ANALYSIS OF IGF-BINDING PROTEIN TO DETERMINE ANIMAL WELFARE

Description

[0001] The invention relates to methods and kits for determining animal welfare, i.e., animal-friendly treatment or "animal welfare". In particular, the invention provides a method for analyzing the expression of IGF (Insulin-Like Growth Factor) binding proteins, especially IGFBP-2 and IGFBP-3, as markers for animal welfare. By examining these markers, it is possible to differentiate between short-term and long-term impairments to animal welfare, i.e., between impairments that arise in the short term during the transport of animals and impairments that arise, for example, from disease and / or inadequate husbandry conditions.

[0002] The majority of Germans want the assurance that the meat on their plate comes from an animal that was well cared for during its life. This broad consensus is reflected in the current "Animal Welfare" initiative. Participants in this initiative commit to adhering to certain standards, such as space requirements, natural light, and monitoring of barn climate and watering systems. The complex concept of animal welfare (also known as well-being or animal-friendly husbandry) describes the state of an individual's physical and mental health and its ability to respond to and cope with environmental stimuli (Broom, 1986). Indicators of poor well-being can include a shortened lifespan, impaired growth, reduced reproductive performance, injuries, illnesses, or behavioral abnormalities. Some of these characteristics may already be observable on-site.However, animal welfare can also be reduced without any externally recognizable impairment of the animal (Broom, 1991).

[0003] The British Farm Animal Welfare Council (FAWC) developed the concept of the "Five Freedoms" in the 1980s. It forms the basis for various measurement and assessment systems for animal welfare. The Five Freedoms concept incorporates the aforementioned value systems and offers an approach to operationalization, i.e., the practical measurement of animal welfare in livestock farming. The Five Freedoms are: Freedom from hunger and thirst: Animals have access to fresh water and healthy, nutritious food. Freedom from husbandry-related ailments: Animals have suitable housing (e.g., a shelter in the pasture), adequate resting areas, etc. Freedom from pain, injuries, and disease: The animals are cared for through preventative measures, rapid diagnosis and treatment, and the avoidance of amputations (or anesthesia). Freedom from fear and stress: Fear and stress are avoided through procedures and management, e.g., by avoiding the use of herding aids. Freedom to express normal behavior patterns: The animals have the opportunity to exhibit normal behavior, e.g., through sufficient space, group housing, etc.

[0004] The measurement of various aspects of animal welfare is usually based on indicators. A distinction is made between: Resource-related indicators, which provide information on husbandry practices and space requirements; management-related indicators, which record practices such as dehorning cattle or castration of male piglets, but also feeding and animal handling; and animal-based indicators, which are measured directly on the animal. Examples of animal-based indicators include footpad dermatitis in broiler chickens, lameness in dairy cows, and lung findings on carcasses of fattening pigs (Wikipedia).

[0005] Animal welfare is, above all, a socially constructed concept. This becomes clear in the example of a free-range pig with sunburn. Free-range farming stems from a human ideal, but sunburn, as an animal-related parameter, is the animal's decisive reaction or response to assess or adjust this ideal. Therefore, the target values ​​of animal-related parameters must be determined, and their deviations in response to disturbances must be examined.

[0006] The concentration of IGF-1 in milk (Ruffer, 2003) has already been discussed in the literature as a marker for udder health. The suitability of the IGF-I system for predicting health and fertility in multiparous dairy cows was investigated by Mysegades (2014). Walker et al. (2008) discuss an assessment of estrus intensity in dairy cows as an indicator of chronic stress.

[0007] Biomarkers, as animal-related indicators, could contribute to a better assessment of animal welfare in the future. This often raises the question of which factors lead to a decline in animal welfare. With this in mind, the inventors set themselves the task of developing a biomarker for animal welfare that allows for a distinction between short-term and long-term impairments.

[0008] This problem is solved by the present invention, which is described below and in the claims.

[0009] The invention provides a method for determining animal welfare, in which a distinction between short-term and long-term impairment of animal welfare is possible, in which the expression of IGFBP-2 and IGFBP-3 in a sample of an animal is analyzed, wherein the method is used to determine the transport time and / or the transport conditions of an animal, wherein a low expression of IGFBP-2 and IGFBP-3 is associated with a short-term impairment of animal welfare due to the transport of the animal, and wherein the duration of the stress intensifies the extent of the change in IGFBP concentrations, wherein the animal is a pig.

