Mirnas from processed milk products as indicators of stress in bovine

Abstract

The present invention relates miRNA biomarkers from a processed milk product as indicators of welfare in bovines. In particular, the present invention relates to the identification of heat stress in processed milk products from bovines, which in turn means actions can be taken to manage and improve productive animal welfare at the individual or the herd level.

Description

[0001] miRNAs FROM PROCESSED MILK PRODUCTS AS INDICATORS OF STRESS IN BOVINE

[0002] Technical field of the invention

[0003] This present invention relates to the use of micro-RNAs in processed milk products derived from a dairy cattle as indicators of animal welfare, such as indicators of stress such as heat 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] Currently, different indicators are used to evaluate if an animal has experienced or is experiencing stress conditions. These include,

[0007] - 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;

[0008] - 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, and stage of 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.

[0009] 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).

[0010] Miretti et al. discloses microRNAs as biomarkers for animal health and welfare in Livestock (Front. Vet. Sci. 7:578193). 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).

[0011] Li et al. (BMC Genomics (2018) 19:975) discloses characterization of miRNA profiles in the mammary tissue of dairy cattle in response to heat stress.

[0012] The present applicants found that 24 miRNAs are biomarkers in milk of acute stress in cattle. There is also a need to establish whether miRNAs can also act as biomarkers of stress at the herd level, and / or in processed milk products. This is important as if cattle welfare can be assessed with miRNA biomarkers at the herd level and in processed products, the welfare status of the animal can be easily communicated and confirmed to the consumer.

[0013] Hence, an improved method to non-invasively determine animal welfare in processed milk products would be advantageous, and in particular a more efficient and / or reliable method to determine animal stress, such as heat stress, would be advantageous. The present invention addresses this need.

[0014] Summary of the invention

[0015] The present invention relates to the identification of miRNA biomarkers from a processed milk sample, as indicators of welfare (or as used herein a welfare parameter) in cattle. In particular, the present invention relates to the identification of stress, particularly heat 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.

[0016] Thus, an object of the present invention relates to the provision of miRNA biomarkers which can be indicative of welfare parameters in cattle, preferably dairy cattle where the sample is taken from a processed milk product.

[0017] In particular, it is an object of the present invention to provide miRNA biomarkers from biological sample derived exosomes, which can indicate heat stress in (dairy) cattle and which can be used across different breeds of (dairy) cattle.

[0018] Example 1 shows miRNA expression in bulk milk from a herd of cattle in heat stress. Example 2 shows miRNA expression in products processed from bulk milk obtained from a herd of cattle in heat stress.

[0019] Thus, one aspect of the invention relates to a method of determining from a processed milk product, wherein said processed milk product has been subjected to at least heat treatment at around 65°C or higher, whether at least one welfare parameter of at least one herd of cattle from which the milk was obtained has been or is compromised, the method comprising determining in the processed milk product the presence or level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 respectively; wherein if said one or more miRNA is detected at least one welfare parameter of the at least one herd of cattle has been or is compromised

[0020] Another aspect of the invention relates to a method of determining from a processed milk product whether at least one welfare parameter of at least one herd of cattle from which the milk was obtained has been or is compromised, the method comprising determining in the processed milk product the presence or level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18 or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18 respectively; wherein if said one or more miRNA is detected at least one welfare parameter of the at least one herd of cattle has been or is compromised.

[0021] In one embodiment of the method, the method comprises i. determining in the processed milk product that has been subjected to at least heat treatment at around 65°C or higher, the level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, and 24 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, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 respectively; and ii. comparing the level of the at least one miRNA to a reference level, wherein if said one or more miRNA is altered compared to said reference level at least one welfare parameter of the at least one herd of cattle has been or is compromised.

[0022] In one embodiment of the method, the method comprises i. determining in the processed milk product the level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18 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, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18 respectively; and ii. comparing the level of the at least one miRNA to a reference level, wherein if said one or more miRNA is altered compared to said reference level at least one welfare parameter of the at least one herd of cattle has been or is compromised.

[0023] In one embodiment, the at least one miRNA is SEQ ID NO: 16.

[0024] In one embodiment, the at least one miRNA is SEQ ID NO: 17.

[0025] In one embodiment, the at least one miRNA is SEQ ID NO: 16 and SEQ ID NO: 17.

[0026] In one embodiment, the welfare parameter of the at least one herd 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 heat stress. That is, the detection of or a change in levels of at least one miRNA biomarker as defined in one of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 in a processed milk sample that has been subjected to at least heat treatment at about 65°C or higher is indicative that the at least one herd of cattle has been or is subject to heat stress

[0027] In one embodiment, the welfare parameter of the at least one herd 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 heat stress. That is, the detection of or a change in levels of at least one miRNA biomarker as defined in one of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18, preferably SEQ ID NO: 16 and / or 17, is indicative that the at least one herd of cattle has been or is subject to heat stress. Once it is determined that at least one welfare parameter of at least one herd of cattle has been compromised it is then possible to improve the welfare of the animal.

[0028] 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 heat stress). Accordingly, in this embodiment, the method can be used to determine if the at least one herd of cattle is or was exposed to both heat stress and a further alternative stress.

[0029] In another aspect of the invention there is provided a method of improving the welfare of at least one herd 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, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24, 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, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 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, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 (or a variant thereof) is below said one or more reference level at least one welfare parameter of the at least one herd of cattle is or has been compromised; and iii. improving the welfare of the at least one herd of cattle if at least one welfare parameter of the at least one herd of cattle is or has been compromised.

[0030] As used herein “by improving welfare” is meant improving at least one welfare parameter, as defined herein. The improvement may be relative to a herd of cattle where the welfare or at least one welfare parameter has not been improved by the methods described below

[0031] In another aspect of the invention, there is provided a method of determining whether at least one welfare parameter of at least one herd of cattle is improved, the method comprising i. determining in a first processed milk product obtained from the at least one herd of cattle the level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15,

[0032] 16, 17, 18, 19, 20, 21 , 22, 23 and 24 or a functional variant thereof, and wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16,

[0033] 17, 18, 19, 20, 21 , 22, 23 and 24 respectively; wherein if said at least one miRNA is detected at least one welfare parameter of the at least one herd of cattle is or has been compromised; ii. improving the welfare of the at least one herd of cattle if at least one welfare parameter of the at least one herd of cattle is or has been compromised; iii. determining in a second processed milk product obtained from the at least one herd of cattle the level of at least one miRNA selected from SEQ ID NO: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 respectively; and iv. comparing the level of the at least one miRNA in the second processed milk product to the first processed milk product, wherein if the level of the miRNA in the second processed milk product has decreased compared to the first processed milk product, the welfare of the herd of cattle has improved wherein said first and second processed milk products have both been subjected to at least heat treatment at around 65°C or higher.

[0034] In another aspect of the invention, there is provided a method of determining whether at least one welfare parameter of at least one herd of cattle is improved, the method comprising i. determining in a first processed milk product obtained from the at least one herd of cattle the level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18 or a functional variant thereof, and wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18 respectively; wherein if said at least one miRNA is detected at least one welfare parameter of the at least one herd of cattle is or has been compromised; ii. improving the welfare of the at least one herd of cattle if at least one welfare parameter of the at least one herd of cattle is or has been compromised; iii. determining in a second processed milk product obtained from the at least one herd of cattle the level of at least one miRNA selected from SEQ ID NO: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18 a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18 respectively; and iv. comparing the level of the at least one miRNA in the second processed milk product to the first processed milk product, wherein if the level of the miRNA in the second processed milk product has decreased compared to the first processed milk product, the welfare of the herd of cattle has improved.

[0035] According to the aspect above, the second processed milk product (in step iii) will be obtained after the welfare of the herd of cattle is altered (in step ii).

