Equine insulin dysregulation

Abstract

Described is a method of diagnosing the presence or absence of insulin dysregulation in an equine animal. The method comprises (a) carrying out an assay to determine the level of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate and argininate, in a sample that has been obtained from the equine animal; and (b) comparing the level of the one or more biomarkers determined in step (a) to a reference result; wherein a difference between the level of the one or more biomarkers determined in step (a) and the reference result is used to determine the presence or absence of insulin dysregulation. Related methods of diagnosing the presence or absence of susceptibility to a condition associated with insulin dysregulation in an equine animal, methods of monitoring the level of one or more biomarkers in an equine animal and methods of assessing the effectiveness of a treatment for insulin dysregulation in an equine animal are also disclosed.

Description

[0001]

[0002] Background to the invention

[0003] One of the major endocrine conditions affecting equine animals is the equine metabolic syndrome (EMS). The original definition of the syndrome which linked insulin resistance, obesity and predisposition to laminitis was developed in the early 2000s (Johnson 2002, Hoffman et al. 2003, T reiber et al. 2005, T reiber et al. 2006). Laminitis is a very painful condition, that is common in equine animals, which involves disruption of the laminae tissues that connect the bones of the hoof to the hoof wall. This can result in sinking or rotation of the coffin bone (also known as the pedal bone) away from the hoof wall, which may ultimately lead to a need for euthanasia. Evidence to confirm the direct link between insulin and laminitis came from Australian researchers who showed that laminitis could be induced in ponies and horses by maintaining persistently high plasma insulin concentrations of around 1000 pill / mL for prolonged periods (Asplin etal. 2007, de Laat ef al. 2010). This led to the term the ‘equine metabolic syndrome’ becoming widely used to describe the phenotype of an animal with obesity and insulin resistance with a predisposition towards laminitis (Frank et al. 2010).

[0004] Initially, this label was applied to equids with: (i) increased adiposity, either in specific locations (regional adiposity) or generally (obesity); (ii) insulin resistance, characterised by hyperinsulinaemia or abnormal glycaemic and insulinaemic responses to oral or intravenous glucose and / or insulin challenges; and (ill) clinical or subclinical laminitis that developed in the absence of recognised causes such as grain overload, colic, colitis or retained placenta. More recently, however, there has been an increased acceptance that lean animals may have (even if they have never been obese) or retain (if they were previously obese) the EMS phenotype, and therefore an increased risk of laminitis. Accordingly, obesity and increased regional adiposity have now been excluded as being a necessary component of EMS. In short, obese animals may or may not be EMS animals, and lean animals may or may not be EMS animals. The recent consensus statement of the European College of Equine Internal Medicine (Durham et al. 2019) made this clear - the key consistent feature of EMS is the presence of insulin dysregulation (ID). The presence of obesity or other metabolic alterations in EMS animals is more variable.

[0005] Insulin dysregulation, therefore, is a collective term that indicates a disturbance of the balanced relationship between the plasma concentrations of insulin, glucose and lipids (Durham et al. 2019). This includes tissue insulin resistance, as well as basal and postprandial hyperinsulinemia. ID plays a central role in EMS, and EMS in turn is associated with an increased risk of laminitis. In fact, laminitis is the primary clinical consequence of EMS (Durham et al. 2019). Recent work has confirmed that the severity of the insulin dysregulation is associated with the extent of any laminitis risk independent of the body condition score (Knowles et al. 2023a, 2023b).

[0006] Insulin dysregulation is also associated with pituitary pars intermedia dysfunction (PPID) - over a third of horses with PPID are insulin dysregulated, and horses with PPID were found to be 2.7 times more likely to have hyperinsulinemia than age-matched controls (McGowan 2018). PPID, also known as equine Cushing’s disease, is a disease that is most commonly seen in older equine animals. The condition is characterised by a loss of inhibition of the pituitary gland (specifically the pituitary pars intermedia), which results in overproduction of pituitary hormones, including adrenocorticotropin (ACTH), alpha melanocyte stimulating hormone (a-MSH), P-endorphin and corticotrophin-like intermediate peptide (CLIP) (McGowan 2018). PPID is associated with a wide array of clinical signs such as increased coat length (hypertrichosis) and abnormal hair shedding patterns, laminitis, muscle wastage, abnormal fat distribution, increased drinking and urination (polydipsia and polyuria), increased susceptibility to infections and infertility.

[0007] Although insulin dysregulation is associated with a number of endocrine conditions in equine animals, it can be difficult to reliably diagnose. Body condition, as discussed above, cannot be used as a reliable indicator of ID status. Insulin dysregulation in equine animals may also be associated with old age. In a given animal, one or more different components of ID can be present, and different tests are recommended to evaluate these different ID components. Recent work in ID ponies has suggested that the insulin response to oral sugars may be one of the most important predictors of laminitis risk (Meier et al. 2019). The frequently sampled IV glucose tolerance test (FSIGTT) and the euglycemic hyperinsulinaemic clamp are primarily used to evaluate tissue insulin resistance, but these tests are costly and are not practical for routine use. Oral challenge tests assess an animal’s response to oral ingestion of hydrolysable carbohydrates (sugar and starch), which may be linked with laminitis risk. One such test, the oral sugar test (OST), is a practical test that can be performed easily in the field by clinicians and owners, although the recommended Karo Light Corn Syrup is not available in all countries. Moreover, diagnostic methods that rely on basal insulin concentrations can often result in misdiagnosis, as different reference ranges are required for different insulin diagnostic tests, and insulin concentrations can be affected by various factors including background diet, season, etc., meaning that the interpretation of test results can be challenging.

[0008] There is therefore a need for methods for diagnosing and monitoring ID (and susceptibility to related conditions that are associated with ID, such as EMS, laminitis, diabetes, obesity and PPID) that are more reliable and potentially cheaper and easier to carry out. Summary of the invention

[0009] The present inventors have developed a specific panel of biomarkers that each enable the consistent differentiation of equine animals with insulin dysregulation from equine animals without insulin dysregulation, regardless of diet or other potentially confounding factors. Accordingly, the invention provides a method of diagnosing the presence or absence of insulin dysregulation in an equine animal, based on the biomarkers present in a sample that has been obtained from the animal.

[0010] In one embodiment, the method of diagnosing the presence or absence of insulin dysregulation in an equine animal comprises:

[0011] (a) carrying out an assay to determine the level of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate and argininate, in a sample that has been obtained from the equine animal;

[0012] (b) comparing the level of the one or more biomarkers determined in step (a) to a reference result; wherein a difference between the level of the one or more biomarkers determined in step (a) and the reference result is used to determine the presence or absence of insulin dysregulation.

[0013] In one embodiment, step (a) comprises determining the ratio of the level of one or more of the biomarkers to the level of a standard marker in the sample that has been obtained from the equine animal, or determining the ratio of the levels of two or more of the biomarkers in the sample that has been obtained from the equine animal.

[0014] As insulin dysregulation is associated with a number of other conditions, these biomarkers are also indicative of an increased risk of or a susceptibility to one or more conditions associated with insulin dysregulation. Accordingly, the invention also provides a method of diagnosing the presence or absence of susceptibility to a condition associated with insulin dysregulation in an equine animal, based on the biomarkers present in a sample that has been obtained from the animal.

[0015] In one embodiment, the method of diagnosing the presence or absence of susceptibility to a condition associated with insulin dysregulation in an equine animal comprises:

[0016] (a) carrying out an assay to determine the level of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate and argininate, in a sample that has been obtained from the equine animal;

[0017] (b) comparing the level of the one or more biomarkers determined in step (a) to a reference result; wherein a difference between the level of the one or more biomarkers determined in step (a) and the reference result is used to determine the presence or absence of susceptibility to a condition associated with insulin dysregulation.

[0018] In one embodiment, step (a) comprises determining the ratio of the level of one or more of the biomarkers to the level of a standard marker in the sample that has been obtained from the equine animal, or determining the ratio of the levels of two or more of the biomarkers in the sample that has been obtained from the equine animal.

[0019] The invention also provides a method of monitoring the level of one or more biomarkers in an equine animal over time.

[0020] In one embodiment, the method of monitoring the level of one or more biomarkers in an equine animal comprises:

[0021] (a) carrying out an assay to determine the level of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate and argininate, in a sample that has been obtained from the equine animal; and

[0022] (b) repeating step (a) at two or more separate time points.

[0023] In one embodiment, step (a) comprises determining the ratio of the level of one or more of the biomarkers to the level of a standard marker in the sample that has been obtained from the equine animal, or determining the ratio of the levels of two or more of the biomarkers in the sample that has been obtained from the equine animal.

[0024] The invention also provides a method of assessing the effectiveness of a treatment for insulin dysregulation in an equine animal and a method of assessing the effectiveness of a treatment for a condition associated with insulin dysregulation in an equine animal.

[0025] In one embodiment, the method of assessing the effectiveness of a treatment for insulin dysregulation comprises:

[0026] (a) carrying out an assay to determine the level of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate and argininate, in a sample that has been obtained from the equine animal; and

[0027] (b) repeating step (a) at two or more separate time points, wherein at least one of the time points is after the equine animal has begun the treatment. In one embodiment, step (a) comprises determining the ratio of the level of one or more of the biomarkers to the level of a standard marker in the sample that has been obtained from the equine animal, or determining the ratio of the levels of two or more of the biomarkers in the sample that has been obtained from the equine animal.

