Mirnas from a biological sample as indicators of stress in bovine

Abstract

The present invention relates miRNA biomarkers from a biological sample, e.g. extracellular vesicle-derived microRNA, as indicators of welfare in bovines. In particular, the present invention relates to the identification of social stress in bovines allowing to propose management actions to improve productive animal welfare at the individual or the herd levels.

Description

[0001] miRNAs FROM A BIOLOGICAL SAMPLE AS INDICATORS OF STRESS IN BOVINE

[0002] Technical field of the invention

[0003] This present invention relates to the use of micro-RNAs from a biological sample from dairy cattle as indicators of animal welfare, such as indicators of stress such as social stress.

[0004] Background of the invention

[0005] International regulatory framework proposes observations of changes in animal health status and behaviour to define if the animal(s) are stressed and to propose improvements in animal welfare management. However, a visual observation alone is subjective and does not consider specific physiological markers of stress.

[0006] Social stress occurs when cattle are subjected to one or more condition(s) that affects the ability of the cattle to express a normal pattern of behaviour, leads to physical discomfort and / or fear and distress. This in turn leads to the welfare of the animal being compromised. That is, social stress leads to the welfare of the animal being compromised. The welfare of an animal can be assessed by a assessing a number of conditions that can broadly be grouped into i. physical environmental conditions, ii. nutritional conditions, iii. health conditions and iv. behavioural interactions, which include interactions with the environment, interactions with other animals and interactions with humans. Social or behavioural stress can occur when one or more of these conditions are negative.

[0007] For example, a negative physical environment that leads to social stress may comprise (i) close confinement or overcrowding which leads to physical discomfort; (ii) unsuitable substrate or wet / soiled ground, which can lead to musculoskeletal pain and skin irritation, (iii) monotony, e.g. ambient lighting can lead to malaise from unnatural constancy; (iv) unpredictable events can lead to anxiety, fear and hypervigilance; and / or (v) physical limits of rest and sleep, which leads to exhaustion. Conversely positive physical conditions are where the animal has space for spontaneous locomotion and / or suitable substrate and well- drained soil that leads to physical comfort; maintenance of within-day environmental variability, which leads to congenial variability; predictability achieved by established routines, which leads to relaxation based ease and calmness; and / or conditions conducive to rest and sleep, which leads to well rested animals. An example of a negative nutritional condition that leads to social stress may comprise voluntary overeating, which leads to a feeling of being bloated or overfull. Conversely, positive nutritional conditions are where the animal eats the correct quantities of food leading to comfort of satiety.

[0008] An example of a negative health condition that leads to social stress may comprise poor physical fitness and muscle de-conditioning which leads to muscle weakness and exhaustion. Conversely, a positive health condition is where the animal has a good level of fitness.

[0009] An example of a negative behavioural indication that leads to social stress may comprise (i) invariant, barren or confined environments (which may be ambient, physical or biotic) and that may lead to boredom, helplessness, depression and withdrawal; (ii) inescapable sensory impositions that may lead to being startled by unexpected events or neophobia; and (iii) choices being markedly restricted, such as environment-focused activity constrained or foraging constrained, leading to hypervigilance, anger, frustration and a negative cognitive bias. These are all examples of how interactions with the environment can affect social stress. Conversely, a positive interaction with the environment may comprise a varied, novel environment, which leads to animals that are interested and pleasantly occupied.

[0010] Another example of a negative behavioural indication that leads to social stress may comprise (i) the constraint of animal to animal interactive activities, which leads to loneliness, depression and a yearning for company, a thwarted desire to play, sexual frustration and a thwarted hunger drive; (ii) significant threats which can lead to anger, anxiety, fear, panic, insecurity and neophobia; and / or (iii) limitations on sleep and rest which can lead to exhaustion. These are all examples of how interactions with other animals can affect social stress. Conversely, a positive interaction with other animals can include (i) bonding or reaffirming bonds, which can lead to affectionate sociability; (ii) rearing young, which can lead to maternal or paternal group rewards; (iii) playing which can lead to excitation or playfulness; (iv) sexual activity which can lead to sexual gratification; (v) hunting, which leads to alert engagement and highly stimulated animals; (vi) the absence of threats (using refuges) which leads to secure, protected and confident animals; and (vii)sufficient sleep / rest which leads to animals that are energised and refreshed. Another example of a negative behavioural indication that leads to social stress may comprise (i) negative human attributes or behaviours that in turn lead to negative animal behaviours, such as negative human attitudes (e.g. uncertain, fearful, indifferent, insensitive, impatient, oppressive, belligerent, domineering, callous, cruel, vindictive);(ii) negative voice conditions (e.g. hesitant, angry, loud, shouting) and human aptitude (e.g. inexperienced) - which in turn leads to negative behaviours in the animal, such as long flight distance, hypervigilant, attack / flight, hyper-reactive, escape avoidance, freezing, cowering, appeasing, withdrawn and non-compliant; and (iii) poor human handling (e.g. erratic, rough, excessively forceful, punishment-focused) which in turn lead to anxiety, fear, neophobia, insecurity and a negative cognitive bias in the animal. These are all examples of how interactions with humans can affect social stress. Conversely, a positive interaction with humans comprises (i) a positive human attitude (e.g. caring, confident, sensitive, patient, kind, empathic), positive voice conditions (e.g. calm, confident, encouraging and pleasantly rhythmic) and (ii) a positive aptitude (e.g. experienced, trained) which leads to positive behaviours in the animal, such as short flight distance, calm, alertness); and (iii) good handling (e.g. gentle, reward-focused, subtle pressure cues, secondary reinforces and timely release of aversive stimuli) which can lead to animals that are calm, confident, at ease and finds bonding with humans rewarding.

[0011] Currently, different indicators are used to evaluate if an animal has or is experiencing stress conditions. These include,

[0012] - observations in changes in animal health status and behaviour, which is currently considered the reference tool to monitor and detect early warning signs in animal health and welfare deterioration;

[0013] - changes in blood parameters, such as glycemia, blood cell counts, levels of hormones and metabolites. However, these parameters are highly variable across different animal breeds, age, stage of the life. Moreover, the level of hormones such as cortisol passing in milk is too variable, and as such classical biomarkers are not suitable stress specific indicators.

[0014] By contrast, the expression of miRNA is specific to cells and tissues and well-regulated. loannidis et al. discloses associations of plasma microRNA expression with age, genetic background and functional traits in dairy cattle (SCIENTIFIC REPORTS| (2018) 8:12955). Miretti et al. discloses microRNAs as biomarkers for animal health and welfare in livestock (Front Vet. Sci. 7:578193).

[0015] Billa et al. discloses that nutrigenomic analyses reveal miRNAs and mRNAs affected by feed restriction in the mammary gland of midlactation dairy cows (PLoS ONE 16(4): e0248680).

[0016] There is a growing body evidence that stress conditions alter miRNA biogenesis, the expression of mRNA targets and the activities of miRNA-protein complexes. However, the precise miRNA biomarkers that indicate stress are not currently known.

[0017] Hence, an improved method to non-invasively determine animal welfare would be advantageous, and in particular a more efficient and / or reliable method to determine animal stress, such as social stress would be advantageous. The present invention addresses this need.

[0018] Summary of the invention

[0019] The present invention relates to miRNA biomarkers from a biological sample, e.g. exosome or extracellular-vesicle-derived microRNA, as indicators of welfare (or as used specifically herein a welfare parameter) in cattle. In particular, the present invention relates to the identification of stress as the welfare parameter, particularly social stress in cattle, which in turn means actions can be taken to manage and improve productive animal welfare at the individual or the herd level. In the context of social stress, the present invention therefore provides a means to determine the extent to which a cow or herd of cattle experience discomfort or “happiness”.

[0020] Animal welfare can be considered to be compromised in one of the “five freedoms” are compromised. The five freedoms are:

[0021] I. freedom from hunger, thirst and malnutrition;

[0022] II. freedom from fear and distress;

[0023] III. freedom from physical and thermal (heat) discomfort;

[0024] IV. freedom from pain, injury and disease; and

[0025] V. freedom to express normal patterns of behaviour.

[0026] In the context of the present invention, an animal can be considered to have or be experiencing social stress if one, preferably two, more preferably three of the five freedoms are compromised. Specifically, social stress may be caused by infringement of the freedom to express normal patterns of behaviours, physical discomfort and / or fear and distress. These three freedoms can be compromised as a result of any number of changes in an animal’s physical, health, behavioural or nutritional conditions, as described above. For example - but not limited to - the three freedoms mentioned above, and as a consequence social stress, may be caused by maternal separation and weaning, social isolation or overstocking, mixing with unfamiliar animals or housing animals in unfamiliar environments (social instability), poor handling and / or barren housing.