[0036] In one embodiment of the method, the method comprises i. determining in a first processed milk product obtained from the at least one herd of cattle the level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: 16 or 17 or a functional variant thereof, and wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 16 or 17 respectively; ii. comparing the level of the at least one miRNA to a corresponding reference level, wherein if said at least one miRNA is altered compared to the reference level at least one welfare parameter of the at least one herd of cattle is or has been compromised; iii. improving the welfare of the at least one herd of cattle if at least one welfare parameter of the at least one herd of cattle is or has been compromised; iv. determining in a second processed milk product obtained from the at least one herd of cattle the level of at least one miRNA selected from SEQ ID NO: 16 or 17 a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 16 or 17 respectively; and v. comparing the level of the at least one miRNA in the processed milk product to the corresponding reference level, wherein if the relative change of the miRNA to the reference level in the second processed milk product is reduced compared to the relative change measured the first processed milk product (step ii) the welfare of the herd of cattle has improved.

[0037] According to the embodiment above, the second processed milk product (in step iv) will be obtained after the welfare of the herd of cattle is altered (in step iii).

[0038] In one embodiment, altering or improving the welfare of the at least one herd of cattle comprises one or more of changing heat exposure, improving access to additional water, changing the feed, reducing animal density and the provision of cooling systems.

[0039] Again, in some embodiments the method further comprises determining 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 heat stress). Accordingly, in this embodiment, the method can be used to determine if the herd of cattle is or was exposed to both heat stress and a further, alternative stress.

[0040] 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 herd of cattle, the method comprising i. determining in a processed milk product that has been subjected to at least heat treatment at around 65°C or higher, obtained from the at least one herd of cattle the level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 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, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24; wherein detection of the at least one miRNA is indicative that at least one welfare parameter of the at least one herd of cattle has been or is compromised; and ii. if at least one welfare parameter of the at least one herd of cattle has been or is compromised, then improving the welfare of the at least one herd of cattle; wherein improving the welfare of the at least one herd of cattle improves at least one of milk yield, milk quality, meat yield and meat quality. 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 herd of cattle, the method comprising i. determining in a processed milk product obtained from the at least one herd of cattle the level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18 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, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18; wherein detection of the at least one miRNA is indicative that at least one welfare parameter of the at least one herd of cattle has been or is compromised; and ii. if at least one welfare parameter of the at least one herd of cattle has been or is compromised, then improving the welfare of the at least one herd of cattle; wherein improving the welfare of the at least one herd of cattle improves at least one of milk yield, milk quality, meat yield and meat quality.

[0041] In one embodiment of the method, the method comprises i. determining in a first processed milk product obtained from the at least one herd of cattle the level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18 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, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18; wherein detection of the at least one miRNA is indicative that at least one welfare parameter of the at least one herd of cattle has been or is compromised; and ii. comparing the level of the at least one miRNA to a corresponding reference level, wherein if said one or more miRNA is altered compared to said reference level at least one welfare parameter of the at least one herd of cattle has been or is compromised; and iii. if at least one welfare parameter of the at least one herd of cattle has been or is compromised, then improving the welfare of the at least one herd of cattle; wherein improving the welfare of the at least one herd of cattle improves at least one of milk yield, milk quality, meat yield and meat quality.

[0042] In one embodiment, the at least one miRNA is SEQ ID NO: 16.

[0043] In one embodiment, the at least one miRNA is SEQ ID NO: 17.

[0044] In one embodiment, the at least one miRNA is SEQ ID NO: 16 and SEQ ID NO: 17.

[0045] In one embodiment, the welfare parameter of at least one herd 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 heat stress. That is, detection of at least one miRNA biomarker as defined in one of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24; preferably, SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18, in particular SEQ ID NO: 16 or 17 is indicative that the herd of cattle has been or is subject to heat stress.

[0046] In one embodiment, improving the welfare (i.e. alleviating or reducing heat stress) of the at least one herd of cattle comprises one or more measure to alleviate or reduce the social stress of the animal(s). For example, 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, and a less stressful environment.

[0047] In another aspect of the invention, there is provided a method of determining from a processed milk product at least one welfare parameter of at least one herd of cattle from which the milk was obtained, the method comprising determining the presence or level of at least one miRNA in the processed milk product, wherein the miRNA is selected from SEQ ID NO: 16 or 17 or a functional variant thereof, wherein said functional variant has at least 60% overall sequence identity to SEQ ID NO: 16 or 17 respectively; wherein when the at least one miRNA is detected in the processed milk product, at least one welfare parameter of the at least one herd of cattle has been or is compromised. In one embodiment of the method, the method comprises determining the level of at least one miRNA in the processed milk product, wherein the miRNA is selected from SEQ ID NO: 16 or 17 or a functional variant thereof, wherein said functional variant has at least 60% overall sequence identity to SEQ ID NO: 16 or 17 respectively; wherein when the level of an miRNA is altered compared to a reference level, at least one herd of cattle has been or is compromised.

[0048] According to any of the aspects and embodiments of the invention, if at least one of SEQ ID NO: 16 or 17 is detected in a processed milk product at least one welfare parameter of the at least one herd of cattle from which the milk was obtained is or has been compromised.

[0049] According to any of the aspects and embodiments of the invention, if the level of at least of SEQ ID NO: 16 or 17 in a processed milk product is altered compared to one or more reference level at least one welfare parameter of the at least one herd of cattle from which the milk was obtained is or has been compromised.

[0050] In one embodiment, if the level of at least of SEQ ID NO: 16 or 17 is altered by at least 1 log2FC or -1log2FC (1 log fold change) compared to a reference level, at least one welfare parameter of the at least one herd of cattle from which the milk was obtained is or has been compromised.

[0051] According to any of the aspects and embodiments of the invention, if the level of at least one of SEQ ID NO: 16 or 17 in a processed milk product is significantly altered compared to one or more reference level at least one welfare parameter of the at least one herd of cattle from which the milk was obtained has been compromised.

[0052] According to any of the aspects and embodiments of the invention, if the level of at least one of SEQ ID NO: 16 or 17 in a processed milk product is increased or decreased compared to one or more reference level at least one welfare parameter of the at least one herd of cattle from which the milk was obtained is or has been compromised.

[0053] In one embodiment, the welfare parameter of at least one herd of cattle may be or is compromised by at least one type of stress, examples of which include but are not limited to heat stress, social stress, metabolic stress, disease stress or combinations thereof. In one embodiment, the stress is heat stress. In an alternative embodiment the stress is social stress.

[0054] In one embodiment, the processed milk product is derived from a plurality of breeds of cattle.

[0055] 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.

[0056] In one embodiment, the processed milk product is produced from milk obtained from a single herd of cattle. That is, the processed milk product is produced from pooled milk from a plurality of - i.e. two or more - animals. In an alternative embodiment, the processed milk product is produced from milk pooled from a plurality of herds of cattle. That is, the processed milk product is pooled from a plurality of - i.e. two or more - herds.

[0057] The processed milk product may be obtained 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 days before being subjected to methods of the present invention. In some embodiments, the processed milk product is frozen and thawed before being subjected to said method.

[0058] In some embodiments, the processed milk product has been processed by any one or more of pasteurisation, spray drying, homogenisation, filtration, or centrifugation. In some embodiments, the processed milk product is produced from milk that has been processed by any one or more of pasteurisation, spray drying, homogenisation, filtration, or centrifugation.