[0028] Similarly, in one embodiment, the method of assessing the effectiveness of a treatment for a condition associated with insulin dysregulation comprises:

[0029] (a) carrying out an assay to determine the level of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate and argininate, in a sample that has been obtained from the equine animal; and

[0030] (b) repeating step (a) at two or more separate time points, wherein at least one of the time points is after the equine animal has begun the treatment.

[0031] In one embodiment, step (a) comprises determining the ratio of the level of one or more of the biomarkers to the level of a standard marker in the sample that has been obtained from the equine animal, or determining the ratio of the levels of two or more of the biomarkers in the sample that has been obtained from the equine animal.

[0032] Treatments for insulin dysregulation or a condition associated with insulin dysregulation may be intended to alter the level of one or more of the biomarkers of the invention in the equine animal in order to bring it in line with or closer to the level of that biomarker that is typical for a healthy equine animal. The effectiveness of a treatment may therefore be determined by assessing its ability to achieve a desired alteration to the level of one or more of the biomarkers of the invention in the samples that have been obtained from the equine animal. If a treatment achieves a desired alteration to the level of one or more of the biomarkers of the invention in the equine animal, e.g., if a treatment increases the level of propionylglycine in the equine animal or reduces the level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the equine animal, then the treatment may be determined to be effective. Conversely, if a treatment does not achieve a desired alteration to the level of one or more of the biomarkers of the invention in the equine animal, e.g., if a treatment does not increase the level of propionylglycine in the equine animal or does not reduce the level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the equine animal, then the treatment may be determined to be ineffective. Description of the drawings

[0033] Figure 1 shows the results of a metabolomic analysis of the levels of propionylglycine (Fig 1A), allantoin (Fig 1 B), nicotinamide riboside (Fig 1C), allantoic acid (Fig 1 D), hexadecatrienoic acid (Fig 1 E), argininate (Fig 1 F), argininosuccinate (Fig 1G), creatinine (Fig 1 H), phenylalanine (Fig 11), tryptophan (Fig 1 J), leucine (Fig 1 K), acetylcarnitine (Fig 1 L), and 1-palmitoyl-2-oleoyl-GPC (Fig 1 M) in samples obtained from ID and non-ID animals. The animals were fed three different treatment diets, and blood samples were taken before consumption (T1) and at 30 (T2), 60 (T3) and 90 (T4) minutes after consumption of the diets. The three panels within each figure correspond to the three diets and the four time points correspond to the four different blood samples (T1-T4). P1 indicates the ID animals and P2 indicates the non-ID animals. The level of each biomarker in each animal is measured relative to the median value for the level of that metabolite in the overall sample population ( / .e., all animals at all sampling occasions), which is set as a value of 1 .0.

[0034] Detailed description of the invention

[0035] The inventors have developed a panel of biomarkers that are present in different levels and / or ratios in equine animals with insulin dysregulation (ID animals), relative to equine animals without insulin dysregulation (non-ID animals). This panel was developed by collecting blood samples from ID and non-ID animals and identifying the levels of different biomarkers within these samples. Blood samples were taken from multiple ID and non-ID animals before and after the administration of various control diets, and certain biomarkers were consistently identified as differing in levels between the two groups, indicating that the levels of these biomarkers are indicative of insulin dysregulation in a manner that is independent of the diet that the animal has received and the time point at which the sample was obtained. Accordingly, these biomarkers can be used to distinguish between ID animals and non-ID animals.

[0036] The biomarkers that differ in levels between ID and non-ID animals include propionylglycine, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate and argininate. Accordingly, as used herein, the term “biomarkers of the invention” refers to the group of biomarkers consisting of: propionylglycine, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate and argininate. Nicotinamide riboside is a precursor of nicotinamide adenine dinucleotide (NAD) and is an important metabolic intermediate. Allantoin and allantoic acid form part of the purine degradation pathway, and are intermediates in the degradation of the adenine moiety of NAD. The urea cycle is responsible for the processing of ammonia that is produced from amino acid catabolism. The catabolism of certain amino acids, such as methionine, threonine, isoleucine and valine, also produces propionyl-CoA, which can be converted to propionylglycine. The term “urea cycle-associated metabolites” includes any metabolites that are involved in the urea cycle or in secondary pathways associated with the urea cycle. Specific urea cycle-associated metabolites include arginine, argininosuccinate, argininate, ornithine, 3-amino-2- piperidone, 2-oxoarginine, citrulline, homoarginine, homocitrulline, proline, dimethylarginine, N- acetylarginine, N-delta-acetylornithine, trans-4-hydroyxproline, N-methylproline, N,N,N-trimethyl- alanylproline betaine, prolylhydroxylproline aspartate, fumarate and urea. In a particular embodiment, the urea cycle-associated metabolites are argininosuccinate and argininate. The term “argininate” includes both the protonated form of argininic acid and the non-protonated argininate ion. Similarly, the term “hexadecatrienoic acid” includes both the protonated form and the nonprotonated form (commonly referred to as hexadecatrienoate) of the acid.

[0037] These biomarkers can be used to diagnose the presence or absence of insulin dysregulation or susceptibility to a condition associated with insulin dysregulation in an equine animal. In addition, the biomarkers can also be used to diagnose the presence or absence of susceptibility to a condition associated with insulin dysregulation in an equine animal. As used herein, the term “condition associated with insulin dysregulation” includes but is not limited to laminitis, equine metabolic syndrome, diabetes, obesity and pituitary pars intermedia dysfunction (PPID).

[0038] The biomarkers of the invention can also be used to monitor the development of insulin dysregulation and conditions associated with insulin dysregulation in equine animals that have not been diagnosed with these conditions. Once an equine animal is diagnosed with insulin dysregulation or a condition associated with insulin dysregulation, the biomarkers of the invention can be used to monitor the progression of these conditions, and / or to assess the effectiveness of any treatments that have been administered.

[0039] Assaying for biomarkers

[0040] The present invention provides methods of diagnosing the presence or absence of insulin dysregulation, methods of diagnosing the presence or absence of susceptibility to a condition associated with insulin dysregulation in an equine animal, methods of monitoring the level of one or more biomarkers in an equine animal, and methods of assessing the effectiveness of a treatment for insulin dysregulation or for a condition associated with insulin dysregulation.

[0041] The methods of the invention comprise a step of carrying out an assay to determine the level of one or more of the biomarkers of the invention in a sample that has been obtained from the equine animal (also referred to as “the equine animal to be diagnosed”). In one aspect, the assay step may comprise determining the level of two, three, four, five, six, seven, eight, nine, ten, eleven or twelve of the biomarkers of the invention in the sample.

[0042] As used herein, the term “level”, in the context of the level of one or more biomarkers in a sample, can refer to an absolute or a relative value indicating the level of the one or more biomarkers. For example, the level of one or more biomarkers in a sample may be expressed as a concentration of the one or more biomarkers in the sample or the total amount of the one or more biomarkers in the sample. In one embodiment, the sample that has been obtained from the equine animal may be a dried blood spot. In this case, the level of a biomarker in the sample may be expressed as the concentration of the biomarker (if the volume of the blood spot is known) or as the total amount of the biomarker in the dried blood spot. The level of one or more biomarkers in a sample may be expressed in any appropriate unit, depending on the assay that is used to determine the level.

[0043] In one embodiment, the assay step may comprise determining the concentration of one or more of the biomarkers in the sample. For example, the assay step may comprise measuring the concentration of one or more of (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve of) the biomarkers of the invention in the sample. In one embodiment, the assay step may comprise determining the total amount of one or more of the biomarkers in the sample.

[0044] As set out in more detail below, the level of one or more biomarkers in a sample may be expressed as a ratio of the level of one or more of the biomarkers in the sample to the level of another of the biomarkers in the sample. Accordingly, the assay step may comprise determining the ratio of the levels of two or more of the biomarkers in the sample that has been obtained from the equine animal

[0045] Alternatively, the level of one or more biomarkers in a sample may be expressed as a ratio of the level of one or more of the biomarkers in the sample to the level of a standard marker in the sample. In one aspect, the assay step may comprise determining the level of one or more of the biomarkers relative to the level of one or more standard markers in the sample. For example, the assay step may comprise determining the ratio of the level one or more of the biomarkers to the level of one or more standard markers in the sample. Accordingly, the methods of the invention may further comprise a step of carrying out an assay for one or more standard markers in the sample that has been obtained from the equine animal.

[0046] The standard marker may be any marker present in the sample that can be used as a comparator for the level of one or more of the biomarkers. For example, the standard marker may be selected to be a marker that is often or always present in the sample, that is easy to measure and / or that is present in the sample at a consistent level, i.e., that is not variable in level depending on the presence or absence of insulin dysregulation. The use of certain standard markers may require the samples to be collected from animals in a fasted state or without recent provision of forage and / or complementary feeds.

