performance and / or overall health. The test animals were selected from livestock or poultry.

[0080] According to this aspect of the invention, the animal's performance and / or overall health should be quantitative values. For example, indicators of performance and / or overall health.

[0081] Methods according to any aspect of the invention involve identifying multiple CpG (cytosine-guanine phosphate) sites in the animal genome for whose DNA methylation levels are correlated both tissue-specifically and tissue-independently with animal performance and / or growth rate. That is, measuring DNA methylation at these locations (CpG sites) allows for accurate prediction of animal performance and / or growth rate.

[0082] The term "methylation ratio" refers to the number of methylated cytosines divided by the total number of cytosines covered at a specific site.

[0083] The term “reading coverage of CpG sites” should be understood as the number of readings aligned with known CpG sites in the reference sequence.

[0084] The methylation ratio and read coverage of CpG sites in the genome can be determined using bisulfite sequencing in step (a).

[0085] The training sample used in step (a) that is associated with the performance and / or growth rate of a specific tissue may be intestinal tissue, muscle tissue, organ tissue or skin tissue.

[0086] Specifically, the coverage cutoff value defined in step (b) can be at least 3. More specifically, the coverage cutoff value defined in step (b) can be 3 or higher.

[0087] The method according to at least this aspect of the invention may further include step (d): optimizing the fit of the penalized regression model such that the number of CpG sites is reduced to below 100. To optimize the fit of the penalized regression model in step (d), the algorithm preferably applies ridge regression in combination with lasso regression. Advantageously, an α value of 0 to 1 is used to balance the ridge regression and lasso regression methods. The penalized regression model is a linear model.

[0088] According to a further aspect of the invention, DNA methylation profiling analysis is provided for evaluating the effect of at least one component of an animal feed on the performance and / or overall health of a test animal consuming the feed.

[0089] According to another aspect of the invention, an array based on DNA methylation is provided for implementing methods according to any aspect of the invention. Example

[0090] The foregoing describes preferred embodiments, which, as those skilled in the art will understand, can be subject to changes or modifications in design, construction, or operation without departing from the scope of the claims. For example, such changes are contemplated to be covered by the scope of the claims.

[0091] Example 1 Evaluation of nutrient digestibility in pigs Nutrient digestibility in pigs can currently be measured in several ways, but each has its drawbacks (Zhang and Adeola, 2017, Animal Nutrition 3:344e352). A recalcitrant marker can be added to the feed, and then the feed can be measured relative to the nutrients in the feces compared to the marker to evaluate the amount digested by the animal. However, hindgut fermentation and endogenous losses (such as host intestinal cell shedding) cannot explain and contribute to inaccuracies in the calculated digestibility. Cannulation in the ileum allows for the acquisition of digesta samples at one of the final points of digestion in the small intestine before hindgut fermentation can occur, which could contribute to more accurate digestibility calculations. However, prior to digestibility studies, animals require surgical placement of the cannula, raising both ethical concerns and questions about how the cannulation itself might affect animal physiology and, consequently, digestibility.

[0092] Therefore, it is necessary to establish epigenetic markers of 'good' versus 'poor' digestibility from the blood epigenome of pigs to generate a less invasive yet still accurate method for determining nutrient digestibility. Blood was collected from growing pigs aged 9 to 14 weeks. Piglets were obtained from various tests that assessed nutrient digestibility in response to different feed components or feed additives (e.g., probiotics or enzymes). Blood was sorted into 'good' and 'poor' digestibility categories based on various methods used in the study, including fecal marker digestibility measurements and chyme measurements from cannulation. DNA was extracted from at least 100 replicate samples from each category. The DNA was bisulfite-converted and sent for whole-genome bisulfite sequencing according to the methods outlined below. CpGs with significantly differentially methylated 'good' and 'poor' gut health categories were used in random forest analysis to define unique epigenetic markers distinguishing good from poor gut health. CpG sites on these profiles were used to generate probes for livestock methylation bead-based arrays.

[0093] In a future digestibility feeding trial, one group of 9-week-old pigs was fed the enzyme while another group served as a control and received no treatment for 4 weeks. A 5 ml blood sample was collected weekly from each pig for analysis. DNA was extracted and subjected to bisulfite conversion, followed by analysis on a bead-based array. The results showed that the nutrient digestibility of the pigs fed the enzyme was significantly higher than that observed in the control group.

[0094] DNA extraction DNA was extracted using the PureLink Genomic DNA Isolation Minikit (Invitrogen), including RNase treatment following the manufacturer's instructions. DNA quantity was measured using a PicoGreen assay, and DNA quality was evaluated via NanoDrop (Thermo Scientific) to ensure an A260 / 280 ratio ≤ 1.8. Small sample sizes were analyzed on agarose gels to ensure each sample contained high molecular weight DNA.

[0095] Bisulfite conversion and BeadChip analysis Genomic DNA samples were subjected to bisulfite conversion using the EZ DNA Methylation-Gold™ Kit (Zymo Research). Methylation levels were then quantified using the Methylation EPIC BeadChip Kit (Illumina), which can quantify more than 50,000 methylation sites on the genome at single nucleotide resolution.