[0059] In some embodiments the processed milk product has been subjected to at least heat treatment at around 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 82,83,84,85,86,87,88,89,90,91 ,92,93,94,95,96,97,98,99,100,101 ,102,103,104,105,106,10 7,108,109,110,111 ,112,113,114,115,116,117,118,119,120,121 ,122,123,124,125,126,127, 128,129,130,131 ,132,133,134,135,136,137,138,139,140,141 ,142,143,144,145,146,147,1 48,149,150,151 ,152,153,154,155,156,157,158,159,160,161 ,162,163,164,165,166,167,16 8,169,170,171 ,172,173,174,175,176,177,178,179,180,181 ,182,183,184,185,186,187,188, 189,190,191 ,192,193,194,195,196,197,198,199,200,201 ,202,203,204,205,206,207,208,2 09,210,211 ,212,213,214,215,216,217,218,219,220,221 ,222,223,224,225,226,227,228,22 9,230,231 ,232,233,234,235,236,237,238,239,240,241 ,242,243,244,245,246,247,248,249 or 250°C. In some embodiments, the processed milk product has been subjected to heat treatment between 50-250°C, between 60-250°C, between 60-200°C, between 60-180°C, between 60°C-150°C, between 60-120°C and / or between 60-100°C. In a preferred embodiment the processed milk product has been subjected to heat treatment between 60- 180°C, more preferably between 60-150°C, even more preferably between 60-120°C.

[0060] Preferably, the processed milk sample has been subjected to at least heat treatment at around 65°C or higher and / or 90°C or higher. More preferably the processed milk sample has been subjected to at least heat treatment at around 65°C or higher.

[0061] In some embodiments the processed milk product is a dairy product such as yoghurt, buttermilk, cream, cheese, butter or milk powder or frozen milk products such as ice cream. The processed milk product may be further used in food products such as chocolate.

[0062] The method may comprise determining the level of both SEQ ID NO: 16 and 17 or a functional variant thereof wherein the functional variant has at least 60% overall sequence identity to SEQ ID Nos: 16 and 17 respectively.

[0063] The reference level may be the level of the one or more miRNA in a processed milk product produced from milk obtained from at least one herd of cattle where the welfare of the 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.

[0064] The method may further comprise determining in a processed milk product produced from milk obtained from said herd of cattle the level of a physiological parameter, wherein the physiological parameter may be selected from one or more of cortisol levels, substance P levels, glucose levels, protein profiles, triglyceride levels, and haptoglobin levels, heat shock protein 70, a serum protein profile, blood urea nitrogen, blood uric acid, serum chloride, serum phosphorous, serum triglyceride and serum magnesium.

[0065] In one embodiment the method further comprises determining the level of heat shock protein 70 in a processed milk product produced from milk obtained from at least one 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 herd of cattle is or has been compromised.

[0066] In one embodiment the method further comprises determining the serum protein profile in a processed milk product produced from milk obtained from at least one 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 herd of cattle is or has been compromised.

[0067] In one embodiment the method further comprises determining the level of blood urea nitrogen in a processed milk product produced from milk obtained from at least one 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 herd of cattle is or has been compromised.

[0068] In one embodiment the method further comprises determining the level of blood uric acid in a processed milk product produced from milk obtained from at least one 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 herd of cattle is or has been compromised.

[0069] In one embodiment the method further comprises determining the level of serum chloride in a processed milk product produced from milk obtained from at least one 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 herd of cattle is or has been compromised.

[0070] In one embodiment the method further comprises determining the level of serum phosphorous in a processed milk product produced from milk obtained from at least one herd of cattle, comparing said level to 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 herd of cattle is or has been compromised.

[0071] In one embodiment the method further comprises determining the level of serum magnesium in a processed milk product produced from milk obtained from at least one herd of cattle, comparing said level to 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 herd of cattle is or has been compromised.

[0072] In one embodiment the method further comprises determining the level of triglyceride in a processed milk product produced from milk obtained from at least one herd of cattle, comparing said level to corresponding reference level of 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 herd of cattle is or has been compromised.

[0073] In another aspect of the invention, there is provided a device or system adapted to determine from a processed milk sample whether at least one welfare parameter of at least one 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 in a processed milk product produced from milk obtained from at least one herd of cattle; a processor configured with a reference table, said reference table corresponding to reference levels of one or more of the miRNA; wherein the device or system is adapted to

[0074] - receive a processed milk product produced from milk obtained from at least one herd of cattle;

[0075] - determine the levels of the one or more miRNAs in the processed milk product;

[0076] - compare the determined levels of the one or more miRNAs to the reference value; and

[0077] - determine if the welfare of the at least one herd of cattle has been or is compromised. In another aspect of the invention, there is provided a method of determining from a processed milk product that has be subjected to at least heat treatment at around 65°C or higher, if the welfare of at least one herd of cattle from which the milk was obtained has been compromised, the method comprising: i. providing levels of one or more miRNAs from a processed milk product obtained from at least one herd of cattle selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24; ii. providing a mathematical model comprising reference levels of one or more corresponding miRNAs selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24; iii. determining if the provided levels deviate 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 herd of cattle has been or is compromised.

[0078] In another aspect of the invention, there is provided a method of determining from a processed milk product if the welfare of at least one herd of cattle from which the milk was obtained has been compromised, the method comprising: i. providing levels of one or more miRNAs from a processed milk product obtained from at least one herd of cattle selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18, preferably SEQ ID NO: 16 and / or 17; ii. providing a mathematical model comprising reference levels of one or more corresponding miRNAs selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18, preferably SEQ I D NO: 16 and / or 17; iii. determining if the provided levels deviate 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 herd of cattle has been or is compromised.

[0079] In another aspect of the invention, there is provided a kit for evaluating at least one welfare parameter of at least one herd of cattle comprising the device of the invention and instructions for use. Preferably the at least one welfare parameter is heat stress. In another aspect of the invention there is a kit for determining from a processed milk sample that has be subjected to at least heat treatment at around 65°C or higher whether at least one herd of cattle from which the product is or was obtained is or was stressed, particularly heat stressed, wherein said kit comprises: i. a means for collecting a processed milk product produced from milk obtained from at least one 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, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 ; and iii. instructions for use.

[0080] In another aspect of the invention there is a kit for determining from a processed milk sample whether at least one herd of cattle from which the product is or was obtained is or was stressed, particularly heat stressed, wherein said kit comprises: i. a means for collecting a processed milk product produced from milk obtained from at least one 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, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18, preferably SEQ ID NO: 16 and / or 17 ; and iii. instructions for use.

[0081] The present invention will now be described in more detail in the following passages.

[0082] Brief Description of the Figures

[0083] Figure 1 shows the expression of miR-11980-5p, miR-92a-2-3p, miR-25-5p and miR-2436- 3p in a milk sample before heat stress was applied to cattle (T-1) and after heat stress was applied (T4). TO shows expression shortly after heat stress conditions began, T5 and T12 show the recovery phase after cooling systems were put back in place.

[0084] Figure 2 shows the expression of miR-2288-3p, miR-500-5p, miR-339a-5p, miR-339b-5p and miR-532-3p in a milk sample before heat stress was applied to cattle (T-1) and after heat stress was applied (T4). TO shows expression shortly after heat stress conditions began, T5 and T12 show the recovery phase after cooling systems were put back in place. Figure 3 shows the expression of miR-2285n-1-5p, miR-1777a-3p, miR-502b-5p, miR- 6525-5p, miR-664b-3p in a milk sample, before heat stress was applied to cattle (T-1) and after heat stress was applied (T4). TO shows expression shortly after heat stress conditions began, T5 and T12 show the recovery phase after cooling systems were put back in place.

[0085] Figure 4 shows the expression of miR-30f-5p, miR-30a-5p, miR-30b-5p and miR-30c-5p in a milk sample before heat stress was applied to cattle (T-1) and after heat stress was applied (T4). TO shows expression shortly after heat stress conditions began, T5 and T12 show the recovery phase after cooling systems were put back in place.