[0010] In humans, it is already relatively well established that disturbances in growth hormone (GH)-induced growth can be detected by fluctuations in the concentrations of insulin-like growth factor I (IGF-I) and IGF-binding proteins (IGFBP)-2 and -3. Such disturbances have been described at the cellular level in humans in connection with chronic illness or acute stress (Messotten & van den Berghe, 2008). GH exerts its anabolic effects directly via the GH receptor and indirectly by stimulating the expression of IGF-I and IGFBP-3. Thus, higher concentrations of IGF-I and IGFBP-3 indicate "normal" GH-induced growth.

[0011] Human patients who are acutely ill have significantly lower IGFBP-3 concentrations than healthy subjects ( Fig. 1A ), whereas the IGFBP-2 concentrations of the affected individuals are significantly higher than those of the control group ( Fig. 1BThis results in a significantly lower IGFBP-3 / IGFBP-2 ratio in the affected group ( Fig. 1C The ratio of IGFBP-3 to IGFBP-2 is therefore a sensitive parameter in humans for the early detection of disorders of the somatotropic axis and associated diseases (Messotten and van den Berghe, 2008).

[0012] However, as Bielohuby et al. (2014) show, there are significant differences in the regulation of the somatotrophic system, e.g. the regulation of IGF-1, IGFBP-2 and IGFBP-3, between humans and different animal species, so that a direct transfer of the results is not possible.

[0013] The inventors were able to show that, even in pigs, a reduced ratio of the expression (or concentration) of IGFBP-3 to the expression of IGFBP-2 is associated with an impairment of animal welfare.

[0014] This can also be expressed in absolute values ​​for IGFBP-3 and IGFBP-2: low expression of IGFBP-3 and high expression of IGFBP-2 are associated with long-term impairment of animal welfare. However, this reduced ratio of IGFBP-3 to IGFBP-2 only becomes apparent in cases of long-term impairment of animal welfare. Such a determination of long-term impairment of animal welfare is not covered by the present claims. The following description therefore serves only as an illustration.

[0015] High expression of IGFBP-3 and low expression of IGFBP-2, on the other hand, are associated with animal welfare. A high ratio of IGFBP-3 to IGFBP-2 expression indicates long-term animal welfare. However, the determination of long-term animal welfare is not part of the claimed invention.

[0016] The ratio or level of expression is generally assessed in comparison to a control group or, preferably, to known reference values. These reference values ​​are to be determined by analyzing healthy control animals of the same species, preferably the same breed. Age, weight, and sex should also be comparable as far as possible (e.g., deviations of 30% or less, 20% or less, 10% or less for age and weight). For the control group, standardized husbandry conditions and other treatments (e.g., no transport, preferably avoiding any impairment of animal welfare as far as possible) can be used as a basis, which may, for example, comply with specific guidelines (e.g., guidelines of an organic label). To determine a reference value, the average value or a pooled sample of a group of animals (e.g., 5 or more, 10 or more, 20 or more, 50 or more) is examined.

[0017] A reduced ratio of IGFBP-3 to IGFBP-2 expression is assumed, in particular, if this ratio is less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the reference value. The changes should be statistically significant.

[0018] Long-term impairment can result, for example, from a prolonged illness such as lameness, mastitis, malnutrition, insufficient space in the enclosure, poor air quality, etc. Such impairments typically develop over one or more weeks, months, or years, for example, 7 or more days, 14 or more days, 30 or more days, 60 or more days, or 360 or more days.

[0019] The inventors surprisingly found that low expression of IGFBP-2 and IGFBP-3 is associated with a short-term impairment of animal welfare. Here too, expression is generally assessed in comparison to a control group or to known reference values.

[0020] According to the invention, the short-term impairment is caused by transport. Such impairments typically occur for one or more hours or up to a few days, for example, 1 hour to 3 days, 2 hours to 2 days, 3 hours to 1.5 days, 4 to 24 hours, 6 to 24 hours, 8 to 24 hours, 12 to 24 hours, 14 to 24 hours, 16 to 24 hours, 18 to 24 hours, or 20 to 24 hours.

[0021] The method according to the invention thus enables a differentiation between short-term and long-term impairment of animal welfare by determining the expression of IGFBP-2 and IGFBP-3.

[0022] The invention provides a method for determining short-term impairment of animal welfare due to animal transport, in which the expression of IGFBP-2 and IGFBP-3 in an animal sample is analyzed. Low expression of IGFBP-2 and IGFBP-3, preferably compared to a control group or reference value, is associated with short-term impairment of animal welfare due to animal transport.