[0047] The standard marker may be an amino acid, or a fatty acid. In one embodiment, the standard marker may be selected from bicarbonate, bilirubin, calcium, chloride, cholesterol, creatinine, glucose, magnesium, phosphorus, potassium, sodium, urea, phenylalanine, tryptophan, leucine, acetylcarnitine, and 1-palmitoyl-2-oleoyl-GPC. In another embodiment, the standard marker may be a protein selected from albumin, transferrin, fibrinogen, haptoglobin, globulin, alanine transaminase, amylase, alkaline phosphatase, aspartate aminotransferase, creatine kinase, gamma-glutamyl transferase, lactate dehydrogenase and sorbitol dehydrogenase, or may be the total protein present in the sample. In a preferred embodiment, the standard marker is glucose or creatinine. Figures 1 H- M show that the levels of creatinine, phenylalanine, tryptophan, leucine, acetylcarnitine, and 1- palmitoyl-2-oleoyl-GPC in samples obtained from equine animals are relatively consistent between ID animals and non-ID animals. Accordingly, these markers represent potential suitable standard markers.

[0048] In one aspect, the assay step may comprise determining the relative levels of two or more of the biomarkers in the sample. For example, the assay step may comprise determining the ratio of the levels of two or more of the biomarkers in the sample. In one embodiment, the assay step may comprise determining the ratio of the level of propionylglycine to the level of allantoin, allantoic acid, hexadecatrienoic acid, nicotinamide riboside, or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the sample.

[0049] Suitable methods for measuring the level of a biomarker or a standard marker in a sample are well known in the art, and any appropriate technique may be used in the present methods. For instance, the step of carrying out an assay for one or more of the biomarkers of the invention may comprise a chromatography step, particularly a liquid chromatography (LC) step, such as high-performance liquid chromatography (HPLC) or ultra-high-performance liquid chromatography (UHPLC). The chromatography step may comprise separating and quantifying the biomarkers by applying the sample to a chromatographic column coupled to a detector. Suitable columns are well known in the art, and include hydrophobic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) columns. In some aspects, the step of carrying out an assay for one or more of the biomarkers may comprise a mass spectrometry (MS) step. The mass spectrometry step may be coupled to the chromatography step, such as in the form of an HPLC-MS, LC-MS or LC- MS / MS method. Techniques based on nuclear magnetic resonance (NMR) may also be used to determine the level of a specific biomarker or standard marker in a sample. The inventors have demonstrated that the differences in the levels of the biomarkers of the invention between samples that have been obtained from ID and non-ID equine animals are independent of the time at which the samples were obtained and of the diet that the equine animals were given. In the examples, animals were fed with three different diets and samples were obtained at 4 different time points, including before consumption of the diet and 30, 60 and 90 minutes after consumption. In all cases, significant differences were observed between the levels of the biomarkers of the invention between the samples that had been obtained from the ID and non-ID animals.

[0050] Accordingly, the sample upon which the assay step is conducted may be a sample that has been obtained from the equine animal to be diagnosed at any time point. In some embodiments, the sample is a sample that has been obtained prior to the consumption of food, or 30 minutes, 60 minutes or 90 minutes after the consumption of food. In some embodiments, the methods of the invention may comprise a step of carrying out an assay to determine the level of one or more of the biomarkers of the invention in multiple samples that have been obtained from the equine animal to be diagnosed at one or more different time points. For example, the samples may be samples that have been obtained from the equine animal to be diagnosed prior to the consumption of food, 30 minutes after the consumption of food, 60 minutes after the consumption of food and / or 90 minutes after the consumption of food. If the assay step involves determining the level of one or more of the biomarkers in multiple samples that have been obtained from the equine animal, the methods of the invention may comprise comparing the level of the one or more biomarkers determined in each sample to a reference result. Alternatively, the methods of the invention may comprise a step of taking an average (e.g., mean) of the level of the one or more biomarkers determined in each sample, and comparing the average (e.g., mean) level to a reference result.

[0051] The equine animal to be diagnosed may be administered any appropriate diet during the period before the sample is obtained. In some embodiments, the sample may be a sample that has been obtained from an equine animal that has received a control diet during the period before the sample is obtained. The control diet may be a low non-structural carbohydrate (LNSC) diet, such as a diet containing less than 20% non-structural carbohydrates (typically defined as simple sugars and starch). Suitable LNSC diets are well known in the art. The control diet may be administered to the equine animal to be diagnosed for a time period of at least one week before the sample is obtained, such as for at least two weeks, at least three weeks or at least a month before the sample is obtained. Reference results

[0052] The diagnosis methods of the invention comprise a further step of comparing the level of the one or more biomarkers determined in the assay step to a reference result. The reference result is intended to represent the level of one or more of the biomarkers of the invention in a control equine animal. The term “level” in this context has the same meaning as defined above in relation to the assay step. Accordingly, any differences that are identified between the level of the one or more biomarkers determined in the assay step and the reference result may be used to determine the presence or absence of ID and / or susceptibility to conditions associated with ID.

[0053] As used herein, the term “control equine animal” refers to an equine animal to which the equine animal to be diagnosed can be compared. Specifically, the step of comparing the level of the one or more biomarkers determined in the assay step to a reference result may comprise comparing the level of one or more of the biomarkers of the invention in a sample that has been obtained from the equine animal to be diagnosed to the level of that biomarker in a sample that has been obtained from a control equine animal.

[0054] In some embodiments, the control equine animal may be a healthy equine animal. As used herein, the term “healthy equine animal” or “healthy animal” refers to an equine animal that does not suffer from, has not been diagnosed with, or has been confirmed not to have insulin dysregulation, laminitis, equine metabolic syndrome, diabetes, obesity or pituitary pars intermedia dysfunction. If the control equine animal is a healthy equine animal, then a difference between the level of the one or more biomarkers determined in the assay step and the reference result may be used to determine the presence of ID and / or susceptibility to conditions associated with ID.

[0055] In some embodiments, the control equine animal may be an unhealthy equine animal. As used herein, the term “unhealthy equine animal” or “unhealthy animal” refers to an equine animal that suffers from, has been diagnosed with or has otherwise been confirmed to have insulin dysregulation, laminitis, equine metabolic syndrome, diabetes, obesity or pituitary pars intermedia dysfunction. If the control equine animal is an unhealthy equine animal, then a difference between the level of the one or more biomarkers determined in the assay step and the reference result may be used to determine the absence of ID and / or susceptibility to conditions associated with ID.

[0056] The reference result may be a result from an assay to determine the level of one or more of the biomarkers in a sample that has been obtained from a control equine animal. The assay conducted on the sample that has been obtained from the control equine animal may correspond to the assay step described above that is conducted on a sample that has been obtained from the equine animal to be diagnosed. Similarly, the level of one or more of the biomarkers in the sample that has been obtained from the control equine animal may be expressed in the same manner as the level of one or more of the biomarkers in the sample that has been obtained from the equine animal to be diagnosed. Accordingly, in one embodiment, the reference result may comprise a value for the concentration of the one or more biomarkers in the sample that has been obtained from the control equine animal or a value for the total amount of the one or more biomarkers in the sample that has been obtained from the control equine animal.

[0057] In one embodiment, the reference result may comprise the ratio of the level of one or more of the biomarkers of the invention to the level of a standard marker in the sample that has been obtained from the control equine animal. The standard marker that is used in the context of the reference result can be any of the suitable standard markers outlined above. Preferably, the standard marker used in the context of the reference results is the same as the standard marker used in the context of the assay step, i.e., the standard marker used in the assay conducted on the sample that has been obtained from the control equine animal is the same as the standard marker used in the assay conducted on the sample that has been obtained from the equine animal to be diagnosed.

[0058] In one embodiment, the reference result may comprise the ratio of the levels of two or more of the biomarkers of the invention in the sample that has been obtained from the control equine animal. For example, the reference result may comprise the ratio of the level of propionylglycine to the level of allantoin, allantoic acid, hexadecatrienoic acid, nicotinamide riboside, or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the sample that has been obtained from the control equine animal.

[0059] In one embodiment, the reference result may comprise a set of reference results. For example, the reference result may comprise a set of reference results including the results of one or more assays to determine the level of one or more of the biomarkers of the invention conducted on samples that have been obtained from two or more control equine animals, such as 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more control equine animals. In some embodiments, the set of reference results may include the results of any assay to determine the level of one or more of the biomarkers of the invention conducted on two or more samples that have been obtained from the same control equine animal at different time points, such as 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more samples that have been obtained from a control equine animal. Again, the level of one or more of the biomarkers in the samples that have been obtained from the control equine animal(s) may be expressed in the same manner as the level of one or more of the biomarkers in the sample that has been obtained from the equine animal to be diagnosed. As outlined above, the set of reference results may comprise values for the concentration of the one or more biomarkers in the samples that have been obtained from the control equine animal(s) or values for the total amount of the one or more biomarkers in the samples that have been obtained from the control equine animal(s). In one embodiment, the set of reference results may comprise the ratios of the level of one or more of the biomarkers of the invention to the level of a standard marker in the samples that have been obtained from the control equine animal(s). In one embodiment, the set of reference results may comprise the ratios of the levels of two or more of the biomarkers of the invention in the samples that have been obtained from the control equine animal(s). If one or more assays to determine the level of one or more of the biomarkers of the invention are conducted on samples that have been obtained from multiple control equine animals, the reference result may comprise an average value for the level of each biomarker in the samples obtained from the control equine animals. For example, the reference result may comprise the median level of each biomarker in the samples obtained from the control equine animals, or the mean level of each biomarker in the samples obtained from the control equine animals.