[0086] Figure 5 shows the abundance of miR-30a-5p and miR-30b-5p in extracellular vesicles derived from raw milk, pasteurised milk and spray dried milk. TO shows expression shortly after heat stress conditions began, T5 and T12 show the recovery phase after cooling systems were put back in place.

[0087] Figure 6 shows the abundance of miR-30a-5p and miR-30b-5p in whole milk samples derived from raw milk, pasteurised milk and spray dried milk. TO shows expression shortly after heat stress conditions began, T5 and T12 show the recovery phase after cooling systems were put back in place.

[0088] Figure 7 shows the abundance of miR-30a-5p and miR-30b-5p in extracellular vesicles derived from raw milk (non-treated milk), pasteurised milk and spray dried milk. Data is shown separately for non-treated (NT) milk, after pasteurisation (AP) and after spray drying (AS). TO shows expression shortly after heat stress conditions began, T5 and T12 show the recovery phase after cooling systems were put back in place.

[0089] Figure 8 shows the abundance of miR-30a-5p and miR-30b-5p in whole milk samples derived from raw milk (non-treated milk), pasteurised milk and spray dried milk. Data is shown separately for non-treated (NT) milk, milk after pasteurisation (AP) and milk after spray drying (AS). TO shows expression shortly after heat stress conditions began, T5 and T12 show the recovery phase after cooling systems were put back in place. Detailed description of the invention

[0090] Definitions

[0091] Prior to discussing the present invention in further detail, the following terms and conventions will first be defined:

[0092] Head of cattle

[0093] At least one head of cattle refers to one animal of unspecified age or gender.

[0094] Herd of cattle

[0095] A herd of cattle refers to a group of cattle that live alongside one another. A herd may be either wild or domestic. A wild herd of cattle refers to a group of untamed animals that live and travel together, whereas a domestic herd refers to group of animals kept together and maintained by humans.

[0096] Dairy cattle

[0097] 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.

[0098] Bulk milk sample

[0099] A bulk milk sample refers to a pooled milk sample derived from a plurality of cows. Any number of milk samples may comprise a bulk milk sample. A bulk milk sample may be derived from a single herd of cattle or a plurality of herds of cattle. Preferably, a bulk milk sample comprises milk only from cattle that were milked on the same day.

[0100] Functional Variant

[0101] 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, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18, 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.

[0102] Altered Compared to (Reference Level)

[0103] In the context of the present invention an alteration is a variation in the level of a miRNA compared or relative to a reference level, preferably a corresponding reference level. Therefore, the language ‘altered’ or ‘alteration’ indicates that the value has changed relatively or comparatively to the given reference level. This change may be an increase or decrease.

[0104] The degree of alteration may be an increase or decrease of 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 an alteration may be a variation 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.

[0105] In one embodiment, altered indicates a statistically significant alteration of an miRNA level relative to a reference level.

[0106] Increase

[0107] 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.

[0108] In one embodiment, an increase may be a statistically significant increase in an miRNA level compared to a reference level.

[0109] Decrease

[0110] 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.

[0111] In one embodiment, a decrease may be a statistically significant decrease in an miRNA level compared to a reference level.

[0112] 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.

[0113] Welfare

[0114] 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’:

[0115] I. freedom from hunger, thirst and malnutrition;

[0116] II. freedom from fear and distress;

[0117] III. freedom from physical and thermal (heat) discomfort;

[0118] IV. freedom from pain, injury and disease; and

[0119] V. freedom to express normal patterns of behaviour.

[0120] If one of those five freedoms (“welfare parameters”) are not fulfilled, a freedom or welfare parameter is or has been considered to be compromised. For example, if an animal is exposed to heat stress, said parameter (freedom no. Ill) is considered to be compromised. 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 - and in prtaicular freedom III. By “has been compromised” may mean that the welfare was compromised 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 compromised at least 20 days, preferably at least 21 days ago. The phrasing “at least one welfare parameter” and “welfare” may be used interchangeably.

[0121] When a welfare parameter is compromised, it means that the animal is not receiving the level of welfare 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 of the animal or animals.

[0122] 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 heat stress), and is able to express behaviours that are important for its physical and mental state.

[0123] 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.

[0124] Such measures can lead to criteria and indicators that help to evaluate how different methods of managing animals influence their welfare.

[0125] Many aspects of the environment can impact the welfare of animals. The risk of heat stress for cattle is influenced by environmental factors including air temperature, relative humidity, wind speed, animal density (area and volume available per animal), shade availability, animal factors including breed, age, body condition, metabolic rate and stage of lactation, and coat colour and density. As such, it can be considered that heat stress is caused by exposing an animal to one or more environmental factor selected from high or low air temperatures, increased humidity, increased wind speed, increased animal density (area and volume available per animal), decreased shade availability. In a preferred embodiment, compromised welfare is determined as cattle being exposed to heat stress. Heat stress may be defined by the “temperature-humidity index (THI)”.

[0126] The temperature-humidity index (THI) is a single value representing the combined effects of air temperature and humidity associated with the level of thermal stress. This index has been developed as a weather safety index to monitor and reduce heat-stress-related losses. Different animal species and humans have different sensitivities to ambient temperature and the amount of moisture in the air. During hot and humid weather the natural capability of cattle to dissipate heat load by sweating and panting is compromised and heat stress occurs. At THI values of 75 to 78, the animal organism is under heat stress, but the mechanisms of thermoregulation still manage to cope, while at THI over 78 it is assumed that the stress is so high that it is impossible to maintain the thermoregulatory mechanisms or normal body temperature.

[0127] Thus, in an embodiment, heat stress is defined as at least one cattle of a herd having been or being exposed to a THI of 75 or above, preferably 76 or above, such as in the range 75- 78 or 76-78.

[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. Reference level

[0133] 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.

[0134] 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 miRNAs in a processed milk product produced from milk obtained from a herd of cattle having normal welfare or an average level from several cattle having normal welfare, preferably from dairy cattle.

[0135] In another embodiment, said reference level is the level of the one or more miRNAs in a processed milk product produced from milk obtained from (dairy) cattle, which is not or has not been exposed to heat stress or an average level from several (dairy) cattle not exposed to heat stress.

[0136] 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 a processed milk product produced from milk obtained from a herd of (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.

[0137] 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 group of cattle (i.e. control, cattle having normal welfare) can be correctly identified as having normal welfare.

[0145] 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.

[0146] Sensitivity

[0147] Sensitivity = TP / (TP + FN) = number of true positive assessments / number of all samples from cattle with compromised welfare. 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 apparent for a person skilled in the art that it may be advantageous to select a higher sensitivity at the expense of lower specificity in some cases, to identify as many positive subjects as possible.

[0153] 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%.

[0154] 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%.

[0155] In a preferred embodiment, compromised welfare is determined as cattle being exposed to heat stress. 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 processed milk product produced from milk obtained from a herd of cattle, as indicators of whether a welfare parameter has been compromised, such as a herd of cattle having been exposed to heat stress.

[0157] Thus, an aspect of the invention relates to a method for evaluating at least one welfare parameter in at least one processed milk product, wherein said processed milk product has been subjected to at least heat treatment at around 65°C or higher, the method comprising:

[0158] - determining in the processed milk product produced from the milk obtained from the at least one herd of cows, the presence or level of one or more miRNAs selected from the group consisting of SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 or a functional variant, wherein the functional variant has at least 60% overall sequence identity to SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24; and

[0159] - wherein if said one or more miRNA levels is detected, it is indicative of that at least one welfare parameter of the cattle is compromised; and

[0160] - wherein if said one or more miRNA levels is not detected, it is indicative of that at least one welfare parameter of the cattle is not compromised.