[0027] Thus, an object of the present invention relates to the provision of miRNA biomarkers, which can be indicative of at least one welfare parameter in cattle, preferably dairy cattle.

[0028] In particular, it is an object of the present invention to provide miRNA biomarkers from a biological sample, and in particular biological sample derived extracellular vesicles, which can indicate at least social stress in (dairy) cattle and which can be used across different breeds of (dairy) cattle.

[0029] Example 1 shows the level of milk derived biomarkers in cattle exposed to social stress.

[0030] In one embodiment, the welfare parameter of at least one head of cattle is or was comprised by at least one type of stress, examples of which include but are not limited to social stress, heat stress, metabolic stress, disease stress or combinations thereof. In one embodiment, the stress is social stress. That is, detection of at least one miRNA biomarker as defined in one of SEQ ID NO: 1 , 2, 3, 4, 5, and 6 (or a variant thereof as defined herein) is indicative that the animal has or is subject to social stress. Once it is determined that at least one welfare parameter of at least one head of cattle has been compromised it is then possible to improve the welfare of the animal.

[0031] The method may also comprise the detection of one or more further biomarker, where that one or more further biomarker is associated with a different welfare parameter - that is, a different type of stress (e.g. not social stress).

[0032] In another aspect of the invention there is provided a method of improving the welfare of at least one head of cattle, the method comprising i. determining in a biological sample the level of at least one miRNA selected from SEQ ID NO: 1 ,2, 3, 4, 5, and 6, or a functional variant thereof wherein the functional variant has at least 60% sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, and 6 respectively; ii. comparing the level of the at least one miRNA to a reference level, wherein if said one or more miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5, and 6 (or a variant thereof) is below said one or more reference level at least one welfare parameter of the at least one head of cattle is or has been compromised; and iii. improving the welfare of the at least one head of cattle if at least one welfare parameter of the at least one head of cattle is or has been compromised.

[0033] As used herein “by improving welfare” is meant improving at least one welfare parameter, as defined herein. The improvement may be relative to a head of cattle where the welfare or at least one welfare parameter has not been improved by the methods described below.

[0034] In another aspect of the invention, there is provided a method of improving - or determining whether there been an improvement in at least one welfare parameter of at least one head of cattle, the method comprising i. determining in a first biological sample the level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: 1 , 2, 3, 4, 5, and 6 or a functional variant thereof wherein the functional variant has at least 60% sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5 and 6; and ii. comparing the level of the at least one miRNA to a reference level, wherein if said one or more miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5, and 6 (or variant thereof) is below said one or more reference level at least one welfare parameter of the at least one head of cattle has been compromised; and where at least one welfare parameter has been compromised, the method further comprises iii. improving the welfare of the at least one head of cattle; iv. determining in a second biological sample the level of at least one miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5 and 6 or a functional variant thereof wherein the functional variant has at least 60% sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, and 6 respectively; and v. comparing the level of the at least one miRNA in the first biological sample to the level in the second biological sample, wherein if the level of one of SEQ ID NO: 1 , 2, 3, 4, 5, and 6 or a functional variant thereof wherein the functional variant has at least 60% sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, and 6 respectively in the second biological sample is higher than the corresponding level in the first biological sample or higher than the level in the reference sample the welfare of the head of cattle has improved (likely as a result of the improvements made to the welfare).

[0035] In one embodiment, the welfare parameter of at least one head of cattle is or was comprised by at least one type of stress, examples of which include but are not limited to social stress, heat stress, metabolic stress, disease stress or combinations thereof. In one embodiment, the stress is or was social stress (for example as defined above).

[0036] In one embodiment, improving the welfare (i.e. alleviating or reducing social stress) of the at least one head of cattle comprises one or more measure to alleviate or reduce the social stress of the animal(s). For example, regrouping (from maternal separation), improving housing conditions, such as removing or reducing barren housing, removing or reducing overcrowding, reducing mixing with unfamiliar animals or re-housing in a familiar environment, reducing or improving handling, improving food and water quality / quantity, alleviating disease, providing established routines and / or avoiding extreme weather.

[0037] Furthermore, in this embodiment, improving the welfare of the at least one head of cattle may further comprise one or measure to reduce or alleviate stress, such as but not limited to changing heat exposure, improving access to additional water, changing the type of feed, through the provision of cooling systems, or to reduce social stress by reducing animal density, or increasing space allowance, providing brushes as enrichment, and improving social stability.

[0038] In a further aspect of the invention, there is provided a method of improving at least one of milk yield, milk quality, meat yield or meat quality of at least one head of cattle, the method comprising i. determining in a first biological sample the level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: 1 , 2, 3, 4, 5, and 6 or a functional variant thereof wherein the functional variant has at least 60% sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, and 6; ii. comparing the level of the at least one miRNA to a reference level, wherein if said one or more miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5, and 6 is below said one or more reference level at least one welfare parameter of the at least one head of cattle has been or is compromised; and iii. improving the welfare of the at least one head of cattle; wherein improving the welfare of the at least one head of cattle improves at least one of milk yield, milk quality, meat yield and meat quality.

[0039] In one embodiment, the welfare parameter of at least one head of cattle is or was compromised by at least one type of stress, examples of which include but are not limited to social stress, heat stress, metabolic stress, disease stress or combinations thereof. In one embodiment, the stress is social stress. That is, detection of at least one miRNA biomarker as defined in one of SEQ ID NO: 1 , 2, 3, 4, 5, and 6 is indicative that the animal has or is subject to social stress.

[0040] In one embodiment, improving the welfare (i.e. alleviating or reducing social stress) of the at least one head of cattle comprises one or more measure to alleviate or reduce the social stress of the animal(s). For example, regrouping (from maternal separation), improving housing conditions, such as removing or reducing barren housing, removing or reducing overcrowding, reducing mixing with unfamiliar animals or re-housing in a familiar environment, reducing or improving handling, improving food and water quality / quantity, alleviating disease, providing established routines and / or avoiding extreme weather.

[0041] In another aspect of the invention, there is provided a method of determining at least one welfare parameter of at least one head of cattle, the method comprising determining the level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: 1 , 2, 3, 4, 5, and 6 or a functional variant thereof, wherein said functional variant has at least 60% overall sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, and 6 respectively; wherein when the level of SEQ ID NO: 1 , 2, 3, 4, 5, and 6 or a functional variant thereof is decreased by at least 1 log2FC (-1 log fold change) or more compared to a reference level, at least one welfare parameter of the at least one head of cattle has been or is compromised.

[0042] In a preferred embodiment the biological sample is a milk sample. In another embodiment, the biological sample is selected from a blood sample, a meat sample or a urine sample, optionally a blood sample. In a further embodiment, the biological sample is not milk or a milk-derived sample. In another aspect of the invention, there is provided a method of determining from a bulk milk sample or dairy product whether at least one welfare parameter of at least one herd of cattle, has been or is compromised, the method comprising determining in the bulk milk or dairy product the level of at least one miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5 and 6 or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, and 6 respectively; and comparing the level of the at least one miRNA to a reference level, wherein if said one or more miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5 and 6 is below said reference level at least one welfare parameter of the at least one herd of cattle has been or is compromised. In one embodiment, the welfare parameter of at least one herd of cattle is or was comprised by social stress.

[0043] In one embodiment, the bulk milk sample is from a single herd of cattle. That is, the biological sample is pooled from a plurality of - i.e. two or more - animals. In an alternative embodiment, the bulk milk sample is from a plurality of herds of cattle. That is, the biological sample is pooled from a plurality of - i.e. two or more - herds.

[0044] In some embodiments, the bulk milk sample has been processed by any one or more of pasteurisation, spray drying, homogenisation, filtration, or centrifugation.

[0045] In some embodiments the bulk milk sample is in a dairy product such as skimmed milk, yoghurt, buttermilk, cream, cheese, butter or milk powder.

[0046] Preferably, the dairy product is a processed milk product, any may be selected from skimmed milk, yoghurt, buttermilk, cream, cheese, butter, or milk powder.

[0047] In some embodiments methods of the present invention further comprise determining the level of substance P wherein if the level of substance P is increased compared to a reference level at least one welfare parameter of the at least one head of cattle has been compromised.