[0060] If the control equine animals comprise one or more healthy equine animals and one or more unhealthy equine animals, the reference result may comprise an average value for the level of each biomarker in the samples obtained from the healthy control equine animals and an average value for the level of each biomarker in the samples obtained from the unhealthy control equine animals. For example, the reference result may comprise the median level of each biomarker in the samples obtained from the healthy control equine animals and the median level of each biomarker in the samples obtained from the unhealthy control equine animals, or the mean level of each biomarker in the samples obtained from the healthy control equine animals and the mean level of each biomarker in the samples obtained from the unhealthy control equine animals.

[0061] The control equine animal(s) may be of the same category and / or the same age or life stage as the equine animal to be diagnosed. Animals from the same category may be animals from the same taxonomic family, genus or species, and / or from the same breed. Equine animals may be categorised into one of the following life stages: foal (from 0-6 months); weanling (from 6-12 months), yearling (from 1-2 years), adolescent (from 2-4 years), adult (from 4-20 years) senior (from 20 years onwards). For example, if the equine animal to be diagnosed is an adult horse, the set of reference results may comprise the results of an assay to determine the level of one or more of the biomarkers of the invention conducted on sample(s) that have been obtained from one or more control adult horses.

[0062] In some embodiments, the methods of the invention may comprise a further step of generating the reference result. For example, the methods of the invention may comprise a step of carrying out one or more assays to determine the level of one or more of the biomarkers of the invention in sample(s) that have been obtained from one or more control equine animals. If samples have been obtained from multiple control equine animals or if multiple assays have been conducted, for example on samples obtained from the same control equine animal at different time points, the results can then be compiled into a set of reference results. As in the assay step described above conducted on the sample that has been obtained from the equine animal to be diagnosed, this step of assaying sample(s) that have been obtained from one or more control equine animals may comprise measuring the concentration of one or more of the biomarkers of the invention; determining the level of one or more of the biomarkers of the invention relative to the level of a standard marker; and / or determining the relative levels of two or more of the biomarkers.

[0063] The reference result does not need to be prepared every time the methods of the invention are performed. Instead, a skilled person can rely on an established reference result.

[0064] In one embodiment, the reference result may comprise a pre-determined threshold for one or more of the biomarkers of the invention. The pre-determined threshold for each biomarker may be a concentration that is considered to be healthy ( / .e., not indicative of ID or an associated condition) or typical for a healthy equine animal or a concentration that is considered to be unhealthy ( / .e., indicative of ID or an associated condition) or typical for an unhealthy equine animal. Again, any differences that are identified between the level of the one or more biomarkers determined in sample that has been obtained from the equine animal to be diagnosed and the pre-determined threshold may be used to determine the presence or absence of ID and / or susceptibility to conditions associated with ID.

[0065] In some embodiments, the pre-determined threshold for each biomarker may be based on the level of that biomarker in one or more control equine animals, e.g., one or more control equine animals that are of the same category and / or of the same age or life stage as the equine animal to be diagnosed. The pre-determined threshold for each biomarker may be determined by assessing the level of that biomarker in one or more control equine animals of the same category and optionally the same life stage as the equine animal to be diagnosed, and taking the average (e.g., mean) value. For example, if the equine animal to be diagnosed in an adult horse, the pre-determined threshold for each biomarker may be the average (e.g., mean) level of that biomarker in a healthy adult horse or an unhealthy adult horse. The pre-determined thresholds do not need to be determined every time the method of the invention is performed. Instead, the skilled person can rely on established thresholds.

[0066] In some embodiments, the pre-determined threshold can be a fixed value, i.e., a single point, or a fixed range of values. The pre-determined threshold may vary between different genera or species of equine animals and / or between equine animals of different life stages. The skilled person will appreciate that equine animals of different genera or species or different life stages may typically have higher or lower levels of the biomarkers, and thus will be capable of making any necessary adjustments to pre-determined threshold values based on the level of the respective biomarkers in an equine animal of a specific species and life stage.

[0067] Diagnosis

[0068] The present inventors have shown that the biomarkers of the invention can be used to distinguish ID equine animals from non-ID equine animals. On this basis, they have developed a method of diagnosing the presence or absence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation in an equine animal.

[0069] The biomarkers of the invention differ in level between ID equine animals and non-ID equine animals. Accordingly, the step of comparing the level of the one or more biomarkers determined in the assay step ( / .e., the level of the one or more biomarkers determined in the assay conducted on a sample that has been obtained from the equine animal to be diagnosed) to the reference result allows for the diagnosis of the presence or absence of insulin dysregulation and / or the presence or absence of susceptibility to a condition associated with insulin dysregulation. In some embodiments, the diagnosis of the presence or absence of insulin dysregulation and / or the presence or absence of susceptibility to a condition associated with insulin dysregulation may be based on the level of two, three, four, five, six, seven, eight, nine, ten, eleven or twelve of the biomarkers of the invention in the sample.

[0070] A difference between the level of the one or more biomarkers determined in the assay step and the reference result can be used to determine the presence or absence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. Alternatively put, a diagnosis of the presence or absence of insulin dysregulation and / or the presence or absence of susceptibility to a condition associated with insulin dysregulation can be based on an indication that one or more of the biomarkers of the invention is present in a sample that has been obtained from the equine animal to be diagnosed at a level that is lower or higher than the reference result.

[0071] Propionylglycine is present at a lower level in ID equine animals, relative to non-ID equine animals. Thus, if the control equine animal is a healthy equine animal, i.e., if the reference result comprises the result from an assay to determine the level of one or more of the biomarkers in a sample that has been obtained from a healthy equine animal, a lower level of propionylglycine in the sample that has been obtained from the equine animal to be diagnosed, relative to the reference result, is indicative of the presence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. This lower level of propionylglycine may be a level that is lower relative to the result of an assay conducted on sample(s) that have been obtained from one or more control equine animals, or lower relative to a pre-determined threshold for propionylglycine.

[0072] In this context, the term “lower” refers to a level of propionylglycine in the sample that has been obtained from the equine animal to be diagnosed that is lower than the reference result by at least a specific margin, such as by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%. Alternatively put, a level of propionylglycine in the sample that has been obtained from the equine animal to be diagnosed that is lower than the reference result (obtained from a healthy equine animal) by at least 5%, such as by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% may be indicative of the presence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. In some embodiments, a level of propionylglycine in the sample that has been obtained from the equine animal to be diagnosed that is lower than the reference result (obtained from a healthy equine animal) by a statistically significant margin may be indicative of the presence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation.

[0073] If the control equine animal is a healthy equine animal, i.e., if the reference result comprises the result from an assay to determine the level of one or more of the biomarkers in a sample that has been obtained from a healthy equine animal, then a level of propionylglycine in the sample that has been obtained from the equine animal to be diagnosed that is not lower than the reference result by at least 5%, such as by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% may be indicative of the absence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. In some embodiments, a level of propionylglycine in the sample that has been obtained from the equine animal to be diagnosed that is not lower than the reference result (obtained from a healthy equine animal) by a statistically significant margin may be indicative of the absence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. In one embodiment, a level of propionylglycine in the sample that has been obtained from the equine animal to be diagnosed that is within 50% of the reference result (obtained from a healthy equine animal), i.e., that does not differ from the reference result by more than 50% in either direction, may be indicative of the absence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. A level of propionylglycine in the sample that has been obtained from the equine animal to be diagnosed that is within 45% of the reference result (obtained from a healthy equine animal), such as within 40%, within 35%, within 30%, within 25%, within 20%, within 15%, within 10%, or within 5% of the reference result, may be indicative of the absence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation.

[0074] Conversely, if the control equine animal is an unhealthy equine animal, i.e., if the reference result comprises the result from an assay to determine the level of one or more of the biomarkers in a sample that has been obtained from an unhealthy equine animal, then a level of propionylglycine in the sample that has been obtained from the equine animal to be diagnosed that is not higher than the reference result by at least 5%, such as by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% may be indicative of the presence of insulin dysregulation and / or susceptibility to a condition associated with the presence of insulin dysregulation. In some embodiments, a level of propionylglycine in the sample that has been obtained from the equine animal to be diagnosed that is not higher than the reference result (obtained from an unhealthy equine animal) by a statistically significant margin may be indicative of the presence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. In one embodiment, a level of propionylglycine in the sample that has been obtained from the equine animal to be diagnosed that is within 50% of the reference result (obtained from an unhealthy equine animal), i.e., that does not differ from the reference result by more than 50% in either direction, may be indicative of the presence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. A level of propionylglycine in the sample that has been obtained from the equine animal to be diagnosed that is within 45% of the reference result (obtained from an unhealthy equine animal), such as within 40%, within 35%, within 30%, within 25%, within 20%, within 15%, within 10%, or within 5% of the reference result, may be indicative of the presence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation.

[0075] Allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate and argininate, are present at a higher level in ID equine animals, relative to non-ID equine animals. Thus, if the control equine animal is a healthy equine animal, i.e., if the reference result comprises the result from an assay to determine the level of one or more of the biomarkers in a sample that has been obtained from a healthy equine animal, a higher level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle- associated metabolite, such as argininosuccinate or argininate, in the sample that has been obtained from the equine animal to be diagnosed, relative to the reference result, is indicative of the presence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. Again, this higher level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, may be a level that is higher relative to the result of an assay conducted on sample(s) that have been obtained from one or more control equine animals, or higher relative to a pre-determined threshold for that biomarker of the invention.