[0161] In another aspect the invention relates to a method for evaluating at least one welfare parameter in at least one processed milk product, the method comprising:

[0162] - determining in the processed milk product produced from the milk obtained from the at least one herd of cows, the presence or level of one or more miRNAs selected from the group consisting of SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18, preferably SEQ ID NO: 16 or 17 or a functional variant, wherein the functional variant has at least 60% overall sequence identity to SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18; and

[0163] - wherein if said one or more miRNA levels is detected, it is indicative of that at least one welfare parameter of the cattle is compromised; and

[0164] - wherein if said one or more miRNA levels is not detected, it is indicative of that at least one welfare parameter of the cattle is not compromised.

[0165] In one embodiment of the method, the method comprises: - determining in a processed milk product produced from the milk obtained from the at least one herd of cows, the presence or level of one or more miRNAs selected from the group consisting of SEQ ID Nos: 16 or 17 or a functional variant, wherein the functional variant has at least 60% overall sequence identity to SEQ ID Nos: 16 or 17;

[0166] - comparing said one or more miRNA levels to one or more corresponding reference levels;

[0167] • wherein if said one or more miRNA levels is altered compared to the reference levels, it is indicative of that at least one welfare parameter of the cattle is compromised; and

[0168] • wherein if said one or more miRNA levels is not altered compared to the reference levels, it is indicative of that at least one welfare parameter of the cattle is not compromised.

[0169] Welfare

[0170] 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 heat stress, social stress, metabolic stress, disease stress, and combinations thereof, preferably the parameter is heat stress. In e.g. example 1 , miRNA biomarkers in relation to heat stress have been identified.

[0171] Thus, in a preferred embodiment, the compromised welfare parameter is caused by heat stress.

[0172] Cattle

[0173] 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.

[0174] The method of the invention may be used on a single group or herd of cattle, or a plurality of groups or herds of cattle. Individual cattle are referred to as a “head of cattle”.

[0175] In a related embodiment, said processed milk product is produced from bulk milk obtained from a single herd of cattle or a plurality of herds of cattle. 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.

[0176] Milk sample

[0177] Different types of milk samples can be used to produce the processed milk product of the present 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, dairycontaining products, such as an infant formula, cheese or yoghurt or being derived from such food product or food ingredient.

[0178] In a preferred embodiment the product is a mixed sample from several individual milk samples, this can be considered “bulk milk”.

[0179] In an embodiment, the level of miRNA is the level of miRNA in milk-derived exosomes. In example 1 , the level of miRNA has been determined from milk-derived exosomes.

[0180] In a preferred embodiment the miRNA is derived from a milk-derived exosome that expresses CD9 and / or TSG101.

[0181] Biomarkers

[0182] As shown in example 1 and 2, 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 one of the miRNAs, preferably two miRNA biomarkers.

[0183] 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.

[0184] In an embodiment, the one or more miRNAs are selected from the group listed in Table 2. The regulation is as indicated in Figures 1-4 and the corresponding text.

[0185] In another embodiment, the one or more miRNAs are selected from the group listed in Table 2, with the proviso that the cattle is Brown Swiss. The regulation is as indicated in Figures 1-4 and the corresponding text. In yet another embodiment, the one or more miRNAs are selected from the group listed in Table 2, with the proviso that the cattle is Holstein. The regulation is as indicated in Figures 1-4 and the corresponding text.

[0186] 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.

[0187] According to any of the aspects and embodiments of the invention, it may be advantageous to include miRNAs, which in combination are involved in two or more, such as three or more, preferably such as four or more or more preferably all of the pathways selected from the group consisting of cell cycle / cell cycle arrest, fatty acid synthesis, pain and stress, insulin signalling and reproduction. In a preferred embodiment, the miRNAs are involved in at least regulating the cell cycle - or regulate the cell cycle, more preferably cell cycle arrest. 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.

[0188] Determining improvement

[0189] By comparing different samples of processed milk products obtained at different time points (minutes or hours), it may be possible to evaluate if any improvement in animal welfare has taken place.

[0190] Thus, an aspect of the invention relates to a method for evaluating from a processed milk sample whether at least one welfare parameter of at least one herd of cattle has improved, the method comprising: i. determining in a processed milk product produced from milk obtained from at least one herd of cattle, the level of one or more miRNAs selected from the group consisting of SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24, or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24; ii. determining in a second processed milk product produced from milk obtained from the same at least one herd of cattle, the level of one or more miRNAs selected from the group consisting of SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24, or a functional variant thereof wherein the functional variant has at least 60% overall sequence identity to of SEQ ID Nos: 1 ,

[0191] 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24; wherein said second processed milk product is produced later in time than the first processed milk product; iii. comparing the level of the one or more miRNAs in the second processed milk product to the level in the first processed milk product; wherein if the level of the miRNA in the second processed milk product is reduced compared to the level of the miRNA measured in the first processed milk product (step i) the welfare of the herd of cattle has improved; wherein said first and second processed milk products have both been subjected to at least heat treatment at around 65°C or higher.

[0192] Another aspect of the invention relates to a method for evaluating from a processed milk sample whether at least one welfare parameter of at least one herd of cattle has improved, the method comprising: iv. determining in a processed milk product produced from milk obtained from at least one herd of cattle, the level of one or more miRNAs selected from the group consisting of SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18, , preferably SEQ ID NO: 16 or 17 or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID Nos: 1 , 2,

[0193] 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18; v. determining in a second processed milk product produced from milk obtained from the same at least one herd of cattle, the level of one or more miRNAs selected from the group consisting of SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18, , preferably SEQ ID NO: 16 or 17 or a functional variant thereof wherein the functional variant has at least 60% overall sequence identity to of SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18; wherein said second processed milk product is produced later in time than the first processed milk product; vi. comparing the level of the one or more miRNAs in the second processed milk product to the level in the first processed milk product; wherein if the level of the miRNA in the second processed milk product is reduced compared to the level of the miRNA measured in the first processed milk product (step i) the welfare of the herd of cattle has improved.

[0194] In one embodiment of the method, the method comprises:

[0195] - determining in a processed milk product produced from milk obtained from at least one herd of cattle, the level of one or more miRNAs selected from the group consisting of SEQ ID NO: 16 or 17 or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID Nos: 16 or 17;

[0196] - comparing the level of the one or more miRNAs in the processed milk product to a reference level;

[0197] - determining in a second processed milk product produced from milk obtained from the same at least one herd of cattle, the level of one or more miRNAs selected from the group consisting of SEQ ID NO: 16 or 17 or a functional variant thereof wherein the functional variant has at least 60% overall sequence identity to of SEQ ID NO: 16 or 17; wherein said second processed milk product is produced later in time than the first processed milk product;

[0198] - comparing the level of the one or more miRNAs in the second processed milk product to the same corresponding reference level;

[0199] - comparing said one or more levels in the first processed milk product to the one or more corresponding levels in the second processed milk product; wherein if the relative change of the miRNA to the reference level in the second processed milk product is reduced compared to the relative change measured in the first processed milk product (step ii) the welfare of the herd of cattle has improved.

[0200] In some embodiments, rather than comparing miRNA levels in each product to the reference level, the miRNA levels in the first and second processed milk products may be compared directly.

[0201] In an embodiment, initiatives have taken place between the sampling of the first product and the sampling of the second product, to improve the one or more welfare parameters of the cattle.

[0202] In yet another embodiment, the welfare parameter is heat stress. Heat stress occurs when body temperature rises above normal due to an inability to dissipate heat effectively. This can happen when the environmental temperature is high, air movement is low, or humidity is high. Cattle rely on various mechanisms to regulate their body temperature, including panting, sweating, and behavioural changes. Signs and symptoms of heat stress in cattle include but are not limited to, an increased respiratory rate, increases salivation, loss of appetite, reduced biological production and increased susceptibility to disease.