[0048] In any of the above-described aspects, the method may comprise determining the level of all of SEQ ID NO: 1 , 2, 3, 4, 5, and 6 or a functional variant thereof, wherein said functional variant has at least 60% overall sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5 and 6 respectively. In an embodiment, if the level of SEQ ID NO: 1 or a functional variant thereof is below said one or more reference level at least one welfare parameter of the at least one head of cattle has been compromised. Specifically, if the level of SEQ ID NO: 1 or a functional variant thereof is below said one or more reference level, the animal(s) has been exposed to social stress or is currently socially stressed.

[0049] In one embodiment, the cattle is dairy cattle, preferably bovine. In another embodiment, the breed of cattle is selected from Holstein and / or Brown Swiss.

[0050] The reference level may be the level of the one or more miRNA in a biological sample from at least one head or herd of cattle where the welfare of the head or herd of cattle is not or has not been compromised or wherein the reference level is an average level from cattle where the welfare of the cattle is not or has not been compromised (for example, as described above).

[0051] The method may further comprise determining in a biological sample from said head (or herd) of cattle, the level of a physiological parameter, wherein the physiological parameter may be selected from one or more of substance P levels, glucose levels, protein profiles, triglyceride levels, heat shock protein 70, a serum protein profile, blood urea nitrogen, blood uric acid, serum chloride, serum phosphorous, serum triglyceride and serum magnesium.

[0052] In one embodiment the method comprises determining the level of heat shock protein 70 in a biological sample from said head (or herd) of cattle, comparing said level to a corresponding reference level of heat shock protein 70 (a reference level is defined herein), wherein where the level of heat shock protein 70 is increased compared to the reference level it is indicative that at least one welfare parameter (i.e. the welfare) of the at least one head of cattle has been compromised.

[0053] In one embodiment the method comprises determining the serum protein profile in a biological sample from said head (or herd) of cattle, comparing said level to a corresponding reference serum protein profile (a reference level is defined herein), wherein if total proteins are increased compared to the reference level it is indicative that at least one welfare parameter (i.e. the welfare) of the at least one head of cattle has been compromised. In one embodiment the method comprises determining the level of blood urea nitrogen in a biological sample from said head (or herd) of cattle, comparing said level to a corresponding reference level of blood urea nitrogen (a reference level is defined herein), wherein where the level of blood urea nitrogen is increased compared to the reference level it is indicative that at least one welfare parameter (i.e. the welfare) of the at least one head of cattle has been compromised.

[0054] In one embodiment the method comprises determining the level of blood uric acid in a biological sample from said head (or herd) of cattle, comparing said level to a corresponding reference level of the blood uric acid (a reference level is defined herein), wherein where the level of the blood uric acid is decreased compared to the reference level it is indicative that at least one welfare parameter (i.e. the welfare) of the at least one head of cattle has been compromised.

[0055] In one embodiment the method comprises determining the level of serum chloride in a biological sample from said head (or herd) of cattle, comparing said level to a corresponding reference level of serum chloride (a reference level is defined herein), wherein where the level of serum chloride is increased compared to the reference level it is indicative that at least one welfare parameter (i.e. the welfare) of the at least one head of cattle has been compromised.

[0056] In one embodiment the method comprises determining the level of serum phosphorous in a biological sample from said head (or herd) of cattle, comparing said level to a corresponding reference level of serum phosphorous (a reference level is defined herein), wherein where the level of serum phosphorous is increased compared to the reference level it is indicative that at least one welfare parameter (i.e. the welfare) of the at least one head of cattle has been compromised.

[0057] In one embodiment the method comprises determining the level of serum magnesium in a biological sample from said head (or herd) of cattle, comparing said level to a corresponding reference level of serum magnesium (a reference level is defined herein), wherein where the level of serum magnesium is decreased compared to the reference level it is indicative that at least one welfare parameter (i.e. the welfare) of the at least one head of cattle has been compromised. In one embodiment the method comprises determining the level of serum triglyceride in a biological sample from said head (or herd) of cattle, comparing said level to a corresponding reference level of serum triglyceride (a reference level is defined herein), wherein where the level of triglyceride is decreased compared to the reference level it is indicative that at least one welfare parameter (i.e. the welfare) of the at least one head of cattle has been compromised.

[0058] In one embodiment the method comprises determining the level of substance P in a biological sample from said head (or herd) of cattle, comparing said level to a corresponding reference level of substance P (a reference level is defined herein), wherein where the level of substance P is increased compared to the reference level it is indicative that at least one welfare parameter (i.e. the welfare) of the at least one head of cattle has been compromised.

[0059] In one embodiment, the welfare parameter of at least one head of cattle is or was comprised by at least one type of stress, examples of which include but are not limited to social stress, heat stress, metabolic stress, disease stress or combinations thereof. In one embodiment, the stress is social stress.

[0060] In another aspect of the invention, there is provided a device or system adapted to determine if at least one welfare parameter of at least one head or herd of cattle has been or is compromised, the device comprising: a unit able to determine the level of one or more miRNAs described herein; a processor configured with a reference table, said reference table corresponding to reference levels of one or more of the miRNAs according to SEQ ID NO: 1 , 2, 3, 4, 5 or 6;

[0061] - wherein the device or system is adapted to receive the biological sample; determine the levels of the one or more miRNAs; compare the determined levels of the one or more miRNAs to the reference table; and determine if the welfare of the at least one head of cattle has been or is compromised. In another aspect of the invention, there is provided a computer implemented method of determining if the welfare of at least one head or herd of cattle has been compromised, the method comprising: providing levels of one or more miRNAs from a cattle biological sample selected from SEQ ID NOs: 1 , 2, 3, 4, 5 and 6; providing a mathematical model comprising reference levels of one or more corresponding miRNAs selected from SEQ ID NOs: 1 , 2, 3, 4, 5 and 6; determining if the provided levels deviates significantly from the reference levels, using the mathematical model; and providing to a system or a user a determination of whether the welfare of the at least one head of cattle has been or is compromised.

[0062] In a preferred embodiment the biological sample is a milk sample. In another embodiment, the biological sample is selected from a blood sample, a meat sample or a urine sample, optionally a blood sample. In a further embodiment, the biological sample is not milk or a milk-derived sample.

[0063] In some embodiments the milk sample is a bulk milk sample. In another embodiment the sample is a or from a processed milk product.

[0064] In alternative embodiments the milk sample has been processed by any one or more of pasteurisation, spray drying, homogenisation, filtration, or centrifugation.

[0065] In another aspect of the invention, there is provided a kit for evaluating at least one welfare parameter of at least one head of cattle comprising the device of the invention and instructions for use.

[0066] In another aspect of the invention there is a kit for evaluating at least one welfare parameter in at least one head of cattle wherein said kit comprises: i. a means for collecting a biological sample obtained from at least one head or herd of cattle; and ii. a unit for measuring the level of at least one miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5 and 6; and iii. instructions for use. In a further embodiment of any of the above-described methods, uses and devices or systems, the miRNA is alternatively that defined in SEQ ID NO: 1 or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 1.

[0067] In a further embodiment of any of the above-described methods, uses and devices or systems, the miRNA is alternatively that defined in SEQ ID NO: 2 or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 2.

[0068] In a further embodiment of any of the above-described methods, uses and devices or systems, the miRNA is alternatively that defined in SEQ ID NO: 3 or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 3.

[0069] In a further embodiment of any of the above-described methods, uses and devices or systems, the miRNA is alternatively that defined in SEQ ID NO: 4 or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 4.

[0070] In a further embodiment of any of the above-described methods, uses and devices or systems, the miRNA is alternatively that defined in SEQ ID NO: 5 or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 5.

[0071] In a further embodiment of any of the above-described methods, uses and devices or systems, the miRNA is alternatively that defined in SEQ ID NO: 6 or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 6.

[0072] The present invention will now be described in more detail in the following passages.

[0073] Brief Description of the Figures Figure 1 shows levels of miRNA before and after social stress was experienced by cattle. Data was analysed using a threshold of Wilcoxon p-value less than 0.01 on data that had been corrected for any batch effect. Figure 1A shows data for bta-miR-2467-3p, Figure 1b shows data for bta-miR-2448-3p, Figure 1c shows data for bta-miR-150, Figure 1d shows data for bta-miR-2285by, Figure 1e shows data for bta-miR-382 and Figure 1f shows data for bta-miR-15a.

[0074] Detailed description of the invention

[0075] Definitions

[0076] Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

[0077] Head of cattle

[0078] At least one head of cattle refers to one animal of unspecified age or gender.