[0076] In this context, the term “higher” refers to a level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the sample that has been obtained from the equine animal to be diagnosed that is higher than the reference result for that biomarker of the invention by at least a specific margin, such as by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%. Alternatively put, a level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the sample that has been obtained from the equine animal to be diagnosed that is higher than the reference result for that biomarker of the invention by at least 5%, such as by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% may be indicative of the presence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. In some embodiments, a level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle- associated metabolite, such as argininosuccinate or argininate, in the sample that has been obtained from the equine animal to be diagnosed that is higher than the reference result (obtained from a healthy equine animal) for that biomarker of the invention by a statistically significant margin may be indicative of the presence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation.

[0077] If the control equine animal is a healthy equine animal, i.e., if the reference result comprises the result from an assay to determine the level of one or more of the biomarkers in a sample that has been obtained from a healthy equine animal, then a level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the sample that has been obtained from the equine animal to be diagnosed that is not higher than the reference result for that biomarker of the invention by at least 5%, such as by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% may be indicative of the absence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. In some embodiments, a level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the sample that has been obtained from the equine animal to be diagnosed that is not higher than the reference result (obtained from a healthy equine animal) for that biomarker of the invention by a statistically significant margin may be indicative of the absence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. In one embodiment, a level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the sample that has been obtained from the equine animal to be diagnosed that is within 50% of the reference result (obtained from a healthy equine animal) for that biomarker of the invention, i.e., that does not differ from the reference result by more than 50% in either direction, may be indicative of the absence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. A level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the sample that has been obtained from the equine animal to be diagnosed that is within 45% of the reference result (obtained from a healthy equine animal) for that biomarker of the invention, such as within 40%, within 35%, within 30%, within 25%, within 20%, within 15%, within 10%, or within 5% of the reference result, may be indicative of the absence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation.

[0078] Conversely, if the control equine animal is an unhealthy equine animal, i.e., if the reference result comprises the result from an assay to determine the level of one or more of the biomarkers in a sample that has been obtained from an unhealthy equine animal, then a level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the sample that has been obtained from the equine animal to be diagnosed that is not lower than the reference result for that biomarker of the invention by at least 5%, such as by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% may be indicative of the presence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. In some embodiments, a level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the sample that has been obtained from the equine animal to be diagnosed that is not lower than the reference result (obtained from an unhealthy equine animal) for that biomarker of the invention by a statistically significant margin may be indicative of the presence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. In one embodiment, a level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the sample that has been obtained from the equine animal to be diagnosed that is within 50% of the reference result (obtained from an unhealthy equine animal) for that biomarker of the invention, i.e., that does not differ from the reference result by more than 50% in either direction, may be indicative of the presence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. A level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the sample that has been obtained from the equine animal to be diagnosed that is within 45% of the reference result (obtained from an unhealthy equine animal) for that biomarker of the invention, such as within 40%, within 35%, within 30%, within 25%, within 20%, within 15%, within 10%, or within 5% of the reference result, may be indicative of the presence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation.

[0079] If an equine animal is indicated to have insulin dysregulation or to be susceptible to a condition associated with insulin dysregulation, the methods of the present invention may further comprise a step of providing a recommendation for an intervention.

[0080] In some embodiments, the intervention may be a visit to a veterinary professional. The recommendation may therefore be that a visit to a veterinary professional is required. In some embodiments, the intervention may be a notification that the animal is at risk of developing a condition associated with insulin dysregulation, such as laminitis, and should be monitored. The recommendation may therefore be that an assay for one or more of the biomarkers of the invention in a sample that has been obtained from the equine animal is carried out again within a certain time frame from the recommendation, e.g., within six months, four months, three months, two months or one month. In some embodiments, the intervention may be a treatment regimen for insulin dysregulation or a condition associated with insulin dysregulation. The recommendation may therefore be that a treatment regimen is adopted. This treatment regimen may include dietary management, exercise, and / or appropriate medication, as well as core management changes. Core management changes may be intended to treat the underlying condition and / or to remove the inciting cause. For example, this may include the removal of feeds that may have been involved in the development of ID or a condition associated with ID and feeds that have the potential to exacerbate the severity of ID or a condition associated with ID. Animals may be removed from pasture and provided with a diet low in non-structural carbohydrates (NSC). Core management changes may further include one or more or all of treatment to reduce blood insulin, treatment to minimise lamellar damage, provision of analgesia, treatment to promote weight loss, general husbandry and shoeing.

[0081] In some embodiments, the methods of the invention further comprise implementing the recommendation for an intervention, i.e., taking the equine animal to a veterinary profession, or starting treatment for insulin dysregulation or a condition associated with insulin dysregulation.

[0082] Monitoring methods

[0083] The invention also provides a method of monitoring the level of one or more of the biomarkers of the invention in an equine animal over time. The method of monitoring the level of one or more biomarkers in an equine animal comprises:

[0084] (a) carrying out an assay to determine the level of one or more biomarkers in a sample that has been obtained from the equine animal, wherein the biomarkers are selected from the group consisting of: propionylglycine, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate and argininate; and

[0085] (b) repeating step (a) at two or more separate time points.

[0086] In this monitoring method, the two or more separate time points may be separated by a period of at least one week, such as at least two weeks, at least three weeks, at least one month, at least two months, at least three months, at least four months, at least six months, at least nine months, at least twelve months or more. The method is not restricted to repeating the assay of step (a) at only two time points. The assay may be repeated at three or more time points, such as at four, five, six, seven, eight, nine, ten or more time points.

[0087] The method of monitoring will provide two or more measurements of the level of one or more of the biomarkers of the invention in the samples that have been obtained from the equine animal, wherein each measurement corresponds to the level of a given biomarker in a sample that has been obtained from the equine animal at a different time point. In some embodiments, the method may further comprise a step of comparing at least two of the measurements of the level of one or more of the biomarkers in the samples that have been obtained from the equine animal.

[0088] If an equine animal does not suffer from, has not been diagnosed with or has been confirmed not to have insulin dysregulation and / or a condition associated with insulin dysregulation, then the method of monitoring the level of one or more of the biomarkers of the invention in an equine animal may also be seen as a method of monitoring the development of insulin dysregulation and / or a condition associated with insulin dysregulation in the equine animal. The results of the comparison between different measurements of the level of one or more of the biomarkers of the invention in the samples that have been obtained from the equine animal may indicate that the equine animal is developing insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation.

[0089] As noted above, propionylglycine is present at a lower level in ID equine animals, relative to non-ID equine animals, and allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate and argininate, are present at a higher level in ID equine animals, relative to non-ID equine animals. Accordingly, in some embodiments, a decrease in the level of propionylglycine between measurements in two samples that have been obtained from an equine animal, i.e., where the level of propionylglycine in the sample obtained at a later time point is lower than the level of propionylglycine in the sample obtained at an earlier time point, may indicate that the animal is developing insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. Conversely, the absence of a decrease in the level of propionylglycine between measurements in two samples that have been obtained from an equine animal may indicate that the animal is not developing insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation.

[0090] Similarly, an increase in the level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, between measurements in two samples that have been obtained from an equine animal, i.e., where the level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle- associated metabolite, such as argininosuccinate or argininate, in the sample obtained at a later time point is higher than the level of that biomarker of the invention in the sample obtained at an earlier time point, may indicate that the animal is developing insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. The absence of an increase in the level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, between measurements in two samples that have been obtained from an equine animal may indicate that the animal is not developing insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation.

[0091] In this context, the term “decrease” refers to a decrease in the level of one or more of the biomarkers of the invention as compared to the level in a sample obtained at an earlier time point (e.g., a level that is lower in a sample obtained at a second or subsequent point as compared to a level in a sample obtained at a first time point). The decrease may be by at least a specific margin, such as by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%, between measurements in two samples that have been obtained from the equine animal (e.g., between consecutive measurements). Propionylglycine is present at a lower level in ID equine animals, relative to non-ID equine animals. Thus, a decrease in the level of propionylglycine by at least 5%, such as by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% between measurements in two samples that have been obtained from the equine animal may indicate that the animal is developing insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. In some embodiments, a decrease in the level of propionylglycine by a statistically significant margin between measurements in two samples that have been obtained from the equine animal may indicate that the animal is developing insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. Conversely, the absence of a decrease in the level of propionylglycine by at least 5%, such as by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% between measurements in two samples that have been obtained from the equine animal may indicate that the animal is not developing insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. The absence of a decrease in the level of propionylglycine by a statistically significant margin between measurements in two samples that have been obtained from the equine animal may indicate that the animal is not developing insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation.

[0092] In this context, the term “increase” refers to an increase in the level of one or more of the biomarkers of the invention as compared to the level in a sample obtained at an earlier time point (e.g., a level that is higher in a sample obtained at a second or subsequent point as compared to a level in a sample obtained at a first time point). The increase may be by at least a specific margin, such as by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%, e.g., between measurements in two samples that have been obtained from the equine animal (e.g., between consecutive measurements). Allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate or argininate, are present at a higher level in ID equine animals, relative to non-ID equine animals. Thus, an increase in the level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid or a urea cycle-associated metabolite, such as argininosuccinate or argininate, by at least 5%, such as by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% between measurements in two samples that have been obtained from the equine animal may indicate that the animal is developing insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. In some embodiments, an increase in the level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid or a urea cycle-associated metabolite, such as argininosuccinate or argininate, by a statistically significant margin between measurements in two samples that have been obtained from the equine animal may indicate that the animal is developing insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation.