[0203] Table 1 : Common parameters to determine heat stress:

[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, 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 herd of cattle, the method comprising i. determining in a first processed milk product produced from milk obtained from at least one herd of cattle, the level of one or more miRNAs selected from the group consisting of SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24; ii. improving the welfare of the at least one herd of cattle if at least one welfare parameter of the at least one herd of cattle is or has been compromised; iii. determining in a second processed milk product produced from milk obtained from the same at least one herd of cattle, the level of one or more miRNAs selected from the group consisting of SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 or a functional variant thereof wherein the functional variant has at least 60% overall sequence identity to of SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,

[0206] 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24; iv. comparing the level of the one or more miRNAs in the second processed milk product to the level in the first processed milk product; wherein if the level of the miRNA in the second processed milk product is reduced compared to the level of the miRNA measured in the first processed milk product (step i) at least one of milk yield, milk quality, meat yield and meat quality from at least one herd of cattle the welfare of the herd of cattle has improved; wherein said first and second processed milk products have both been subjected to at least heat treatment at around 65°C or higher.

[0207] 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 herd of cattle, the method comprising i. determining in a first processed milk product produced from milk obtained from at least one herd of cattle, the level of one or more miRNAs selected from the group consisting of SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12,

[0208] 13, 14, 15, 16, 17 and 18, preferably SEQ ID NO: 16 or 17 or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18, preferably SEQ ID NO: 16 and / or 17; ii. improving the welfare of the at least one herd of cattle if at least one welfare parameter of the at least one herd of cattle is or has been compromised; iii. determining in a second processed milk product produced from milk obtained from the same at least one herd of cattle, the level of one or more miRNAs selected from the group consisting of SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18, preferably SEQ ID NO: 16 or 17 or a functional variant thereof wherein the functional variant has at least 60% overall sequence identity to of SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18, preferably SEQ ID NO: 16 or 17; iv. comparing the level of the one or more miRNAs in the second processed milk product to the level in the first processed milk product; wherein if the level of the miRNA in the second processed milk product is reduced compared to the level of the miRNA measured in the first processed milk product (step i) at least one of milk yield, milk quality, meat yield and meat quality from at least one herd of cattle the welfare of the herd of cattle has improved.

[0209] In one embodiment of the method, the method comprises i. determining in a processed milk product produced from milk obtained from at least one herd of cattle, the level of one or more miRNAs selected from the group consisting of SEQ ID NO: 16 or 17 or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID Nos: 16 and / or 17; ii. comparing the level of the one or more miRNAs in the processed milk product to a reference level; iii. improving the welfare of the at least one herd of cattle if at least one welfare parameter of the at least one herd of cattle is or has been compromised; iv. determining in a second processed milk product produced from milk obtained from the same at least one herd of cattle, the level of one or more miRNAs selected from the group consisting of SEQ ID NO: 16 and / or 17 or a functional variant thereof wherein the functional variant has at least 60% overall sequence identity to of SEQ ID NO: 16 and / or 17; wherein said second processed milk product is produced later in time than the first processed milk product v. comparing the level of the one or more miRNAs in the second processed milk product to the corresponding reference level; vi. comparing said one or more levels in the first processed milk product to the one or more corresponding levels in the second processed milk product; wherein if the relative change of the miRNA to the reference level in the second processed milk product is reduced compared to the relative change measured in the first processed milk product (step ii) at least one of milk yield, milk quality, meat yield and meat quality from at least one herd of cattle the welfare of the herd of cattle has improved.

[0210] 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.

[0211] 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,

[0212] 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.

[0213] Meat quality is a complex concept that encompasses a variety of factors including:

[0214] - Appearance : high quality meat has a bright appealing colour, uniform texture and no signs of discoloration.

[0215] - Texture : high quality meat is tender and easy to chew, whereas low quality meat is chewy and / or tough.

[0216] - Flavour : high quality meat has a richer and more complex flavour than low quality meat.

[0217] Uses

[0218] 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, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24, preferably SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18, more preferably SEQ ID Nos: 16 and / or 17 or a functional variant thereof to determine from a processed milk product produced from milk obtained from at least one herd of cattle whether at least one welfare parameter of the cattle has been or is compromised.

[0219] In an embodiment, the welfare parameter is heat stress. Device

[0220] The method of the invention may also be implemented in a device or system.

[0221] Thus, yet another aspect of the invention relates to a device or system adapted to determine from a processed milk product that has been subjected to at least heat treatment at around 65°C or higher whether at least one welfare parameter of at least one herd of cattle, preferably bovine, from which the milk was obtained, has been or is compromised, the device comprising: a unit able to determine the level of one or more miRNAs in a processed milk product, wherein the processed milk product is produced from milk obtained from at least one herd of cattle; a processor configured with a reference table, said reference table corresponding to reference levels of one or more of the miRNA according to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 or a functional variant thereof ; wherein the device or system is adapted to

[0222] - receive a processed milk product;

[0223] - determine the levels of the one or more miRNAs in the processed milk product;

[0224] - compare the determined levels of the one or more miRNAs to the reference table; and

[0225] - determine if the welfare of the at least one herd of cattle has been or is compromised.

[0226] In another aspect of the invention relates to a device or system adapted to determine from a processed milk product whether at least one welfare parameter of at least one herd of cattle, preferably bovine, from which the milk was obtained, has been or is compromised, the device comprising: a unit able to determine the level of one or more miRNAs in a processed milk product, wherein the processed milk product is produced from milk obtained from at least one herd of cattle; a processor configured with a reference table, said reference table corresponding to reference levels of one or more of the miRNA according to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18, or a functional variant thereof ; wherein the device or system is adapted to - receive a processed milk product;

[0227] - determine the levels of the one or more miRNAs in the processed milk product;

[0228] - compare the determined levels of the one or more miRNAs to the reference table; and

[0229] - determine if the welfare of the at least one herd of cattle has been or is compromised.

[0230] In an embodiment, the reference table comprises corresponding miRNAs values from at least one herd of cattle where the corresponding welfare parameter has not been compromised.

[0231] 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, and the breed from which the processed milk product is derived.

[0232] Computer implemented method

[0233] 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 from a processed milk product that has been subjected to at least heat treatment at around 65°C or higher whether at least one welfare parameter of at least one herd of cattle, preferably bovine, from which the milk was obtained has been compromised or not, the method comprising: providing levels of one or more miRNAs from a processed milk product produced from milk obtained from at least one herd of cattle selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, and 24 or a functional variant thereof; providing a mathematical model comprising reference levels of one or more corresponding miRNAs selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, and 24 prefeor a functional variant thereof; 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 herd of cattle has been or is compromised. In another aspect the invention relates to a computer implemented method of determining from a processed milk product whether at least one welfare parameter of at least one herd of cattle, preferably bovine, from which the milk was obtained has been compromised or not, the method comprising: providing levels of one or more miRNAs from a processed milk product produced from milk obtained from at least one herd of cattle selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18, preferably SEQ ID NOs: 16 or 17 or a functional variant thereof; providing a mathematical model comprising reference levels of one or more corresponding miRNAs selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18, preferably SEQ ID NOs: 16 or 17 or a functional variant thereof; 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 herd of cattle has been or is compromised.

[0234] In an embodiment, the reference table comprises corresponding miRNA values from a herd of cattle where the corresponding welfare parameter has not been compromised.

[0235] 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.

[0236] 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.

[0237] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0238] The invention will now be described in further detail in the following non-limiting examples.

[0239] Examples

[0240] Example 1- miRNAs as indicators of cattle welfare in bulk milk Aim of study:

[0241] 1 . To investigate which miRNA biomarkers of heat stress can be detected in bulk milk derived from herds that have experienced acute heat stress.