[0096] Data processing Sequencing reads were trimmed and mapped using BSMAP1 version 2.5, with the chicken (Gallus gallus) genome assembly version 5.0 used as a reference sequence. After duplicate removal using picard2, methylation ratios were determined using the Python script (methratio.py) included with the BSMAP package. To analyze the ability to classify samples based on methylation data related to different antibiotic treatments, the R package Random Forest was used, and a Random Forest-based classification was built using the most variable CpG in each group.

[0097] Custom chip array data processing was performed using SeSAMe version 1.14.2 in R version 4.1.2. The DNA methylation level at each site was calculated as a methylation β value. The β value was defined as methylation signal / (methylation signal + unmethylation signal). It was calculated using the getBetas function. Quality checks were performed on the samples to remove probes with a detection p-value < 0.05 from all samples. The SeSAMe procedure (Zhou et al. 2018, Nucleic Acids Res.; 46(20):e123.) was used to generate normalized β values ​​and for quality control. Low-intensity detection calls and preparation (based on p-values) were performed using pOOBAH. Background subtraction based on normal exponential deconvolution using out-of-band probe noob (Triche et al. 2013, Nucleic Acids Res.; 41(7):e90.) and optionally with additional bleed-through subtraction were also implemented.

[0098] The methylation profile of the samples was then input into a constructed random forest classifier to identify the accurate feed groups.

[0099] The results revealed a methylation profile in piglets fed with enzymes that was consistent with positive properties of nutrient digestibility, while control piglets not fed with enzymes were more consistent with poor digestibility.

[0100] Example 2 To evaluate the feed efficiency of different nutrient sources in response feeds in broilers.

[0101] Feed conversion ratio (FCR; feed intake per kg / gain of live weight per kg) is a major determinant of profitability for chicken producers. To date, FCR has generally been measured by weighing an entire pen or rack of chickens and dividing by the total weight of the animals in that pen or rack. This requires many animals and a considerable amount of time to measure the variation. To measure feed efficiency in broilers in real time with fewer animals, epigenetic markers for FCR were created. Blood and breast meat samples from 1000 broilers were collected from various trials, each with a known and individually measured FCR for each chicken. DNA was extracted from the blood and breast muscle tissue of each broiler, bisulfite-converted, and sent for whole-genome bisulfite sequencing. CpG sites in the sampled broiler population were correlated with the measured FCR ranges to create penalized regression models that allowed prediction of individual broiler FCR from DNA methylation profiles. The correlated CpG sites were then used to create a custom methylation bead-based array for real-time determination of individual broiler FCR.

[0102] A broiler trial was conducted to compare two different sources of methionine as an essential amino acid and a frequently limiting amino acid in broiler diets. A total of 11 dietary treatments were used, including a methionine-deficient diet with no methionine supplementation, and five titrations of two supplemental methionine sources ranging from deficient to adequate to over-supplemented. Only 20 broilers were fed each dietary treatment for a one-week period. At the end of this period, blood was collected from each broiler for DNA extraction. The DNA from each sample was bisulfite-converted and analyzed using a custom bead-based array for FCR determination. Results showed that the lowest FCR (i.e., the most efficient utilization of nutrients for broiler growth) was observed at the fourth supplementation level using the first methionine source compared to the second methionine source. The recommendations for nutrient supplementation levels and sources are therefore possible with only 220 broilers, compared to standard trials that rely on weighing of animals and feed (which may require 10 pens of 20 chickens per treatment and thus 2200 broilers).

[0103] Data Analysis All CpGs associated with sex chromosomes were removed. All CpGs listed as SNPs related to the red junglefowl genome in the dbSNP database (https: / / www.ncbi.nlm.nih.gov / snp / ) were filtered out. Analysis was limited to CpGs showing chain-specific coverage greater than 10 in each sequencing sample. To establish a chicken methylation clock for the FCR, a penalized regression model (implemented in the R package glmnet47) was applied to perform regression analysis on the normalized methylation values ​​of CpG probes relative to the animal's FCR. This method assigns weights to the set of CpG probes and thus selects an optimized set of markers.

[0104] Custom chip array data processing was performed in R version 4.1.2 using sesame version 1.14.2. The DNA methylation level at each site was calculated as a methylation β value. The β value was defined as methylation signal / (methylation signal + unmethylation signal). It could be calculated using the getBetas function. Quality checks were performed on the samples to remove probes with a detection p-value < 0.05 from all samples. The SeSAMe procedure (Zhou et al. 2018, Nucleic Acids Res.; 46(20):e123.) was used to generate normalized β values ​​and for quality control. Detection calls and preparation based on low intensity (based on p-value) were performed using pOOBAH. Background subtraction based on normal exponential deconvolution using out-of-band probe noob (Triche et al. 2013, Nucleic Acids Res.; 41(7):e90.) and optionally with additional permeation subtraction were also implemented.

[0105] The FCR (methylation rate) is calculated based on the methylation profile of individual broilers using the FCR methylation clock.