[0093] Conversely, the absence of an increase in the level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid or a urea cycle-associated metabolite, such as argininosuccinate or argininate, by at least 5%, such as by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% between measurements in two samples that have been obtained from the equine animal may indicate that the animal is not developing insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation. The absence of an increase in the level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid or a urea cycle-associated metabolite, such as argininosuccinate or argininate, by a statistically significant margin between measurements in two samples that have been obtained from the equine animal may indicate that the animal is not developing insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation.

[0094] If the equine animal has already been diagnosed with insulin dysregulation or a condition associated with insulin dysregulation, the method of monitoring the level of one or more of the biomarkers of the invention may be seen as a method of monitoring the progression of insulin dysregulation or a condition associated with insulin dysregulation in the equine animal. The results of the comparison between different measurements of the level of one or more of the biomarkers of the invention in the samples that have been obtained from the equine animal may indicate that the insulin dysregulation or the condition associated with insulin dysregulation is progressing, stable or regressing.

[0095] In this context, the term “progressing” refers to a clinical situation that is developing or worsening over time, e.g., a situation in which the symptoms displayed by the equine animal are getting more severe, more frequent or longer in duration. The term “stable" refers to a clinical situation which is not significantly changing over time, i.e., a situation which is neither worsening nor improving. This may be indicated by symptoms that are consistent or unchanged. The term “regressing” refers to a clinical situation that is improving over time, e.g., a situation in which the symptoms displayed by the equine animal may become less severe, less frequent, or shorter in duration. The skilled person will recognise these different clinical situations and will appreciate their significance in the monitoring of equine animals.

[0096] For example, in some embodiments, a decrease in the level of propionylglycine between measurements in two samples that have been obtained from the equine animal (e.g. the equine animal with insulin dysregulation or a condition associated with insulin dysregulation) may indicate that the insulin dysregulation or the condition associated with insulin dysregulation is progressing. Similarly, an increase in the level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid or a urea cycle-associated metabolite, such as argininosuccinate or argininate, between measurements in two samples that have been obtained from the equine animal (e.g., the equine animal with insulin dysregulation or a condition associated with insulin dysregulation) may indicate that the insulin dysregulation or the condition associated with insulin dysregulation is progressing.

[0097] Conversely, an increase in the level of propionylglycine between measurements in two samples that have been obtained from the equine animal (e.g., the equine animal with insulin dysregulation or a condition associated with insulin dysregulation) may indicate that the insulin dysregulation or the condition associated with insulin dysregulation is regressing. Similarly, a decrease in the level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid or a urea cycle-associated metabolite, such as argininosuccinate or argininate, between measurements in samples that have been obtained from the equine (e.g., the equine animal with insulin dysregulation or a condition associated with insulin dysregulation) animal may indicate that the insulin dysregulation or the condition associated with insulin dysregulation is regressing.

[0098] If the level of one or more of the biomarkers of the invention in the samples that have been obtained from the equine animal neither increases nor decreases between measurements in two samples that have been obtained from the equine animal (e.g., the equine animal with insulin dysregulation or a condition associated with insulin dysregulation), this may indicate that, the insulin dysregulation or the condition associated with insulin dysregulation is stable. The terms “increase” and “decrease” in the context of a method of monitoring are defined in detail above. The level of one or more of the biomarkers of the invention may be determined to have neither increased or decreased between measurements in two samples that have been obtained from an equine animal if neither of the above definitions of an increase and a decrease is satisfied. For example, the level of one or more of the biomarkers of the invention may be determined to have neither increased or decreased between measurements in two samples that have been obtained from an equine animal if the level has changed by less than 50% in either direction, such as by less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10% or less than 5% in either direction. In one embodiment, the level of one or more of the biomarkers of the invention may be determined to have neither increased nor decreased between measurements in two samples that have been obtained from an equine animal if the level has not changed by a statistically significant amount.

[0099] The monitoring of the level of one or more of the biomarkers of the invention in an equine animal may be continuous. For example, the monitoring method may comprise repeating the assay of step (a) and (b) about every week, every two weeks, every month, every six weeks, every two months, every three months, every four months, every six months, every nine months or every year (e.g. over a period of up to six months, a year, two years, three years, four years, five years).

[0100] This type of continuous monitoring is useful when an equine animal is being treated for insulin dysregulation or for a condition associated with insulin dysregulation. In this regard, the invention also provides a method of assessing the effectiveness of a treatment for insulin dysregulation in an equine animal and a method of assessing the effectiveness of a treatment for a condition associated with insulin dysregulation in an equine animal. Insulin dysregulation and conditions associated with insulin dysregulation are typically treated with dietary management and exercise, and / or with medication as well as core management changes. Potential core management changes are defined above. Dietary treatments of insulin dysregulation and conditions associated with insulin dysregulation may involve caloric restriction and / or restriction of non-structural carbohydrates (NSC). Medications that may be administered to treat insulin dysregulation or a condition associated with insulin dysregulation include levothyroxine, SLGT-2 inhibitors, such as velagliflozin, canagliflozin, and ertugliflozin, biguanides, such as metformin, GLP- 1 analogues, such as exenatide, GLP-1 inhibitors and equivalents (Meier et al. 2019, de Laat 2023).

[0101] The method of assessing the effectiveness of a treatment for insulin dysregulation in an equine animal comprises:

[0102] (a) carrying out an assay to determine the level of one or more biomarkers in a sample that has been obtained from the equine animal, wherein the biomarkers are selected from the group consisting of: propionylglycine, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate and argininate;

[0103] (b) repeating step (a) at two or more separate time points, wherein at least one of the time points is after the equine animal has begun the treatment.

[0104] Similarly, the method of assessing the effectiveness of a treatment for a condition associated with insulin dysregulation in an equine animal comprises:

[0105] (a) carrying out an assay to determine the level of one or more biomarkers in a sample that has been obtained from the equine animal, wherein the biomarkers are selected from the group consisting of: propionylglycine, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate and argininate;

[0106] (b) repeating step (a) at two or more separate time points, wherein at least one of the time points is after the equine animal has begun the treatment.

[0107] Accordingly, the method involves assay carrying out an assay to determine the level one or more of the biomarkers of the invention in a sample that has been obtained from the equine animal on at least two time points. The first time point may be before the animal has begun treatment, in order to provide a baseline assessment of the level of the biomarkers of the invention before treatment begins. Again, the time points may be separated by a period of at least one week, such as at least two weeks, at least three weeks, at least one month, at least two months, or more (e.g., over a period of up to six months, a year, two years, three years, four years, five years).

[0108] At least one of the time points must be after the equine animal has begun the treatment. This time point may be selected so as to allow sufficient time after the treatment begins for it to have an effect on the level of the biomarkers of the invention. For example, the time point after the equine animal has begun the treatment may be at least one week after the treatment is begun, such as at least two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks or eight weeks, after the treatment is begun.

[0109] Again, the method is not restricted to repeating the assay of step (a) at only two time points. These steps may be repeated at three or more time points, such as at four, five, six, seven, eight, nine, ten or more time points. The monitoring of the effectiveness of the treatment may continue for the duration of the period that the treatment is administered. For example, the effectiveness of the treatment may be monitored regularly, such as once every two weeks, once every month, once every six weeks, once every two months, once every four months, once every six months or once every year.

[0110] As in the monitoring methods outlined above, the method of assessing the effectiveness of a treatment for insulin dysregulation or a condition associated with insulin dysregulation will provide two or more measurements of the level of one or more of the biomarkers of the invention in the samples that have been obtained from the equine animal, wherein each measurement corresponds to the level of a given biomarker in a sample that has been obtained from the equine animal at a different time point. In some embodiments, the method may further comprise a step of comparing at least two of the measurements of the level of one or more of the biomarkers in the samples that have been obtained from the equine animal. The results of the comparison between different measurements of the level of one or more of the biomarkers of the invention in the samples that have been obtained from the equine animal may indicate that the treatment is effective or ineffective.

[0111] A treatment for insulin dysregulation or a condition associated with insulin dysregulation may be intended to alter the level of one or more of the biomarkers of the invention in the equine animal in order to bring it in line with or closer to the level that is typical for a healthy equine animal. For example, propionylglycine is present at a reduced level in ID equine animals, relative to non-ID equine animals, so a treatment for insulin dysregulation or a condition associated with insulin dysregulation may be intended to increase the level of propionylglycine in the equine animal. Similarly, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle- associated metabolites, such as argininosuccinate and argininate, are present at an increased level in ID equine animals, relative to non-ID equine animals, so a treatment for insulin dysregulation or a condition associated with insulin dysregulation may be intended to decrease the level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the equine animal. The effectiveness of a treatment may therefore be determined by assessing its ability to achieve a desired alteration to the level of one or more of the biomarkers of the invention in the equine animal. If a treatment achieves a desired alteration to the level of one or more of the biomarkers of the invention in the equine animal, e.g., if a treatment increases the level of propionylglycine in the equine animal or reduces the level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the equine animal, then the treatment may be determined to be effective. Conversely, if a treatment does not achieve a desired alteration to the level of one or more of the biomarkers of the invention in the equine animal, e.g., if a treatment does not increase the level of propionylglycine in the equine animal or does not reduce the level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the equine animal, then the treatment may be determined to be ineffective.