[0079] Dairy cattle

[0080] Dairy cattle (also called dairy cows) are cattle bred for the ability to produce large quantities of milk, from which dairy products are made. Dairy cattle generally are of the species Bos Taurus.

[0081] Functional Variant

[0082] The term ‘variant’ or ‘functional variant’ refers to a miRNA sequence where the nucleotides of the miRNA are substantially identical to one of the recited sequences. The variant may be achieved by modifications such as insertion, substitution or deletion of one or more nucleotides. In a preferred embodiment, the variant has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of the recited sequences, such as SEQ ID NOs 1 , 2, 3, 4, 5 and 6 , preferably over the full length of the sequence. In one embodiment, sequence identity is at least 90%. In another embodiment, sequence identity is 100%. Sequence identity can be determined by any one known sequence alignment program in the art. For the avoidance of doubt, a functional variant performs the same function as the non-variant sequence. For example, a functional variant of an miRNA s one that is involved in synaptic regulation the same way as the non-variant sequence. Increase

[0083] In the context of the present invention an increase is an increase in the level of a miRNA by up to or more than 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the reference level. Alternatively, an increase as used herein is an increase by up to or at least 1log2FC, 1.5log2FC, 2log2FC, 3log2FC, 4log2FC, 5log2FC, 6log2FC, 7log2FC 8log2FC, 9log2FC, 10log2FC or more compared to the reference level.

[0084] Decrease

[0085] In the context of the present invention a decrease is a decrease in the level of an miRNA by up to or at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 or 95% compared to the reference level. Alternatively, as used herein a decrease is a decrease by up to or at least -1log2FC, -1.5log2FC, -2log2FC, -3log2FC, - 4log2FC, -5log2FC, -6log2FC, -7log2FC, -8log2FC, -9log2FC, -10log2FC or more compared to the reference level.

[0086] Log2FC stands for Iog2 fold change, which is a statistical measure used to quantify the change in expression of a nucleic acid between two conditions. The fold change is simply the ratio of the expression levels of the nucleic acid in the two conditions and the Iog2 transformation is used to normalise the data for interpretation. A log2FC of 1 indicates that the expression of a miNA has doubled, while a log2FC of 2 indicates that the expression of a miRNA has quadrupled. The log2FC is used to identify miRNAs that are differentially expressed under different conditions.

[0087] In an embodiment bta-miR-2467-3p is decreased by at least -0.5log2FC, -1 log2FC, - 5log2FC, -10log2FC, -15log2FC or -20log2FC. Preferably, bta-miR-2467-3p is decreased by around -15logFC.

[0088] In an embodiment bta-miR-2448-3p is decreased by at least -0.5log2FC, -1log2FC, - 1.5log2FC, -2log2FC, -3log2FC, -4log2FC, -5log2FC, -10log2FC. Preferably, bta-miR-2448- 3p is decreased by around -3logFC. In an embodiment bta-miR-150 is decreased by at least -0.5log2FC, -1log2FC, -1.5log2FC, -2log2FC, -3log2FC, -4log2FC, -5log2FC, -10log2FC. Preferably, bta-miR-150 is decreased by around -4logFC.

[0089] In an embodiment bta-miR-2285by is decreased by at least -0.5log2FC, -1 log2FC, - 1.5log2FC, -2log2FC, -3log2FC, -4log2FC, -5log2FC, -6log2FC, -7log2FC, -8log2FC, - 9log2FC -10log2FC, -15log2FC or -20log2FC. Preferably, bta-miR-2285by is decreased by around -7logFC.

[0090] In an embodiment bta-miR-382 is decreased by at least -0.5log2FC, -1log2FC, -1.5log2FC, -2log2FC, -3log2FC, -4log2FC, -5log2FC, -10log2FC. Preferably, bta-miR-382 is decreased by around -3logFC.

[0091] In an embodiment bta-miR-15a is decreased by at least -0.5log2FC, -1 log2FC, -1.5log2FC, -2log2FC, -3log2FC, -4log2FC, -5log2FC, -6log2FC, -7log2FC, -8log2FC, -9log2FC - 10log2FC, -15log2FC, -20log2FC, -50log2FC, -75log2FC, -100log2FC, -150log2FC, - 200log2FC, -250log2FC or -300log2FC. Preferably, bta-miR-15a is decreased by around - 250logFC.

[0092] Welfare

[0093] Welfare is a broad term, which includes the many elements that contribute to an animal's quality of life, including those referred to in the ’five freedoms’:

[0094] VI. freedom from hunger, thirst and malnutrition;

[0095] VII. freedom from fear and distress;

[0096] VIII. freedom from physical and thermal (heat) discomfort;

[0097] IX. freedom from pain, injury and disease; and

[0098] X. freedom to express normal patterns of behaviour.

[0099] If one of those five freedoms (each of the freedoms may be considered to be a “welfare parameter”) are not fulfilled, a freedom or welfare parameter is or has been considered to be compromised. For example, if an animal is exposed to social stress, said parameter is considered to be compromised It is understood that, not fulfilling one or more of freedom from fear and distress, freedom from pain, injury and disease, and / or freedom to express normal patterns of behaviour can be considered or lead to “social stress”. That is, one, two, or all three of these freedoms may be broken under conditions of social stress. Thus, in the present context, the term “compromised welfare parameter” refers to one of these five freedoms not being fulfilled for the animal or herd of animals. By “has been comprised” may mean that the welfare was comprised in an animal in the past. In particular, it may be that the welfare of the animal was compromised at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days or at least 30 days ago. In one embodiment, the welfare of the animal was comprised at least 20 days, preferably at least 21 days ago. The phrasing “at least one welfare parameter” and “welfare” may be used interchangeably.

[0100] When a welfare parameter is compromised, it means that the animal is not receiving the welfare conditions they should be. Welfare parameters include but are not limited to nutritional welfare, physical welfare, behavioural welfare and environmental welfare. Action can then be taken to improve the welfare state of the animal or animals.

[0101] Animal welfare means the physical and mental state of an animal in relation to the conditions in which it lives and dies. An animal experiences good welfare if the animal is healthy, comfortable, well nourished, safe, is not suffering from unpleasant states such as pain, fear and distress (such as social stress), and is able to express behaviours that are important for its physical and mental state.

[0102] Some measures of animal welfare involve assessing the degree of impaired functioning associated with injury, disease and malnutrition. Other measures provide information on animals' needs and affective states such as hunger, pain and fear, often by measuring the strength of animals' preferences, motivations and aversions. Others assess the physiological, behavioural and immunological changes or effects that animals show in response to various challenges.

[0103] Such measures can lead to criteria and indicators that help to evaluate how different methods of managing animals influence their welfare. Many aspects of the environment can impact the welfare of animals. The risk of social stress for cattle is influenced by environmental factors including overcrowding, regrouping, barren housing, social instability, animal density (area and volume available per animal), poor handling, inadequate food and water, disease, extreme weather and mixing with unfamiliar animals.

[0104] As such, it can be considered that social stress is caused by exposing an animal to one or more environmental factors selected from overcrowding, regrouping, barren housing, social instability, increased animal density (area and volume available per animal), poor handling, inadequate food and water, disease, extreme weather and mixing with unfamiliar animals.

[0105] In a preferred embodiment, compromised welfare is determined as cattle being exposed to social stress. The most common social stressors in cattle are maternal separation and weaning, social isolation and mixing, barren housing, poor handling and cattle overstocking.

[0106] Maternal separation and weaning:

[0107] In 5 to 6 month old calves, abrupt maternal separation produces: Psychological stress of breaking the maternal bond; and / or Nutritional changes associated with their changed diet

[0108] This separation results in behavioural changes in cattle, both calves and cows that may persist for several days and cause a more chronic form of social stress.

[0109] Their vocalisation and ambulation activity may increase, and this may persist at elevated levels for at least three days.

[0110] Mixing and social isolation’.

[0111] Cattle are herd animals that establish social orders with dominant and submissive animals within each group. Factors that cause social stress include competition for resources, stocking density, group size, group composition, especially commingling of primiparous and multiparous cows.

[0112] Introducing a single animal to an established group produces acute behavioural and biological responses, including:

[0113] Reduced epithelial cell tight junctions Altered response to infection

[0114] Increased fear response

[0115] Altered heart rate

[0116] Decreased milk production

[0117] Overstocking-.

[0118] Many studies document the effects of short-term overstocking on cow behaviour. Cattle overstocking can affect the lying and standing behaviour of dairy cattle because competition for stalls is increased, causing a reduction of lying time and a higher standing time outside the stalls. Reducing the feeding space per cow increases competition for feed, causing aggressiveness. Cows can vary their feeding rate in response to increased stocking pressure, and social mixing can lead to a decrease in time the cows spend feeding.