[0023] A 1998 study by McCusker et al. demonstrated that stress leads to reduced expression of IGFBP-3 (McCusker et al., 1998, Controlling Insulin-Like Growth Factor Activity and the Modulation of Insulin-Like Growth Factor Binding Protein and Receptor Binding. Journal of Dairy Science 81(6), 1790-1800). McCusker et al., 1989, revealed that 24-hour fasting leads to a reduction in IGFBP-2 and 3 in the plasma of piglets (McCusker et al., 1989. The Insulin-Like Growth Factor-Binding Proteins of Porcine Serum: Endocrine and Nutritional Regulation. Endocrinology 125(1), 501-509). In line with this finding, Laeger et al., 2014 describe that a short-term fasting period also results in lower expression of IGFBP-2 and IGFBP-3 in the plasma of cows (Laeger et al., 2014, Effects of parturition and feed restriction on concentrations and distribution of insulin-like growth factor-binding proteins in plasma and cerebrospinal fluid of dairy cows. Journal of Dairy Science 97(5), 2876-2885). However, in summary, none of these documents suggests a link between low expression of IGFBP-2 and IGFBP-3 and a short-term impairment of animal welfare due to the transport of an animal.

[0024] In the context of the invention, the animal is a pig.

[0025] The sample can be selected from the group comprising blood, serum, plasma, seminal plasma, cerebrospinal fluid, follicular fluid, urine, sputum, tissue (e.g., meat, such as muscle tissue), drip fluid, or milk. While investigations in humans are generally carried out using blood samples, the inventors were surprisingly able to demonstrate that the method according to the invention also works using tissue samples (e.g., meat) or drip fluid (meat juice). This enables the examination of pigs, in particular, after slaughter without additional steps such as blood sampling, on the live animal. Preferably, blood or meat taken after or during slaughter is examined.

[0026] Particularly for investigating short-term impairment of animal welfare, the investigation according to the invention can be carried out immediately after the potentially stress-inducing event, i.e., immediately after transport (e.g., after arrival at the slaughterhouse). The duration of the stress can intensify the change in IGFBP concentrations (see Figs. 5 and 6 Analysis is also possible after an animal has been slaughtered and the meat (preferably refrigerated, e.g., at 4°C) or other samples have been stored. Sampling after slaughter is preferred to avoid further compromising animal welfare. IGFBPs can also be detected in meat and meat juice; measurement for animal welfare monitoring is possible at any time and can even be carried out at the checkout counter or in the refrigerated section.

[0027] With the method according to the invention, it is of course also possible to analyze further biomarkers in the sample. For example, the expression of IGF-I in the sample can also be analyzed. Higher IGF-I expression is associated with animal welfare. Here, too, the assessment is usually carried out using a suitable reference value.

[0028] Furthermore, the method according to the invention allows for the verification – at least on a random basis – of whether exogenous administration of anabolic hormones (e.g., GH or IGF-1) has taken place. Such administration of exogenous hormones could distort the results of the method according to the invention. However, the administration of GH / IGF-1 leads to far greater fluctuations in the somatotropic system than would be expected from husbandry-related effects. Therefore, undetected distortion is unlikely. The use of GH / IGF in animal husbandry can be ruled out by unannounced on-site inspections and / or blood tests. The IGFBP signature is also intended to be used as a means of evaluating specific husbandry conditions and thereby improving animal welfare. In the EU, the administration of exogenous anabolic hormones for performance enhancement in farm animals such as pigs and cattle is currently prohibited, but permitted in the USA, for example.

[0029] The expression of the biomarkers IGFBP-2 and IGFBP-3 according to the invention can be comparatively investigated, for example, using the method described by Hossenlopp et al. in 1987; however, this method does not provide quantitative data. ELISA or RIA are unreliable due to a lack of specificity for intact IGFBPs. Therefore, the expression of the biomarkers according to the invention is preferably analyzed at the protein level using a quantitative Western ligand blot. The inventors have shown that this method yields significantly more accurate results than ELISAs generally used in the prior art, since the antibodies used therein also bind to non-functional fragments of the binding proteins.