[0112] In one embodiment, a treatment may be determined to be effective if: (i) the level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in a sample that has been obtained from the equine animal at a given time point is decreased, relative to the level of that biomarker of the invention in a sample that has been obtained from the equine animal at an earlier time point; and / or (ii) if the level of propionylglycine in a sample that has been obtained from the equine animal at a given time point is increased, relative to the level of propionylglycine in a sample that has been obtained from the equine animal at an earlier time point. Conversely, a treatment may be determined to be ineffective if: (I) the level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in a sample that has been obtained from the equine animal at a given time point is not decreased, relative to the level of that biomarker of the invention in a sample that has been obtained from the equine animal at an earlier time point; and / or (ii) if the level of propionylglycine in a sample that has been obtained from the equine animal at a given time point is not increased, relative to the level of propionylglycine in a sample that has been obtained from the equine animal at an earlier time point. The terms “increase” and “decrease” are defined in detail above in the context of a method of monitoring the level of one or more biomarkers in an equine animal, and these definitions apply equally here.

[0113] In one embodiment, the method of assessing the effectiveness of a treatment for insulin dysregulation or a condition associated with insulin dysregulation may further comprise a step of comparing the level of the one or more biomarkers determined in the samples that have been obtained from the equine animal to a reference result. The term “reference result” is defined in detail above in relation to the methods of diagnosis, and the same definition applies here. In short, the reference result represents the level of one or more of the biomarkers of the invention in a control equine animal. Accordingly, this step of comparison can indicate whether a given treatment reduces the difference between the level of one or more of the biomarkers of the invention in the samples that have been obtained from the equine animal and the typical level of that biomarker of the invention in the control equine animal. If the control equine animal is a healthy equine animal, the treatment may be determined to be effective if the level of one or more of the biomarkers of the invention in a sample that has been obtained from the equine animal at a given time point is closer to the reference result than the level of that biomarker of the invention in a sample that has been obtained from the equine animal at an earlier time point. Conversely, the treatment may be determined to be ineffective if the level of one or more of the biomarkers of the invention in a sample that has been obtained from the equine animal at a given time point is not closer to the reference result than the level of that biomarker of the invention in a sample that has been obtained from the equine animal at an earlier time point.

[0114] If the control equine animal is an unhealthy equine animal, the treatment may be determined to be effective if the level of one or more of the biomarkers of the invention in a sample that has been obtained from the equine animal at a given time point is not closer to the reference result than the level of that biomarker of the invention in a sample that has been obtained from the equine animal at an earlier time point. Conversely, the treatment may be determined to be ineffective if the level of one or more of the biomarkers of the invention in a sample that has been obtained from the equine animal at a given time point is closer to the reference result than the level of that biomarker of the invention in a sample that has been obtained from the equine animal at an earlier time point.

[0115] If a treatment for insulin dysregulation or a condition associated with insulin dysregulation is determined to be ineffective, the methods of the present invention may further comprise a step of providing a recommendation for an intervention. In some embodiments, the recommendation may be that the existing treatment regimen is adjusted to change the dose of the treatment that the equine animal receives. This change in dose may be an increase ora decrease in the dose of the treatment. For example, if an equine animal is being treated for insulin dysregulation, and the treatment is determined to be ineffective for achieving a desired alteration to the level of one or more of the biomarkers of the invention, the recommendation may be that the existing treatment regimen is adjusted to increase the dose of the treatment that the equine animal receives.

[0116] Again, in some embodiments, the methods of the invention further comprise implementing the recommendation for an intervention, i.e., adjusting the treatment regimen to increase or decrease the dose of the treatment that the equine animal receives. Samples

[0117] The methods of the invention refer to carrying out an assay for one or more biomarkers in a sample that has been obtained from an equine animal. The sample may be a bodily fluid obtained from the equine animal, such as whole blood, plasma, serum, saliva or urine, or may be a dried blood spot.

[0118] In some embodiments, the sample is provided in the form of a dried blood spot. Dried blood spot sampling can be used to assess a range of biomarkers, including the biomarkers of the invention. Dried blood spot samples can be prepared by contacting a small volume of blood onto a protein saver card and allowing the sample to air dry. The drying of the sample halts many of the enzymatic reactions that can cause degradation of metabolites in liquid samples. Suitable protein saver cards are well known in the art, and include Whatman 903 Protein Saver cards, for example. The blood can be obtained by pricking the skin of the equine animal using a catheter, a needle or a blood lancet, for example, or by any other approved means. A number of specific tools are available to assist with providing a blood sample containing an accurate and consistent volume of blood, including Capitainer® B and Mitra® systems. In order to extract the sample from the protein saver card for the assay for the biomarkers of the invention, the card can be contacted with an appropriate solvent, such as water, methanol, acetonitrile and / or isopropanol, and mixed to dissolve the sample. Exemplary protocols for measuring metabolites using dried blood spot cards are well known in the art (McCulloch et aL, 2021).

[0119] The use of dried blood spots for metabolomic analysis has a number of advantages. For instance, smaller volumes of whole blood are required, relative to methods involving analysis of plasma or serum samples. The equipment and expertise required for collection of the sample is also significantly reduced, as it does not require a trained phlebotomist to use a hypodermic needle. Moreover, storage of the sample once it has been collected is cheaper and easier (Allaway et aL, 2022).

[0120] If the sample is a whole blood sample, it may be processed via snap-freezing as soon as possible after collection to prevent any ex vivo metabolic reactions that could affect the results of the assay for the biomarkers of the invention. In some embodiments, the whole blood sample may be preserved using one or more anticoagulants, such as EDTA or heparin. Processing a whole blood sample may comprise fractionating the sample to obtain plasma and / or serum fractions. This fractionation may be done prior to freezing the sample. In some embodiment, the fractionation of the sample may be conducted at a temperature of 10°C or below, or at a temperature of 5°C or below. In some embodiments, the sample may be a sample obtained from an equine animal in a fasted state or from an equine animal without recent provision of forage and / or complementary feeds. For example, in some embodiments, the sample may be a sample obtained from an equine animal that has not been fed for at least 2, 3 , 4 or 5 hours, such as at least 6, 7, 8, 9 ,10, 11 , 12, 18 or 24 hours. In some embodiments, the sample may be a sample obtained from an equine animal at a predetermined time point following the provision of forage and / or complementary feeds. For example, the sample may be a sample obtained from an equine animal at the time of provision of forage and / or complementary feeds, or 30 minutes, 1 , 2, 3 4 or 5 hours after the provision of forage and / or complementary feeds. In some embodiments, the methods of the invention may be applied to a series of samples obtained from an equine animal at different time points. For example, the samples may be samples obtained from an equine animal at the time of provision of forage and / or complementary feeds, and at 1 , 2, 3 4 and 5 hours after the provision of forage and / or complementary feeds.

[0121] The methods of the invention may optionally comprise a step of obtaining a sample from an equine animal. Suitable samples for use in the methods of the invention are defined above.

[0122] Equine animals

[0123] The methods of the invention may be applied to a range of equine animals. In particular, the methods of the invention can be used to diagnose the presence or absence of insulin dysregulation and / or susceptibility to a condition associated with insulin dysregulation in an animal of the genus Equus.

[0124] This genus is part of the Equidae family, which includes numerous extant and extinct species. Species of the genus Equus include horses (Equus caballus), mules (Equus mulus), hinnies, wild horses (Equus ferus), mountain zebras (Equus zebra), African wild asses (Equus africanus), domestic donkey (Equus africanus asinus), Grevy's zebras (Equus grevyi), Asiatic wild asses (Equus hemionus), kiangs (Equus kiang), and plains zebras (Equus quagga). Preferably, the animal is of the species Equus caballus, which includes horses and ponies. The animal of the species Equus caballus may be of any age, gender or castration status, and so the methods of the invention can be applied to (without limitation) foals, weanlings, yearlings, colts, fillies, mares, stallions, geldings or rigs. Any and all breeds of animals of the genus Equus, preferably of the species Equus caballus, can be assessed using methods of the invention. General

[0125] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature.

[0126] The term “comprising” encompasses “including” as well as “consisting”, e.g., a composition “comprising” X may consist exclusively of X or may include something additional, e.g., X + Y.

[0127] The term “about” in relation to a numerical value x is optional and means, for example, x ± 10%.

[0128] The word “substantially” does not exclude “completely”, e.g., a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

[0129] Unless specifically stated, a process or method comprising numerous steps may comprise additional steps at the beginning or end of the method, or may comprise additional intervening steps. Also, steps may be combined, omitted or performed in an alternative order, if appropriate.

[0130] Various embodiments of the invention are described herein. It will be appreciated that the features specified in each embodiment may be combined with other specified features, to provide further embodiments. In particular, embodiments highlighted herein as being suitable, typical or preferred may be combined with each other (except when they are mutually exclusive).

[0131] The invention will be further understood by reference to the following examples, which are provided as an illustration of the invention and are not intended to be limiting.

[0132] Examples

[0133] Example 1

[0134] A study was conducted involving four horses - a 16-year-old female appaloosa, a 16-year-old quarter horse, a 16-year-old male Tennessee walking horse and a 21 -year-old female quarter horse.

[0135] Two weeks prior to sample collection, at the beginning of the spring season, all of the horses undertook an oral sugar test (OST) using the 0.15 mL / kg bodyweight dose. Using published criteria, the horses were confirmed as either having insulin dysregulation (ID) or not having insulin dysregulation (non-ID). Of the four horses tested, two were ID and two were non-ID. In addition, the horses were assessed to confirm they had no signs of pituitary pars intermedia dysfunction (PPID), and their non-fall adrenocorticotropic hormone (ACTH) concentrations were < 30 pg / mL.