Claims

1. A method for evaluating the effect of at least one test component of an animal feed on at least the performance and / or overall health of a test animal consuming an animal feed containing said test component, the method comprising the steps of: (a) Determine the test methylation profile of one or more preselected methylation sites in the DNA of the test animal; (b) Compare the test methylation profile obtained from (a) with at least one control methylation profile from a control animal that consumed animal feed containing no said test component and belonged to the same taxonomic unit as the test animal; and (c) Compare the test methylation profile obtained from (a) with the following (i) A first reference methylation profile obtained from a control animal of good performance and / or overall health that belongs to the same biological taxonomic unit as the test animal; and / or (ii) A second reference methylation profile obtained from control animals with poor performance and / or overall good health, belonging to the same biological taxonomic unit as the test animals; and The results of (a) are used to determine the performance and / or overall health of the test animals; and The significant similarity between the test methylation profile and the control methylation profile in (a) indicates that the test component has no effect on the performance and / or overall health of the test animals. and The significant difference between the test methylation profile and the control methylation profile in (a) indicates that the test component has an effect on the performance and / or overall health of the test animals; and The significant similarity between the test methylation profile in (a) and the first reference methylation profile, and / or the difference between the test methylation profile in (a) and the second reference methylation profile, indicate that the test animal has good performance and / or overall health, similar to control cells; and / or The difference between the test methylation profile in (a) and the first reference methylation profile and / or the significant similarity between the test methylation profile in (a) and the second reference methylation profile indicates that the test animal has poor performance and / or overall health as the control animal. and The test animals were selected from livestock or poultry.

2. The method of claim 1, wherein the overall health of the animal is a measurement of at least one indicator of the animal, and the indicator is selected from body weight or carcass weight, growth rate, animal feed intake, feed conversion ratio (FCR), digestive function, intestinal inflammation status, medical costs, transition period, antibiotic use, mortality rate and reproductive measurements.

3. An in vitro method for establishing the effect of at least one test component of an animal feed on the performance and / or overall health of a test animal consuming said animal feed, said method comprising the following steps: (a) Determine the test methylation level of the set of specific CpG sites in the test DNA of the test animal prior to consumption of the animal feed (T0). (b) Determine the methylation level of the same set of specific CpG sites in the test DNA of the test animal at a specific time point T1 after the consumption of the animal's feed, and (c) Compare the methylation levels of the same set of specific CpG sites from (a) and (b) with the methylation levels of the same CpG sites from a reference that correlates DNA methylation patterns with animal performance and / or overall health. This establishes the role of the test components in animal feed on the performance and / or overall health of the test animals. The test animals were selected from livestock or poultry.

4. The method according to any one of the preceding claims, wherein the livestock is aquatic livestock selected from carp, salmon, trout, tilapia, catfish, saltwater and brackish water fish, softshell turtle, Australian lungfish, shrimp, Chinese mitten crab, marbled crayfish and other decapod crustaceans, bivalves, gastropods, or the livestock is terrestrial livestock selected from cattle, goats, sheep, pigs, horses, donkeys, rabbits and mules, and / or the livestock is poultry selected from chickens, turkeys, ducks, geese and quails.

5. The method according to any one of the preceding claims, used to evaluate the effect of a test component of animal feed on the growth rate and / or feed conversion ratio (FCR) of meat-producing and / or milk-producing test animals consuming animal feed containing said test component.

6. The method according to any one of the preceding claims, wherein the components of the animal feed are selected from compound feeds, medicated feed additives, enzymes, probiotic microorganisms, direct-feeding microorganisms, antimicrobial agents, prebiotics, phytochemicals, immunomodulators, antibodies, plant extracts, essential oils, organic acids, antioxidants, amino acids, oligosaccharides, oleoresins, herbs, spices, saponins, marine plants, vitamins, minerals, chemical preservatives, antibiotics, and fermentation products.

7. The method according to any one of the preceding claims, wherein one or more preselected methylation sites in (a) are methylation sites associated with tissue-specific gene expression, preferably wherein the preselected methylation sites are associated with gene expression in a unique tissue.

8. The method according to any one of the preceding claims, wherein the test methylation profile in step (a) is determined by contacting the genomic material of the test animal with a DNA methylation-based array.

9. The method of claim 8, wherein the DNA methylation-based array is a bead-based array.

10. A method for establishing a reference to the method according to any one of the preceding claims, the method comprising: (a) Determine the performance and / or overall health of a specific animal DNA - methylation ratio and readout coverage of genomic CpG sites in associated training samples; (b) Using the reading coverage cutoff value, define the set of CpG sites with methylation ratios in all training samples from step (a); and (c) By applying a penalized regression model, the methylation rate of step (b) is used as input and the performance and / or overall health associated with the training samples are used as dependent variables; This yields a set of CpG sites, with corresponding weighting factors and the intercept of a penalized regression model as parameters for defining a reference that correlates DNA methylation patterns with animal performance and / or overall health. The test animals were selected from livestock or poultry.

11. The method of claim 10, wherein the methylation ratio and read coverage of the genomic CpG sites are determined in step (a) using bisulfite sequencing.