[0030] To analyze IGF-binding proteins, the proteins are separated using quantitative Western ligand blotting with SDS-PAGE and then transferred to a membrane, e.g., nitrocellulose or, preferably, PVDF. The membrane is blocked and then incubated with labeled IGF (e.g., biotin-labeled), particularly IGF-2. After several washing steps, incubation with a detection reagent follows; in the case of biotin, for example, a streptavidin conjugate. An avidin conjugate can also be used. A streptavidin-peroxidase conjugate is preferred. After washing again and adding a substrate, the IGF-binding proteins are detected and analyzed, e.g., by chemiluminescence, using a KODAK IMAGE station. Quantification is performed using calibration curves, e.g., with recombinant human IGFBPs or, optionally, recombinant IGFBPs of the species under investigation, e.g., pigs.The curve fitting of the individual standards is preferably carried out by a four-parameter nonlinear regression.

[0031] To eliminate the varying transfer efficiencies and the impact of sample preparation artifacts (washing, blocking, incubation), internal standardization is preferably performed, i.e., the sample is spiked with an internal standard. For this purpose, either an IGFBP with a molecular weight distinguishable from the IGFBPs under investigation (preferably 50–100 kDa) from the same or a different species can be used (these can be artificial IGFBPs that have been enlarged or reduced via a cloning vector, or heterologous IGFBPs from other species with distinguishable molecular weights, preferably IGFBP-2), or any biotinylated protein of any other class that does not interfere with detection and has a suitable molecular weight (non-IGFBP, preferably 50–100 kDa). Advantageously, the ability of the distinguishable IGFBP to bind to IGF, e.g.,IGF-2 biotin is bound, so that its detection is based on the same principles as the detection of IGFBP-2 and IGFBP-3. This results in a particularly high accuracy of the assay. The internal standard is then detected via the assay-specific detection system (e.g., streptavidin-conjugated peroxidase).

[0032] Alternatively, in the inventive method, the expression can be analyzed at the RNA level, whereby, for example, it can be purified from meat.

[0033] In one embodiment of the inventive method, the welfare of a population whose animals have been handled under identical or comparable conditions can be determined. Optionally, the welfare of another population whose animals have been handled under different, identical or comparable conditions can also be determined, and the welfare of the populations can be compared. Within the scope of the invention, "one" means "one or more," unless explicitly excluded. Thus, for example, more than two populations can be compared. This comparison is most meaningful when animals of the same breed and, as far as possible, similar age or sex (or as similar a distribution of these parameters as possible) are compared. "Handling" here refers to transport.

[0034] The invention also provides a method for optimizing the handling of an animal, i.e., the transport conditions of an animal, by employing the method according to the invention. Potential impairments to animal welfare can be identified, and attempts can be made to adjust the handling, i.e., the transport conditions, in such a way as to improve animal welfare.

[0035] The invention thus also provides a method for determining the transport time and / or the transport conditions of an animal, in which the method according to the invention is used.

[0036] In another aspect, the invention provides the use of a kit for determining animal welfare using the inventive method, which includes standards for quantifying IGFBP-3 and IGFBP-2, as well as a labeled IGF capable of binding to IGFBP-2 and IGFBP-3, e.g., IGF-2 (which offers increased sensitivity compared to IGF-1). Preferably, the kit includes IGF-2 biotin. It may also include means for performing a quantitative Western blot. Examples of means for performing a quantitative Western blot could be: a PVDF membrane, wash buffer, blocking buffer, substrate solution (or concentrates for buffer and solution). A conjugate for detecting the labeled IGF biotin, e.g., streptavidin peroxidase, may also be included in the kit. The kit may also include a protein suitable for internal standardization, e.g. an IGFBP with a molecular weight that is distinguishable from the IGFBP-2 and IGFBP-3 under investigation.This IGFBP may belong to a different species than the IGFBP-2 and IGFBP-3 used as standards. If the protein suitable for internal standardization is not an IGFBP, it may be biotin-labeled and preferably has a molecular weight of 50–100 kDa. Fig. 1 Concentrations of IGFBP-3 (A), IGFBP-2 (B) and their ratio (C) in healthy and diseased individuals. Modified from Messotten and van den Berghe, 2008. Fig. 2 Exemplary IGFBP profiles in biological fluids from pigs: (A) Serum, (B) Seminal plasma, (C) Cerebrospinal fluid, (D) Follicular fluid Fig. 3 : IGFBP-3 serum concentrations of female pigs of the Mangalitza and German Landrace breeds (A) Fig. 4 Experimental design of a pilot study on the impairment of animal welfare during transport. Fig. 5 Measurement of IGFBP-3 in the pilot study (A) The concentration of IGFBP-3 (ng / ml) compared to sample A (before transport) already decreases significantly 2 h after transport (B) and a further 2 h after the stay in the holding pen (C) or after slaughter (D) (*: p<0.05; **:p<0.01). Fig. 6 Measurement of IGFBP-2 in the pilot study (A) The concentration of IGFBP-2 (ng / ml) compared to sample A decreases significantly in samples B, C and D depending on the time after transport (*p<0.05; **p<0.01). Fig. 7 Commercial pig transport in Canada Fig. 8 Effects of transport over 6 h, 12 h or 18 h on the concentration of IGFBP-2 (A) and IGFBP-3 (B).Eighty pigs per group were examined (*: p<0.05; #: p<0.05; ***:p<0.001). Examples Quantitative Western ligand blot