[0119] Barren housing:

[0120] Barren housing refers to housing arrangements where cattle have limited or no social interaction. This occurs in several situations. For example, when cattle are kept in individual stalls, preventing physical contact with other animals; or when cattle are kept in small groups that doesn’t facilitate the formation of a natural social hierarchy within a herd.

[0121] Barren housing can have negative consequences for cattle welfare. This is because cattle are social animals that prefer to live in herds with established social structures. When cattle are deprived of social interaction through barren housing this can lead to stress and anxiety which can result in: decreased productivity, increased susceptibility to disease and increased aggression.

[0122] Social instability.

[0123] Social instability refers to disruptions in a herd’s social order or hierarchy. This can be caused by mixing a herd with unfamiliar animals, or removing one or more heads of cattle from a herd.

[0124] Poor handling:

[0125] Poor handling refers to any practice that can induce stress, fear or injury to animals, examples of poor handling include:

[0126] Rough handling using excessive force, or sharp objects.

[0127] Loud noises. • Improper use of equipment leading to discomfort and injuries.

[0128] MicroRNA (miRNA)

[0129] MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing around 21 to 23 nucleotides.

[0130] The level of any given miRNA can be measured using techniques known in the art, such techniques include but are not limited to northern blot, microarray assays, RT-PCR, RNA- SEQ and miRNA-SEQ. These are standard assays in the art.

[0131] Exosomes

[0132] Exosomes are membrane-bound extracellular vesicles (EVs) that are produced in the endosomal compartment of most eukaryotic cells. In the present context the terms “exosome”, “extracellular vesicle” and “EV” are used interchangeably.

[0133] Reference level

[0134] In the context of the present invention, the term "reference level" relates to a standard in relation to a quantity, which other values or characteristics can be compared to.

[0135] Reference levels can be selected in different ways, known to the person skilled in the art. In an embodiment, said reference level is the level of the one or more miRNA’s in a biological sample from a cattle having normal welfare or an average level from several cattle having normal welfare. Preferably from dairy cattle.

[0136] In another embodiment, said reference level is the level of the one or more miRNA in a biological sample from a (dairy) cattle, which is not or has not been exposed to social stress or an average level from several (dairy) cattle not exposed to social stress.

[0137] In one embodiment of the present invention, it is possible to determine a reference level by investigating the abundance of one or more of the biomarkers according to the invention in biological samples from (dairy) cattle, which are considered to have normal welfare (i.e. not subject to one or more of the forms of stress discussed herein). By applying different statistical means, such as multivariate analysis, one or more reference levels can be calculated. Based on these results a cut-off may be obtained that shows the relationship between the level(s) detected and (dairy) cattle considered at risk of not having normal welfare. The cutoff can thereby be used to determine the amount of the one or more biomarkers, which corresponds to, for instance, an increased risk of the cattle not having normal welfare.

[0138] The cut-off level could be established using a number of methods, including: multivariate statistical tests (such as partial least squares discriminant analysis (PLS-DA), random forest, support vector machine, etc.), percentiles, mean plus or minus standard deviation(s); median value; fold changes.

[0139] The multivariate discriminant analysis and other risk assessments can be performed on the free or commercially available computer statistical packages (SAS, SPSS, Matlab, R, etc.) or other statistical software packages or screening software known to those skilled in the art.

[0140] In any of the embodiments discussed above, changing the risk cut-off level could change the results of the discriminant analysis for each sample tested.

[0141] Statistics enables evaluation of the significance of each biomarker level. Commonly used statistical tests applied to a data set include t-test, f-test or even more advanced tests and methods of comparing data. Using such a test or method enables the determination of whether two or more samples are significantly different or not.

[0142] The significance may be determined by the standard statistical methodology known by the person skilled in the art.

[0143] The chosen reference level may be changed depending on the specific sample for which the test is applied.

[0144] The chosen reference level may be changed if desired to give a different specificity or sensitivity as known in the art. Sensitivity and specificity are widely used statistics to describe and quantify how good and reliable a biomarker or a diagnostic test is. Sensitivity evaluates how good a biomarker or a diagnostic test is at detecting a disease or state of health, while specificity estimates how likely the tested cattle or group of cattle (i.e. control, cattle having normal welfare) can be correctly identified as having normal welfare. Several terms are used along with the description of sensitivity and specificity; true positives (TP), true negatives (TN), false negatives (FN) and false positives (FP). If a compromised welfare is proven to be present in cattle with compromised welfare, the result of the test is considered to be TP. If compromised welfare is not present in the cattle (i.e. control, without disease), and the test confirms the absence of compromised welfare, the test result is TN. If the test indicates the presence of compromised welfare in cattle with no such compromised welfare, the test result is FP. Finally, if the test indicates no presence of compromised welfare in cattle with compromised welfare, the test result is FN.

[0145] Sensitivity

[0146] Sensitivity = TP / (TP + FN) = number of true positive assessments / number of all samples from cattle with compromised welfare.

[0147] As used herein the sensitivity refers to the measures of the proportion of actual positives which are correctly identified as such - in analogy with a diagnostic test, i.e. the percentage of cattle having welfare below normal who are identified as having welfare below normal.

[0148] Specificity

[0149] Specificity= TN / (TN+ FP) = number of true negative assessments / number of all samples from controls.

[0150] As used herein the specificity refers to measures of the proportion of negatives, which are correctly identified - i.e. the percentage of cattle having welfare at a normal level who are identified as having welfare at a normal level. The relationship between both sensitivity and specificity can be assessed by the ROC curve. This graphical representation helps to decide the optimal model through determining the best threshold or cut-off for a test or a biomarker candidate.

[0151] As will be generally understood by those skilled in the art, methods for screening are processes of decision-making and therefore the chosen specificity and sensitivity depend on what is considered to be the optimal outcome by a given animal handler / farmer / personnel or buyer of the milk from the cattle or dairy products in question.

[0152] It would be obvious for a person skilled in the art that it may be advantageous to select a higher sensitivity at the expense of lower specificity in most cases, to identify as many patients with disease as possible. In a preferred embodiment, the invention relates to a method with a high specificity, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as 100%.

[0153] In another preferred embodiment, the invention relates to a method with a high sensitivity, such as at least 80%, such as at least 90%, such as 100%.

[0154] In a preferred embodiment, compromised welfare is determined as cattle being exposed to social stress.

[0155] Method for evaluating a welfare parameter of cattle

[0156] As also outlined above, the present invention relates to miRNA biomarkers obtained by a non-invasive technique, such as extracellular vesicle- / exosome-derived microRNAs from a biological sample, as indicators of whether a welfare parameter has been compromised, such as the animal having been exposed to social stress. Thus, an aspect of the invention relates to a method for evaluating at least one welfare parameter of cattle, preferably bovine, the method comprising:

[0157] - determining in a biological sample from said cattle, the level of one or more miRNAs selected from the group consisting of SEQ ID NOs: 1 , 2, 3, 4, 5, and 6 or a functional variant thereof wherein the functional variant has at least 60% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, and 6;

[0158] - comparing said one or more levels to one or more corresponding reference levels; wherein if said one or more miRNA levels of SEQ ID NOs: 1 , 2, 3, 4, 5, and 6 or a functional variant thereof, wherein the functional variant has at least 60% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, and 6, are below said one or more reference levels, it is indicative of that at least one welfare parameter of the cattle is compromised; and wherein if said one or more miRNA levels of SEQ ID NOs: 1 , 2, 3, 4, 5, and 6 or a functional variant thereof wherein the functional variant has at least 60% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, and 6 are equal to or above said one or more reference levels, it is indicative of that the at least one welfare parameter of the cattle is not compromised.

[0159] In one embodiment, the welfare parameter of at least one head of cattle is or was comprised by at least one type of stress, examples of which include but are not limited to social stress, heat stress, metabolic stress, disease stress or combinations thereof. In one embodiment, the stress is social stress. That is, detection of at least one miRNA biomarker as defined in one of SEQ I D NO: 1 , 2, 3, 4, 5, and 6 is indicative that the animal has or is subject to social stress.

[0160] The method may also comprise the detection of one or more further biomarker, where that one or more further biomarker is associated with a different welfare parameter - that is, a different type of stress (e.g. not social stress).