[0242] 2. To evaluate the period during which deregulation of the identified miRNAs can be detected in bulk milk after cattle have been returned to normal conditions.

[0243] Materials and Methods:

[0244] In mid- June, farms were provided with dataloggers to detect and store data every 30 minutes on temperature and relative humidity. Dataloggers were placed in a cubicle in the middle of the resting area of the barn, at the height of the animal’s herd. The weather forecast of each zone where a farm is located was monitored using the detailed reports of the National Airforce weather station of Gioia del Colle.

[0245] During the warmest weeks (heat waves), when Temperature Humidity Index (THI) exceeded 69, the cooling systems were turned off for four consecutive days to induce acute heat stress, and then turned on during the morning of the fifth day.

[0246] During this 4 day period, physiological patterns (respiratory rate measured visually, rectal temperature measured by digital rectal thermometer, eye, muzzle, skin and vaginal temperature images were collected by thermography camera) were recorded and bulk milk samples were collected (there was no measure on individual milk samples). As usual in these commercial farms, milk was collected in the morning and in the afternoon. We were interested in the afternoon sample (when miRNA dysregulation was expected to be largest), as well as in the sample that pools morning and afternoon milk (representative of real-life conditions).

[0247] Milk was collected in normothermia before switching off the cooling system (day 1) and on days 2, 3, and 4 during the acute heat stress period.

[0248] Cooling systems were then turned on during the morning of the fifth day and were collected on day 7, 14, and 21 of the restoring phase.

[0249] Fourteen samples of the bulk milk were collected from each farm. Milk was immediately skimmed, stored at +4°C, and frozen within 2 hours. An aliquot of milk was used to test proteins, fat, dry matter, lactose, caseins, SCC, DSCC, fatty acids categories, pH, clotting aptitude, and MUN. Part of the milk was sent to UNIMI for extra vesiculas isolation and miRNAs quantification.

[0250] This longitudinal study required only one arm to evaluate the changes induced by the acute heat-stress phase and the restoring phase. Since bulk milk is the unit of interest, the sample size is defined at the level of commercial farms, not of individual animals. Six commercial farms were sufficient to confirm at farm level the results previously observed at individual cow level.

[0251] The previous study showed significant changes induced by acute heat stress on the average of 37 cows (19 Brown Swiss and 18 Holstein). Under the assumption that the bulk milk of a farm is representative of the average of the individual milk samples, the observed effect on the average of individuals should translate on the bulk milk. Consequently, farms with ~60 animals (>37) should be sufficient to observe the effect in the bulk milk of each farm. Repeating the experiment in six farms is a minimum to generalize the results. Six commercial farms raising an average of around 60 lactating cows of Holstein, Brown Swiss, or mixed Holstein / Brown Swiss animals were included in the study. The lactating cows' barns had cooling systems installed (ventilation) which were switched off for the 4 day heat stress period.

[0252] Animals were observed during the experiment. If, during the experiment, the investigators or the farm veterinarian observed clinical signs of pathological heat stress, the animals with symptoms were promptly excluded from the experiment and placed in conditions to allow the recovery to the normal physiological state.

[0253] Biological sampling was carried out through:

[0254] 1 . Normal milking, without any changes to this procedure.

[0255] 2. Routine veterinary control included blood sampling in a limited number of animals.

[0256] Social interactions and conditions at the farms participating in the experiment were not changed for the animals. The animals were kept in a free stall housing system. The removal of manure was carried out by an automated scraper, at least three times a day. All cows were fed in the same way with same feed, formulated according to specific nutritional needs. The ration was distributed twice a day, i.e. just after the morning and after the evening milking. The space in the free stall made it possible to maintain an adequate level of well-being, animals had free access to water and resting space.

[0257] The 4 day heat stress condition in this study was kept at conditions which are normal for farms that are not equipped with ventilators.

[0258] Results:

[0259] Eighteen miRNAs were found to be deregulated in bulk milk as a result of heat stress in herds of cattle. For each of these miRNA’s, a relative change was observed over the period when heat stress was induced. Therefore, it is possible to measure for a relative change in the concentration of any of these eighteen miRNA’s in a bulk milk sample collected from a herd of cattle to obtain an indicator of the welfare of the herd, in particular as an indicator of heat stress. As we described herein, this relative change is measured relative to a reference level, which may be the level in the same herd prior to heat stress or alternatively a standard for the given breed.

[0260] The Applicants have additionally found that some miRNAs show a significant change in concentration upon induction or relief to / from heat stress.

[0261] For example, miR-2285n-1-5p, miR-30f-5p, miR-1777a-3p, miR-502b-5p, miR-500-5p, miR-339a-5p, miR-339b-5p, miR-532-3p, miR-6525-5p and miR-664b-3p (encoded by SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 11 , 12, 13, 14 and 15, respectively) all showed a significant alteration in concentration during the course of the experiment. These miRNAs are therefore strong indicators of cattle welfare, in particular whether a herd is undergoing or been subject to heat stress.

[0262] In particular, miR-2285n-1-5p, miR-30f-5p, miR-1777a-3p, miR-502b-5p and miR-500-5p (encoded by SEQ ID NO: 1 , 2, 3, 4, 5, and 6) all showed significant alterations in their levels between either T-1 or T1 (the period before heat stress is applied or the day when heat stress is applied) and T4 and T5 (the final day of heat stress and the day following removal of heat stress conditions). These miRNAs are therefore strong indicators for cattle welfare, and are especially useful for methods in measuring herd welfare to potentially detrimental exposures e.g. humidity. Interestingly, miR2288-3p was significantly increased after cattle had been subjected to heat stress for 4 days (T4) as shown in figure 2. Interestingly, this effect on the expression of miR2288-3p was sustained at day 5 (T5), when cooling fans had been switched back on; and day 12 (T12), 6 days after cooling fans had been switched back on. This suggests that exposure to heat stress can have prolonged effects in cattle.

[0263] In contrast to miR2288-3p, the mRNA’s miR2285n-1-5p, miR30f-5p and miR-1777a-3p were both significantly decreased in bulk milk samples after cattle were subjected to heat stress, as shown in figures 3, 4 and 3 respectively. In the case of miR2285n-1-5p this effect was sustained at day 5 (T5), when cooling fans had been switched back on; and day 12 (T12), 6 days after cooling fans had been switched back on. However, for miR30f-5p the effect was sustained at day 5 (T5), when cooling fans had been switched back on but had returned to levels resembling those seen before heat stress was applied by day 12 (T12), 6 days after cooling fans had been switched back on.

[0264] Table 2: miRNAs in bulk milk identified to be deregulated in herds of cattle under heat stress.

[0265] Example 2: miRNAs in products processed from bulk milk as indicators of cattle herd welfare.

[0266] In addition to analysing bulk milk obtained from a herd of cattle, the bulk milk sample was subsequently subjected to processing and analysed further. This experiment was intended to determine if any of the changes in the above eighteen miRNAs is still detectable following processing into products. In this way, it is possible to obtain an indication of herd welfare from processed products derived from the bulk milk collected from at least one herd. Specifically, the bulk milk was split into two fractions, extracellular vesicles and whole milk; and then subjected to the heat treatment parameters and spray drying process shown in Table 3 to obtain a spray dried milk sample.

[0267] Table 3: Conditions used to process bulk milk into spray dried milk.

[0268] Interestingly, miR-30a-5p and miR-30b-5p - encoded by SEQ ID NO: 16 and 17, respectively - were detected in the resulting spray-dried milk. Therefore, miR-30a-5p and miR-30b-5p, can be detected in products processed from the bulk milk to obtain an indication of herd welfare.