[0037] To analyze IGF-binding proteins, serum proteins were separated by SDS-PAGE and subsequently transferred to a PVDF membrane. The membrane was incubated with quenching solution for 20 min, then blocked for 1 h, and subsequently incubated for 2 h with biotin-labeled IGF-2 (ibt-Systems) in blocking buffer (5 µg / ml). After five 5-minute washes, the membrane was incubated for 1 h with a streptavidin peroxidase conjugate. Following further washing (five 5-minute washes) and substrate addition (10 ml Lumina Forte (Milipore), protected from light for 4 min), the IGF-binding proteins were detected by chemiluminescence and imaged using the KODAK IMAGE Station. Quantification was performed using calibration curves with recombinant human IGFBPs. Curve fitting for the individual standards was carried out using four-parameter nonlinear regression. All washing and incubation steps are carried out by shaking, e.g. on a tilt shaker at room temperature (approx. 20-25°C). Examination of porcine samples

[0038] Samples from female Mangalitza (n = 10, age 1–2 years) and German Landrace (n = 12, age < 1 year) pigs were analyzed. The Mangalitza samples originated from the Research Institute for Animal Breeding, Nutrition and Meat Science, Herceghalom, Hungary. The German Landrace pigs came from the experimental pig facility, FBN Dummerstorf. Mean values ​​were compared using the t-test, with the significance level α set at 0.05.

[0039] The modified quantitative Western ligand blot can be used to visualize all IGFBPs present in a given sample or matrix ( Fig. 2A-D The quantitative nature of the data allows for comparisons of larger study groups. The current method is a valuable tool for assessing the status of the somatotropic axis in various livestock matrices.

[0040] In pigs, IGFBP-3 is also the dominant IGF-binding protein, followed by IGFBP-2. Thus, at least at the level of relative expression, the ratios of IGF-binding proteins between humans and pigs appear to be similar. This fulfills a prerequisite for identifying potential disorders of the somatotropic axis in pigs as well.

[0041] The Mangalitza pigs examined also showed significantly higher serum IGFBP-3 concentrations than German Landrace pigs (Fig. 3A). The IGFBP-3 / IGFBP-2 ratio tended to be higher in Mangalitza pigs than in German Landrace pigs (p < 0.1), which could indicate higher animal welfare. The Mangalitza pigs exhibited high fat deposition, slow growth, late maturation, and were kept extensively. The German Landrace pigs showed high muscle deposition, rapid growth, early maturation, and were kept intensively. However, it is also possible that breed or age effects caused the different IGFBP-3 concentrations (Fig. 3B). More conclusive results could be obtained by comparing populations of the same breed and, ideally, also of the same age. Pilot study on the impact of transport on animal welfare

[0042] Sample A was taken from 40 test animals (pigs, Danish Landrace x Pietrain). They were then loaded at the Karrenzin pig fattening facility, transported for two hours to the VION slaughterhouse in Perleberg, and unloaded. Slaughter took place after a two-hour waiting period on the day of transport. Sample B was taken immediately after transport, and sample C after the waiting period. Samples D and E (tissue samples) were taken immediately after slaughter, and sample F (tissue) was taken the following day, approximately 24 hours after slaughter, after storage at 4°C. Samples AD were blood samples. The weight at the start of the experiment was approximately 120 kg. Blood samples and tissue samples were collected from 40 control animals after slaughter.

[0043] The scheme is in Fig. 4 presented the results in Figs. 5 and 6These results show that transport significantly reduces the concentration of both IGFBP-2 and IGFBP-3. This is detectable not only immediately after transport, but also when samples are taken several hours later and after slaughter. Even more than 12 hours after slaughter, the impairment of animal welfare caused by transport was still detectable based on the biomarkers IGFBP-2 and IGFBP-3. Demonstrator project on the effects of commercial pig transport on welfare

[0044] The project was carried out in cooperation with the Dairy and Swine Research and Development Centre, Canada. 240 pigs (Landrace x Large White) with an average slaughter weight of 120.8 ± 0.4 kg were transported for 6h, 12h or 18h ( Fig. 7 The results are shown in Fig. 8.