[0161] Welfare

[0162] Different welfare parameters may be evaluated using the method of the invention. Thus, in an embodiment, the at least one compromised welfare parameter is caused by a parameter selected from the group consisting of social stress, heat stress, metabolic stress, disease stress, and combinations thereof, preferably the parameter is social stress. In example 1 miRNA biomarkers linked to social stress have been identified.

[0163] Thus, in a preferred embodiment, the compromised welfare parameter is caused by social stress.

[0164] Cattle

[0165] Different kind of cattle may be evaluated using the method of the invention. Hence, in an embodiment, the cattle is dairy cattle. In a related embodiment, the cattle is bovine, such as Bos taurus, more preferably bovine dairy cattle.

[0166] The method of the invention may be used on a single cattle or group or herd of cattle. Thus, in an embodiment, said cattle is an individual cattle (a “head of cattle” as referred to herein) or a herd of cattle, preferably a herd of cattle.

[0167] In a related embodiment, said biological sample is from a single cattle or a mixed biological sample from a herd of cattle.

[0168] As also outlined in the example section, the Applicant has identified a number of biomarkers that can be used irrespective of the breed. However, in an embodiment the cattle is of the breed Brown Swiss and / or Holstein. Preferably, the cattle is of the breed Friesian Holstein. Milk sample

[0169] Different types of milk samples can be used in the method of the invention. Thus, in an embodiment, the milk sample is in the form of raw milk, skimmed milk, milk powder, or a food product or food ingredient comprising milk, dairy-containing products, such as an infant formula, cheese or yoghurt or being derived from such food product or food ingredient.

[0170] If the milk sample is a mixed sample from several individual milk samples, it can also be considered “bulk milk”.

[0171] In an embodiment, the level of miRNA, is the level of miRNA in milk-derived extracellular vesicles. In example 1 , the level of miRNA has been determined from milk-derived extracellular vesicles.

[0172] In a preferred embodiment the miRNA is derived from a milk-derived extracellular vesicle that expresses CD9 and / or TSG101.

[0173] Blood sample

[0174] The method can also work efficiently in blood samples.

[0175] Biomarkers

[0176] As shown in example 1 , different individual biomarkers have been identified, which can be used cross-breed. To improve the overall strength of the method, it may be advantageous to use more than one biomarker. Thus, in an embodiment, the method is performed for at least 2 of the miRNAs, such as for at least 3 of the miRNAs, preferably for at least 4 of the miRNAs, more preferably for at least 5 of the miRNAs and most preferably for all 6 biomarkers.

[0177] It may also be an advantage to include at least some of the listed biomarkers in the method based on the pathways they are involved in. In some embodiments biomarkers of the present invention are involved in synaptic regulation; preferably said biomarkers involved in synaptic regulation are selected from one or more of SEQ ID NO: 1 , 2, 3, 5 and 6.

[0178] In another embodiment biomarkers of the present invention are involved in regulating at least the cell cycle, more preferably said biomarkers regulating the cell cycle are selected from one or more of SEQ ID NO: 1 , 3, 5 and 6. The cell cycle is the series of events that takes place in a cell as it grows and divides into two genetically identical daughter cells. It is a highly regulated process that comprises several phases:

[0179] G1 Phase: Metabolic changes prepare the cell for division; the cell grows physically larger and organelles are copied.

[0180] S Phase: DNA is replicated.

[0181] G2 Phase: The cell grows more, makes proteins and organelles, and begins to reorganise its contents in preparation for mitosis.

[0182] M Phase: Nuclear DNA of the cell condenses into visible chromosomes and is pulled apart by the mitotic spindle, a specialised structure made out of microtubules. Mitosis takes place in five stages: prophase, prometaphase, metaphase, anaphase, and telophase.

[0183] Cytokinesis: The cytoplasm is split in two, resulting in two individual cells.

[0184] Cyclins and cyclin-dependent kinases (CDKs) are regulatory molecules that determine how a cell progresses through the cell cycle. When activated by a bound cyclin, CDKs phosphorylate target proteins to activate or deactivate them leading to coordinated progression through the cell cycle.

[0185] As used herein, “regulate” or “regulation” of the cell cycle refers to the mechanisms that control the orderly progression. Positive regulation of the cell cycle drives its progression, while negative regulation of the cell cycle slows progression or arrests the cell cycle.

[0186] Cell cycle arrest refers to a temporary or permanent halting of the cell cycle, preventing cells from progressing through the normal stages of cell division. miRNAs can act as either positive or negative regulators of cell cycle progression. When miRNAs target and downregulate genes encoding cell cycle proteins, they can inhibit cell cycle progression and promote cell cycle arrest. Conversely, when miRNAs target genes encoding proteins that inhibit the cell cycle, they can promote cell cycle progression and proliferation. This delicate balance of miRNA mediated regulation is important for normal cell growth.

[0187] The cell cycle can be measured using a variety of commercially available assays. For example, DyeCycle stains can be used that measure DNA content distribution and are analysed with flow cytometry. Alternatively, antibodies for cell cycle analysis are also available, which can be analysed using flow cytometry or imaging.

[0188] Synapses are junctions that mediate communication between neurones, Synapses are regulated at multiple levels. Neurotransmitter release from the pre-synaptic neurone is highly regulated and influenced by various factors such as calcium influx and gene expression. Signals from the post-synaptic neuron can also influence neurotransmitter release from the pre-synaptic neuron. The number of neurotransmitter receptors and receptor sensitivity is also critical to determining the strength of the signal overall. miRNAs can be involved in synaptic regulation by targeting mRNAs encoding proteins involved in neurotransmitter release, receptor function and post-synaptic signalling pathways amongst other factors.

[0189] In an embodiment, the one or more miRNAs are selected from the group listed in Table 7.

[0190] In a preferred embodiment, the one or more miRNAs are selected from the group listed in Table 7, with the proviso that the cattle is Holstein. The regulation is as indicated in Table 7 and the corresponding text.

[0191] In an embodiment, one or more further miRNAs are selected from the group listed in Table 8.

[0192] Levels of miRNA can be determined using methods known to the person skilled in the art. Thus, in an embodiment, the levels are determined by a method selected from the group consisting of NGS, qPCR, dPCR, and ELISA miRNA.

[0193] Determining improvement

[0194] By comparing different samples obtained at different time points (minutes or hours), it may be possible to evaluate if any improvement in animal welfare has taken place. Thus, an aspect of the invention relates to a method for evaluating if at least one welfare parameter of cattle has improved, the method comprising:

[0195] - determining in a first biological sample from said cattle, the level of one or more miRNAs selected from the group consisting of SEQ ID NOs: 1 , 2, 3, 4, 5 and 6 or a functional variant thereof wherein the functional variant has at least 60% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, and 6 ; - determining in a second biological sample from said cattle, the level of one or more miRNAs selected from the group consisting of SEQ ID NOs: 1 , 2, 3, 4, 5 and 6or a functional variant thereof wherein the functional variant has at least 60% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, and 6; wherein said second biological sample is obtained later in time than the first biological sample;

[0196] - comparing said one or more levels in the first sample to the one or more corresponding levels in the second sample;

[0197] • wherein if said one or more corresponding levels of SEQ ID NOs: 1 , 2, 3, 4, 5, and 6 or a functional variant thereof wherein the functional variant has at least 60% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, and 6 in the second sample are equal to or below said levels in the first sample, it is indicative of that the welfare parameter has not improved; and

[0198] • wherein if said one or more corresponding levels of SEQ ID NOs: 1 , 2, 3, 4, 5 and 6 or a functional variant thereof wherein the functional variant has at least 60% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, and 6 in the second sample are above than said levels in the first sample, it is indicative of that the welfare parameter has improved.

[0199] In one embodiment, the welfare parameter of at least one head of cattle is or was comprised by at least one type of stress, examples of which include but are not limited to social stress, heat stress, metabolic stress, disease stress or combinations thereof. In one embodiment, the stress is social stress. That is, detection of at least one miRNA biomarker as defined in one of SEQ I D NO: 1 , 2, 3, 4, 5, and 6 is indicative that the animal has or is subject to social stress.

[0200] The method may also comprise the detection of one or more further biomarker, where that one or more further biomarker is associated with a different welfare parameter - that is, a different type of stress (e.g. not social stress).

[0201] In some embodiments, rather than comparing miRNA levels in each sample to the reference level, the miRNA levels in the first and second biological samples may be compared directly.

[0202] In an embodiment, initiatives have taken place between the sampling of the first sample and the sampling of the second sample, to improve the one or more welfare parameters of the cattle. In an embodiment, the welfare parameter is social stress. In another embodiment the welfare parameter is heat stress, in some embodiments the welfare parameters are social stress and heat stress.