[0269] Figure 5 shows that miR-30a-5p and miR-30b-5p were detected in extracellular vesicles derived from raw milk, pasteurised milk and spray dried milk at TO (shortly after heat stress conditions began), T5 (when cooling fans had been switched back on) and T12 (6 days after cooling fans had been switched back on. Similarly, miR-30a-5p and miR30b-5p were also detected at TO, T5 and T12 in whole milk samples derived from raw milk, pasteurised milk and spray dried milk (figure 6). This led to the question of whether the processing method affects whether these miRNAs are detected. It was shown that both miR-30a-5p and miR30b-5p were clearly detected in extracellular vesicles (figure 7) and whole milk (figure 8) that was not treated (NT), after pasteurisation (AP) and after spray drying (AS) at all three time points, TO, T5 and T12. This data indicates that miR-30a-5p and miR30b-5p can reliably be used as indicators of cattle welfare in processed milk products. Interestingly, the fact the detection of miR-30a-5p and miR30b-5p was sustained in both extracellular vesicles and whole milk at day 5 (T5), when cooling fans had been switched back on; and day 12 (T12), 6 days after cooling fans had been switched back on suggests that exposure to heat stress can have prolonged effects in cattle that can be detected in processed milk products.

Claims

Claims1. A method of determining from a processed milk product, wherein said processed milk product has been subjected to at least heat treatment at around 65°C or higher, whether at least one welfare parameter of at least one herd of cattle from which the milk was obtained has been or is compromised, the method comprising determining in the processed milk product the level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, and 24 or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 respectively; wherein if said one or more miRNA is detected at least one welfare parameter of the at least one herd of cattle has been or is compromised.

2. A method of determining from a processed milk product whether at least one welfare parameter of at least one herd of cattle from which the milk was obtained has been or is compromised, the method comprising determining in the processed milk product the level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18 or a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18 respectively; wherein if said one or more miRNA is detected at least one welfare parameter of the at least one herd of cattle has been or is compromised.

3. A method of improving the welfare of at least one herd 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 herd of cattle is or has been compromised; andiii. improving the welfare of the at least one herd of cattle.

4. A method of determining whether at least one welfare parameter of at least one herd of cattle is improved, the method comprising i. determining in a first processed milk product obtained from the at least one herd of cattle the level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 or a functional variant thereof, and wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, and 24 respectively; wherein if said at least one miRNA is detected at least one welfare parameter of the at least one herd of cattle is or has been compromised; ii. improving the welfare of the at least one herd of cattle if at least one welfare parameter of the at least one herd of cattle is or has been compromised; iii. determining in a second processed milk product obtained from the at least one herd of cattle the level of at least one miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 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, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, and 24 respectively; and iv. comparing the level of the at least one miRNA in the second processed milk product to the first processed milk product, wherein if the level of the miRNA in the second processed milk product has decreased compared to the first processed milk product, the welfare of the herd of cattle has improved wherein said first and second processed milk products have both been subjected to at least heat treatment at around 65°C or higher.

5. A method of determining whether at least one welfare parameter of at least one herd of cattle is improved, the method comprisingi. determining in a first processed milk product obtained from the at least one herd of cattle the level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18 or a functional variant thereof, and wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18 respectively; wherein if said at least one miRNA is detected at least one welfare parameter of the at least one herd of cattle is or has been compromised; ii. improving the welfare of the at least one herd of cattle if at least one welfare parameter of the at least one herd of cattle is or has been compromised; iii. determining in a second processed milk product obtained from the at least one herd of cattle the level of at least one miRNA selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18 a functional variant thereof, wherein the functional variant has at least 60% overall sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18 respectively; and iv. comparing the level of the at least one miRNA in the second processed milk product to the first processed milk product, wherein if the level of the miRNA in the second processed milk product has decreased compared to the first processed milk product, the welfare of the herd of cattle has improved.

6. A method of improving at least one of milk yield, milk quality, meat yield or meat quality of at least one herd of cattle, the method comprising i. determining in a processed milk product that has been subjected to at least heat treatment at around 65°C or higher obtained from the at least one herd of cattle the level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 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, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24; wherein detection of the at least one miRNA is indicative that at least one welfare parameter of the at least one herd of cattle has been or is compromised; andii. if at least one welfare parameter of the at least one herd of cattle has been or is compromised, then improving the welfare of the at least one herd of cattle; wherein improving the welfare of the at least one herd of cattle improves at least one of milk yield, milk quality, meat yield and meat quality.

7. A method of improving at least one of milk yield, milk quality, meat yield or meat quality of at least one herd of cattle, the method comprising i. determining in a processed milk product obtained from the at least one herd of cattle the level of at least one miRNA, wherein the miRNA is selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18 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, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 and 18; wherein detection of the at least one miRNA is indicative that at least one welfare parameter of the at least one herd of cattle has been or is compromised; and ii. if at least one welfare parameter of the at least one herd of cattle has been or is compromised, then improving the welfare of the at least one herd of cattle; wherein improving the welfare of the at least one herd of cattle improves at least one of milk yield, milk quality, meat yield and meat quality.

8. The method of any preceding claim, wherein the miRNA is SEQ ID NO: 16 and / or SEQ ID NO: 17.

9. The method according to claim 3 or 4, wherein improving or altering the welfare of the at least one herd of cattle comprises one or more of changing heat exposure, improving access to additional water, changing the feed, reducing animal density and the provision of cooling systems.

10. The method according to any preceding claim, wherein the processed milk sample is from a single or plurality of breeds of cattle, wherein preferably the breed of cattle is selected from Holstein and / or Brown Swiss.

11. The method according to any preceding claim wherein the processed milk sample has been processed by at least one of pasteurisation, spray drying, homogenisation, filtration, or centrifugation.

12. The method according to any preceding claim wherein the processed milk sample is in a dairy product such as skimmed milk, yoghurt, buttermilk, cream, cheese, butter, or milk powder.

13. The method according to any preceding claim, wherein the method comprises determining the level of both of SEQ ID NO: 16 or 17 or a functional variant thereof wherein the functional variant has at least 60% overall sequence identity to SEQ ID Nos: 16 or 17 respectively.

14. A device or system adapted to determine if at least one welfare parameter of at least one herd of cattle has been or is compromised, the device comprising: i. a unit able to determine the level of one or more miRNAs selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 in a processed milk product produced from milk obtained from at least one herd of cattle; and ii. a processor configured with a reference table, said reference table corresponding to reference levels of one or more miRNA; wherein the device or system is adapted to; receive a processed milk product produced from milk obtained from at least one herd of cattle; determine the levels of the one or more miRNAs in the processed milk product; compare the determined levels of the one or more miRNAs to the reference value; and determine if the welfare of the at least one herd of cattle has been or is compromised.

15. A computer implemented method of determining if the welfare of at least one herd of cattle has been compromised, the method comprising: i. providing levels of one or more miRNAs from a processed milk product produced from milk obtained from at least one herd of cattle selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19,20, 21 , 22, 23 and 24 , preferably SEQ ID NO: 16 and / or 17 or a functional variant thereof; ii. providing a mathematical model comprising reference levels of one or more corresponding miRNAs selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24, preferably SEQ ID NO: 16 and / or 17 or a functional variant thereof; 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 herd of cattle has been or is compromised.

16. A kit for evaluating at least one welfare parameter of at least one herd of cattle in a processed milk product, the kit comprising the device of claim 13 and instructions for use.

17. A kit for determining from a processed milk product whether at least welfare parameter of at least one herd of cattle from which from which the milk was obtained has been or is compromised, wherein said kit comprises: i. a means for collecting a sample of the processed milk product obtained from at least one 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, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 in the processed milk product sample; and iii. instructions for use.

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