[0045] These results show a clear association between the transport time leading to a decline in animal welfare and a decrease in the expression of IGFBP-2 and IGFBP-3 that is dependent on the duration of the decline. literature

[0046] Bielohuby M. (2014). Validation of serum IGF-I as a biomarker to monitor the bioactivity of exogenous growth hormone agonists and antagonists in rabbits. Disease Models & mechanisms 7:1263-1273.

[0047] Broom, DM (1986): Indicators of poor welfare. Br Vet J 142: 524-526.

[0048] Broom, DM (1991): Animal welfare: Concepts and Measurement. J. Anim. Sci 69:4167–4175

[0049] Goumon S, Brown JA, Faucitano L, Bergeron R, Widowski TM, Crowe T, Connor ML, Gonyou HW. (2013): Effects of transport duration on maintenance behavior, heart rate and gastrointestinal tract temperature of market-weight pigs in 2 seasons. J Anim Sci. 91: 4925-4935.

[0050] Hoeflich A, Wirthgen E, David R, Classen CF, Spitschak M, Brenmoehl J. (2014): Control of IGFBP-2 expression by steroids and peptide hormones in vertebrates. Front. Endocrinol. (Lausanne). 5:43. doi: 10.3389 / fendo.2014.00043.

[0051] Mesotten D. and Van den Berghe G. (2008): Changes within the GH / IGF-I / IGFBP axis in critical illness. In: Contemporary Endocrinology: Acute Cause to Consequence Edited by: G. Van den Berghe, DOI: 10.1007 / 978-1-60327-177-6 10, C. Humana Press, New York, NY.

[0052] Mysegades, W. (2014). Investigations into the suitability of the IGF-I system for predicting health and fertility in multiparous German Holstein dairy cows. Dissertation at the University of Veterinary Medicine Hannover.

[0053] Ruffer, U., (2003). Factors influencing IGF-1 concentrations in quarter initial milk samples from cows with different udder health – a field study. Dissertation at the Justus Liebig University Giessen, self-published by the Institute for Animal Breeding and Husbandry of the Christian-Albrechts University of Kiel.

[0054] Walker SL et al. (2008) Chronic stress, hormone profiles and estrus intensity in dairy cattle. Hormones and Behavior 53: 493-501.

Claims

1. A method of determining animal welfare, wherein a distinction between short-term and long-term impairment of the animal welfare is possible, wherein the expression of IGFBP-2 and IGFBP-3 in a sample from an animal is analyzed, wherein the method is used to determine transport duration and / or transport conditions of an animal, wherein a low expression in comparison to a reference group or, preferably, in comparison to known reference values of IGFBP-2 and IGFBP-3, is associated with a short-term impairment of animal welfare, due to transport of the animal, and wherein the duration of stress increases the extent of the change in the IGFBP concentrations, wherein the animal is a pig, wherein a short-term impairment occurs due to transport over approximately 1 hour to 3 days, while a long-term impairment occurs over approximately 7 or more days.

2. The method of claim 1, wherein the sample is selected from the group comprising blood, serum, plasma, seminal plasma, cerebrospinal fluid, follicular fluid, urine, sputum, tissue, drip fluid, or milk.

3. The method of any of the preceding claims, wherein the analysis is performed after slaughter and the sample is meat or meat juice.

4. The method of any of the preceding claims, wherein the expression of IGF-1 and / or additional IGFBPs in the sample is further analysed.

5. The method of any of the preceding claims, wherein the expression is analysed on a protein level with a quantitative Western Ligand Blot, or analysed on a RNA level.

6. The method of any of the preceding claims, wherein the animal welfare of a population is determined, whose animals have been treated with internally the same or comparable conditions, wherein optionally the animal welfare of an additional population is further determined, whose animals have been treated with other, internally the same or comparable conditions, wherein the animal welfare of the populations is compared.

7. A use of a kit comprising standards for quantification of IGFBP-3 and IGFBP-2 and marked IGF, in a method of any of the preceding claims.

8. The method of any of the claims 1-6 for optimization of the handling of an animal.

9. The method of any of the claims 1-6 for establishing an animal welfare standard and / or quality indicators of animal products.