[0203] As described above, social stress occurs when cattle are subjected to any of maternal separation and weaning, social isolation and mixing, poor handling, barren housing, cattle overstocking, regrouping and / or social instability.

[0204] In yet another embodiment, the initiatives are selected from the group consisting of a change in heat exposure, such as lowering of temperature, installation of fans, possibility of shadowing, installation of sprinklers, improved access to additional water, change in feed, reduction of the animal density, provision of cooling systems as appropriate for the local conditions, changes in moving cattle, improved cattle handling, maintenance of stable herd groups for example by minimising mixing with unfamiliar animals, providing sufficient space per head of cattle, correct use of equipment for cattle handling, comfortable housing, good hygiene and sanitation and a less stressful environment.

[0205] In some embodiments there is a method of improving at least one of milk yield, milk quality, meat yield, meat quality from at least one head of cattle, the method comprising i. determining in a first biological sample from the at least one head of cattle, the level of at least one miRNA as defined in SEQ ID NOs: 1 , 2, 3, 4, 5 and 6 or a functional variant thereof wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, and 6; ii. comparing the level of the at least one miRNA to a reference level; wherein where the level of the at least one miRNA is above said reference the welfare of the at least one head of cattle is compromised; iii. improving the welfare of the at least one head of cattle as described herein; iv. determining in a second biological sample from the same at least one head of cattle, the level of at least one miRNA as defined in SEQ ID NOs: 1 , 2, 3, 4, 5, and 6 or a functional variant thereof wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, and 6; and v. comparing the level in the first biological sample to the level in the second biological sample, wherein if the level of SEQ ID NOs: 1 , 2, 3, 4, 5 and 6 or a functional variant thereof wherein said functional variant has at least 60% overall sequence identity to the non-variant sequence in the second biological sample is higher than the level in the first biological sample or higher than the reference level at least one of milk yield, milk quality, meat yield and meat quality from at least one head of cattle the welfare of the head of cattle has improved.

[0206] Milk yield refers to the amount of milk produced by an animal over a given period of time. Improving milk yield means increasing the amount of milk produced in a given time period.

[0207] Milk quality is concept that encompasses multiple factors such as sensory qualities, composition and bacterial safety. High quality milk is a rich source of protein, fat, calcium and other nutrients such as branched chain fatty acids (BCA’s). High quality milk is also free from contaminants, has a low bacterial count, is a rich creamy white colour with a clean fresh smell and a mild taste,

[0208] Meat yield refers to the amount of meat that can be obtained from an animal after slaughter. Meat yield is typically expressed as the percentage of the live weight or carcass weight of the animal.

[0209] Meat quality is a complex concept that encompasses a variety of factors including:

[0210] - Appearance : high quality meat has a bright appealing colour, uniform texture and no signs of discoloration.

[0211] - Texture : high quality meat is tender and easy to chew, whereas low quality meat is chewy and / or tough.

[0212] - Flavour : high quality meat has a richer and more complex flavour than low quality meat.

[0213] Uses

[0214] Yet another aspect of the invention relates to the use of the level of one or more miRNAs selected from the group consisting of SEQ ID NOs: 1 , 2, 3, 4, 5, and 6 in a cattle biological sample to evaluate the status of a welfare parameter of the cattle.

[0215] In an embodiment, the welfare parameter is social stress. Device

[0216] The method of the invention may also be implemented in a device or system. Thus, yet another aspect of the invention relates to a device or system adapted to determine in a cattle biological sample or biological derived sample if at least one welfare parameter of cattle, preferably bovine, has been compromised or not, the device comprising: a unit able to determine the level of one or more miRNAs in the biological sample or biological-derived sample; a processor configured with a reference table, said reference table corresponding to reference levels of one or more of the miRNAs according to SEQ ID NO: 1 , 2, 3, 4, 5 and / or 6 wherein the device or system is adapted to receive the biological sample or biological-derived sample; determine the levels of the one or more miRNAs; comparing the determined levels of the one or more miRNAs to the reference table; and determining if at least one welfare parameter of the cattle has been compromised or not.

[0217] In an embodiment, the reference table comprises corresponding miRNAs values from cattle where the corresponding welfare parameter has not been compromised.

[0218] In another embodiment, the device or system comprises a user interface, the user interface adapted to receive input from a user relating to one or more of, the miRNA tested, the breed from which the biological sample is derived, and the type of biological sample.

[0219] Computer implemented method

[0220] The method of the invention can also be computer-implemented. Thus, a further aspect of the invention relates to a computer implemented method of determining in a cattle biological sample whether at least one welfare parameter of cattle, preferably bovine, has been compromised or not, the method comprising: providing levels of one or more miRNAs from a cattle biological sample selected from SEQ ID NO: 1 , 2, 3, 4, 5 and 6; providing a mathematical model comprising reference levels of one or more corresponding miRNAs selected from SEQ ID NO: 1 , 2, 3, 4, 5 and 6; determining if the provided levels deviates (significantly) from the reference levels, using the mathematical model; and providing to a system or a user a determination of at least one welfare parameter of the cattle being compromised or not.

[0221] In an embodiment, the reference table comprises corresponding miRNAs values from cattle where the corresponding welfare parameter has not been compromised.

[0222] Yet another aspect of the invention relates to a computer system comprising an input / output device and a processor, the system being enabled to execute the computer implemented method according to the invention on the processor.

[0223] It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

[0224] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0225] The invention will now be described in further details in the following non-limiting examples.

[0226] Examples

[0227] Example 1 - Deregulation of biomarkers in cattle exposed to social stress

[0228] Aim of the Study

[0229] The objective of this study was to investigate the effect of social stress conditions on diverse biomarkers in milk, the biomarkers analysed were: miRNA, substance P, glucose, fat and protein.

[0230] Materials and Methods

[0231] Milk samples were analysed from 18 Friesian Holstein primiparous cows for a total of 36 milk samples (i.e. 2 samples / cow, one at week 8 and one at week 14). Cows were subjected to normal conditions for 9 weeks and then to a gradual worsening of housing conditions between weeks 10-14. Milk samples from the same 18 cows under standard housing conditions (milk of week 8) and worsened housing conditions (milk of week 14) were analysed. After adaptation to the reference conditions, animals were subjected to weekly worsening of housing conditions (overcrowding conditions, barren housing conditions, social instability).

[0232] In normal reference conditions over the first 9 weeks of the study cows had access to 11 cubicles, 11 feeding gates and one fixed brush and the social composition of the group was kept constant. No changes were imposed on the housing condition of the cows during this period.

[0233] How social stress was imposed during weeks 10-14 of the study is described in Table 2.

[0234] Table 2: Social stress imposed between weeks 10-14.

[0235] The facility consisted of pens housing 11 cows each, milk samples were collected from 4 of these 11 cows, the remaining 7 cows were required to induce social stress but were not used to collect milk samples.

[0236] The study was conducted in 3 batches due to operational constraints. Milk was therefore available from 3 batches x 2 pens x 6 cows / batch = 18 cows in each study arm.

[0237] All groups were housed in the same barn, but visual and tactile contacts between the groups was prevented by 2 metre high solid partitions. Mixing between the groups was prohibited. Within a pen, cows had free access to one automatic concentrate dispenser delivering a preset daily amount of concentrates based on individual milk production, and they had ad libitum access to one water trough. Cows received a total mixed ration of maize silage (35 % of dry matter), grass silage (30%), concentrates (20%), grinded whole soy (10%), grinded whole wheat (3%) and minerals (2%) twice a day at 7:00h and 17:00h.

[0238] As described above worsened housing conditions entailed: (1) overcrowding conditions; (2) barren housing conditions and (3) social instability. As a result, the worsening housing conditions did not meet the European minimum recommendations in terms of stocking density for 5 weeks (but reflect conditions which may occur in real life).

[0239] Cows were milked twice a day at around 05:00h and 15:00h.

[0240] The following markers were analysed in the milk samples collected: miRNA, substance P, glucose, fat and protein. Somatic cell count was within normal range, cows did not demonstrate any signs of fever or lameness at the start of experiment. Experimental animals were confirmed pregnant. Veterinary care was available throughout the whole experiment. One experimental animal was replaced in the second week of the third batch due to a miscarriage. Another companion animal was removed on week fourteen from the first batch because she was contagious for paratuberculosis.

[0241] The study confirmed that biomarkers such a miRNAs and substance P and physiological indicators such as milk yield can be used to indicate social stress in cattle.