[0136] The horses were fed three different diets in a random order, with at least one week between each diet. The three diets were:

[0137] (1) control low non-structural carbohydrate (LNSC) fiber-based pellet (Buckeye TM Nutrition, USA);

[0138] (2) control diet plus a pure source of glucose (D-(+)-glucose, dextrose, Sigma Aldrich)

[0139] (3) control diet plus a 50:50 mix of oat starch powder and waxy maize starch (True Nutrition, CA, USA).

[0140] The nutritional information of the three diets is shown in the table below (CP = Crude Protein, WSC = Water Soluble Carbohydrates, ESC = Ethanol Soluble Carbohydrates, NSC = Non-Structural Carbohydrates).

[0141] On the morning of the sampling day (0700), the horses were brought into their individual pens and 16-gauge x 14.6 cm (Covetrus) catheters were placed under local anaesthetic using aseptic technique. While in their individual pens, horses had access to water, but no other feedstuffs. After catheter placement, horses underwent a 30-minute rest period before a blood sample was collected (10 mL serum tubes; Covetrus) and the feed was offered. Diets were consumed by all horses in -10 minutes. Blood was collected pre-feeding and at 30, 60 and 90 minutes following complete consumption of the respective treatment diets. Remnant blood volumes were applied to Whatman ProteinSaver cards. After drying, samples were stored with a desiccant at -80 °C. Samples were sent to Metabolon for broad metabolite profiling.

[0142] Between sample collections, horses were housed in their home paddocks where they had access to limited pasture forage (semi-dry lots), grass hay (11.85% CP, 0.6% starch, 5.9% WSC, 3.75% ESC, and 6.5% NSC on a dry matter basis), and water.

[0143] Metabolomic analysis of the dried blood spots was undertaken. Seven metabolites showed a consistent differentiation between the ID and NID animals at all four time points and on all three diets occasions. These metabolites were propionylglycine, allantoin, nicotinamide riboside, allantoic acid, argininosuccinate, argininate and hexadecatrienoic acid. The results of this metabolomic analysis are shown in Figure 1. The three panels shown for each biomarker correspond to the three treatment diets. Within each panel, the time points T1, T2, T3 and T4 correspond to the samples taken prefeeding and at 30, 60 and 90 minutes after consumption of the diets, respectively. P1 indicates the ID animals and P2 indicates the non-ID animals.

[0144] References

[0145] Allaway et al., Front Vet Sci. (2022) 22:9:887163

[0146] Asplin et a!., Vet J. (2007) 174(3):530-5 de Laat et al., Equine Vet. J. (2010) 42 (2) 129-135 de Laat, “Pathophysiology of Insulin Dysregulation (ID) in Horses and Therapeutic Targets", ACVIM Forum (2023)

[0147] Durham et al., J Vet Intern Med. (2019) 33:335-349

[0148] Frank et al., J Vet Intern Med (2010) 24:467-475

[0149] Hoffman et al., J. Anim. Sci. (2003) 81 :2333-2342

[0150] Johnson, Vet Clin North Am Equine Pract. (2002) 18(2):271-93.

[0151] Knowles et al., Equine Vet J. (2023a) 55:12-23

[0152] Knowles et al., “Factors associated with insulin responses to oral sugars in a mixed-breed cohort of ponies", Equine Vet J. accepted, (2023b)

[0153] McCulloch et al., "Stability of metabolite profiles using dried blood spot cards", Metabolon (2021 ) McGowan, “The Intersection of PPID and Laminitis”, 64th Annual Convention of the American Association of Equine Practitioners (2018)

[0154] Meier et al., BMC Veterinary Research (2019) 15:65

[0155] Treiber et al., J. Anim. Sci. (2005) 83:2357-2364

[0156] Treiber et a / ., J. Nutr. (2006) 136(7 Suppl) 2094S-2098S

Claims

CLAIMS1 . A method of diagnosing the presence or absence of insulin dysregulation in an equine animal, said method comprising:(a) carrying out an assay to determine the level of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate and argininate, in a sample that has been obtained from the equine animal;(b) comparing the level of the one or more biomarkers determined in step (a) to a reference result; wherein a difference between the level of the one or more biomarkers determined in step (a) and the reference result is used to determine the presence or absence of insulin dysregulation.

2. A method of diagnosing the presence or absence of susceptibility to a condition associated with insulin dysregulation in an equine animal, said method comprising:(a) carrying out an assay to determine the level of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate and argininate, in a sample that has been obtained from the equine animal;(b) comparing the level of the one or more biomarkers determined in step (a) to a reference result; wherein a difference between the level of the one or more biomarkers determined in step (a) and the reference result is used to determine the presence or absence of susceptibility to a condition associated with insulin dysregulation.

3. The method of claim 1 or 2, wherein the reference result comprises one or more results from an assay to determine the level of one or more of the biomarkers in a sample that has been obtained from a control equine animal that does not suffer from, has not been diagnosed with or has been confirmed not to have insulin dysregulation, laminitis, equine metabolic syndrome, diabetes, obesity or pituitary pars intermedia dysfunction (PPID).

4. The method of claim 3, wherein a lower level of propionylglycine in the sample that has been obtained from the equine animal, relative to the reference result, is indicative of the presence of insulin dysregulation or susceptibility to a condition associated with insulin dysregulation.

5. The method of claim 3 or 4, wherein a higher level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the sample that has been obtained from the equine animal, relative to the referenceresult, is indicative of the presence of insulin dysregulation or susceptibility to a condition associated with insulin dysregulation.

6. The method of claim 1 or 2, wherein the reference result comprises one or more results from an assay to determine the level of one or more of the biomarkers in a sample that has been obtained from a control equine animal that suffers from, has been diagnosed with or has been confirmed to have insulin dysregulation, laminitis, equine metabolic syndrome, diabetes, obesity or pituitary pars intermedia dysfunction (PPID).

7. The method of claim 6, wherein a level of propionylglycine in the sample that has been obtained from the equine animal that is not higher than the reference result is indicative of the presence of insulin dysregulation or susceptibility to a condition associated with insulin dysregulation.

8. The method of claim 6 or 7, wherein a level of allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and / or a urea cycle-associated metabolite, such as argininosuccinate or argininate, in the sample that has been obtained from the equine animal that is not lower than the reference result is indicative of the presence of insulin dysregulation or susceptibility to a condition associated with insulin dysregulation.

9. The method of any one of claims 3-8, wherein the reference result comprises a value for the concentration of the one or more biomarkers in the sample that has been obtained from the control equine animal.

10. The method of any one of claims 3-9, wherein the reference result comprises the ratio of the level of one or more of the biomarkers to the level of a standard marker in the sample that has been obtained from the control equine animal.11 . The method of claim any one of claims 3-10, wherein the reference result comprises the ratio of the levels of two or more of the biomarkers in the sample that has been obtained from the control equine animal.

12. The method of any one of claims 3-11 , wherein the method further comprises a step of generating the reference result.

13. The method of any one of claims 1-8, wherein the reference result comprises a pre-determined threshold concentration for one or more of the biomarkers.

14. A method of monitoring the level of one or more biomarkers in an equine animal, said method comprising:(a) carrying out an assay to determine the level of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate and argininate, in a sample that has been obtained from the equine animal; and(b) repeating step (a) at two or more separate time points.

15. A method of assessing the effectiveness of a treatment for insulin dysregulation in an equine animal, said method comprising:(a) carrying out an assay to determine the level of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate and argininate, in a sample that has been obtained from the equine animal;(b) repeating step (a) at two or more separate time points, wherein at least one of the time points is after the equine animal has begun the treatment.

16. A method of assessing the effectiveness of a treatment for a condition associated with insulin dysregulation in an equine animal, said method comprising:(a) carrying out an assay to determine the level of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid and urea cycle-associated metabolites, such as argininosuccinate and argininate, in a sample that has been obtained from the equine animal;(b) repeating step (a) at two or more separate time points, wherein at least one of the time points is after the equine animal has begun the treatment.

17. The method of any one of claims 2-13 or 16, wherein the condition associated with insulin dysregulation is laminitis, equine metabolic syndrome, diabetes, obesity or pituitary pars intermedia dysfunction (PPID).

18. The method of any one of claims 1-17, wherein step (a) comprises determining the concentration of the one or more biomarkers in the sample that has been obtained from the equine animal.

19. The method of any one of claims 1-18, wherein step (a) comprises determining the ratio of the level of one or more of the biomarkers to the level of a standard marker in the sample that has been obtained from the equine animal.

20. The method of claim 10 or 19, wherein the standard marker is a fatty acid, an amino acid, or is selected from phenylalanine, tryptophan, leucine, acetylcarnitine, and 1-palmitoyl-2-oleoyl-GPC creatinine, and glucose. 21 . The method of any one of claims 1-20, wherein step (a) comprises determining the ratio of the levels of two or more of the biomarkers in the sample that has been obtained from the equine animal.

22. The method of any one of claims 1-21 , wherein the sample is a bodily fluid obtained from the equine animal, preferably whole blood, saliva, plasma, serum or urine, or wherein the sample is a dried blood spot or a fecal sample.

23. The method of any one of claims 1-22, wherein the equine animal is a horse, a pony, a mule or a donkey.

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