[0242] After being subjected to social stress milk yield was reduced by an average of 1.47kg, showing that social stress has a negative effect on cattle productivity.

[0243] Levels of substance P were elevated in cattle after they had been subjected to social stress through worsening living conditions (Table 5), reinforcing that substance P is an indicator or stress and anxiety in cattle.

[0244] Using a threshold of Wilcoxon p-value less than 0.01 on data corrected for any batch effect, six miRNAs, namely; bta-miR-2467-3p, bta-miR-2448-3p, bta-miR-150, bta, miR-2285by, bta-miR-382 and bta-miR-15a were shown to be downregulated after cattle were subjected to social stress, this is shown in Figure 1. For instance, figure 1A shows that bta-miR-2467- 3p was decreased in cattle after they were subjected to social stress. Figure 1 B shows that bta-miR-2448-3p was reduced in cattle after social stress. Figure 1C shows that bta-miR- 150 was decreased in cattle after social stress. Figure 1 D shows that miR-2285by was decreased in cattle after social stress. Figure 1 E shows that bta-miR-382 was decreased in cattle after social stress and figure 1 F shows that bta-miR-15a was decreased in cattle after social stress. The log fold change of these miRNAs is given in Table 7.

[0245] This provides clear evidence that these bta-miR-2467-3p, bta-miR-2448-3p, bta-miR-150, bta, miR-2285by, bta-miR-382 and bta-miR-15a can be used as indicators of social stress in cattle.

[0246] Table 3: Milk yield and glucose levels in milk derived from cattle exposed to social stress.

[0247] Table 4: Fat and protein levels in milk derived from cattle exposed to social stress.

[0248]

[0249] Table 5: Substance P levels in milk derived from cattle exposed to social stress.

[0250] Table 6: miRNAs downrequlated in milk samples derived from cattle exposed to social stress and pathways said miRNAs have been reported to be involved in.

Claims

Claims1 . A method of determining at least one welfare parameter of at least one head of cattle, the method comprising i. determining in a biological sample the level of at least one miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5 and 6, or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, and 6 respectively; and ii. comparing the level of the at least one miRNA to a reference level, wherein if said one or more miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5 and 6 is below said reference level at least one welfare parameter of the at least one head of cattle has been or is compromised.

2. The method according to claim 1 , wherein the miRNA as defined by any of SEQ ID NO: 1 , 3, 5 and 6 regulates the cell cycle and / or is involved in synaptic regulation.

3. A method of improving the welfare of at least one head of cattle, the method comprising i. determining in a biological sample the level of at least one miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5 and 6 or a functional variant thereof wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, and 6 respectively; ii. comparing the level of the at least one miRNA to a reference level, wherein if said level of one or more miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5, and 6 is below said one or more reference level at least one welfare parameter of the at least one head of cattle is or has been compromised; and iii. improving the welfare of the at least one head of cattle.

4. A method of determining whether at least one welfare parameter of at least one head of cattle is improved, the method comprising i. determining in a first biological sample the level of at least one miRNA selected from SEQ ID NO: 1 ,2, 3, 4, 5, and 6 or a functional variant thereof wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, and 6 respectively;ii. comparing the level of the at least one miRNA to a reference level, wherein if said level of one or more miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5, and 6 is below said one or more reference level at least one welfare parameter of the at least one head of cattle is or has been compromised; iii. improving the welfare of the at least one head of cattle if at least one welfare parameter of the at least one head of cattle is or has been compromised; iv. determining in a second biological sample the level of at least one miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5, and 6, or a functional variant thereof wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 1, 2, 3, 4, 5 and 6 respectively; and v. comparing the level of the at least one miRNA in the first biological sample to the level in the second biological sample, wherein if the level of SEQ ID NO: 1 , 2, 3, 4, 5 and 6, or a functional variant thereof wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5 and 6 respectively in the second biological sample is higher than the level in the first biological sample or lower than the level in the reference sample the welfare of the head of cattle has improved.

5. A method of improving at least one of milk yield, milk quality, meat yield or meat quality of at least one head of cattle, the method comprising i. determining in a first biological sample the level of at least one miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5 and 6, or a functional variant thereof wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 1, 2, 3, 4, 5 and 6; ii. comparing the level of the at least one miRNA to a reference level, wherein if said level of one or more miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5 and 6 is below said one or more reference level at least one welfare parameter of the at least one head of cattle has been or is compromised; and if at least one welfare parameter of the at least one herd of cattle has been or is compromised, then iii. improving the welfare of the at least one head of cattle; wherein improving the welfare of the at least one head of cattle improves at least one of milk yield, milk quality, meat yield and meat quality.

6. The method according to claim 4, 5 or 6 wherein improving the welfare of the at least one head of cattle comprises one or more of improving cattle handling, maintaining stable herd groups, minimising mixing with unfamiliar animals, ensuring sufficient space is available per head of cattle, correct use of equipment for cattle handling, ensuring comfortable housing, ensuring good hygiene and sanitation, changing heat exposure, improving access to additional water, changing the feed, reducing animal density and the provision of cooling systems.

7. A method of determining at least one welfare parameter of at least one head of cattle, the method comprising determining the level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: 1 , 2, 3, 4, 5 and 6, or a functional variant thereof, wherein said functional variant has at least 60% overall sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, and 6 respectively; wherein when the level of SEQ ID NO: 1 , 2, 3, 4, 5, and 6 or a functional variant thereof is decreased by -1 log2FC or more compared to a reference level, at least one welfare parameter of the at least one head of cattle has been or is compromised.

8. A method of determining from a bulk milk sample or from a processed milk sample whether at least one welfare parameter of at least one herd of cattle has been or is compromised, the method comprising iii. determining in a dairy product sample the level of at least one miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5 and 6, or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, and 6 respectively; and comparing the level of the at least one miRNA to a reference level, wherein if said one or more miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5 and 6 is below said reference level at least one welfare parameter of the at least one herd of cattle has been or is compromised.

9. The method according to any preceding claim wherein the method further comprises determining the level of substance P wherein if the level of substance P is increased compared to a reference level at least one welfare parameter of the at least one head of cattle has been compromised.

10. The method according to any preceding claim, wherein the welfare parameter of at least one head or herd of cattle may be or is comprised by at least one of heat stress, social stress, metabolic stress, disease stress or combinations thereof, preferably social stress.

11. The method according to any preceding claim, wherein the cattle is dairy cattle, preferably bovine wherein preferably the breed of cattle is selected from Holstein and / or Brown Swiss.

12. The method of any preceding claim wherein the biological sample is selected from a milk sample, blood sample, a meat sample or a urine sample.

13. A device or system adapted to determine if at least one welfare parameter of at least one head or herd of cattle has been or is compromised, the device comprising: i. a unit able to determine the level of one or more miRNAs according to any preceding claim; ii. a processor configured with a reference table, said reference table corresponding to reference levels of one or more of the miRNAs with at least 60% overall sequence identity to one of SEQ ID NO: 1 , 2, 3, 4, 5 and 6; wherein the device or system is adapted to iii. receive the biological sample; iv. determine the levels of the one or more miRNAs; v. compare the determined levels of the one or more miRNAs to the reference levels of a sequence with at least 60% overall sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5 and 6; and vi. determine if the welfare of the at least one head or herd of cattle has been or is compromised.

14. A computer implemented method of determining if the welfare of at least one head of cattle has been compromised, the method comprising: i. providing levels of one or more miRNAs from a cattle biological sample selected from SEQ I D NO: 1 , 2, 3, 4, 5 and 6, or a variant with at least 60% overall sequence identity thereto;ii. providing a mathematical model comprising reference levels of one or more corresponding miRNAs selected from SEQ ID NO: 1, 2, 3, 4, 5 and 6, or a variant with at least 60% overall sequence identity thereto; iii. determining if the provided levels deviates significantly from the reference levels, using the mathematical model; and iv. providing to a system or a user a determination of whether the welfare of the at least one head of cattle has been or is compromised.

15. A kit for evaluating at least one welfare parameter of at least one head of cattle comprising the device of claim 13 and instructions for use.

16. A kit for evaluating at least one welfare parameter in at least one head of cattle wherein said kit comprises: i. a means for collecting a bulk milk sample obtained from at least one head or herd of cattle; and ii. a unit for measuring the level of at least one miRNA selected from SEQ ID NO : 1, 2, 3, 4 , 5 and 6, or a variant with at least 60% overall sequence identity thereto; and iii. instructions for use.

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