Animal Production Systems

Abstract

The present invention relates to industrial animal production. In particular, the present invention relates to a method for multiplying a herd of animals and a method for determining an analyte in the egg yolk or blood of the animals by ex vivo analysis of blood and / or egg yolk samples.

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention relates to industrial animal production. In particular, the present invention relates to a method for multiplying a herd of animals and a method for determining analytes in the blood and / or egg yolk of animals.

[0002] [Background of the invention] Animals are raised for a variety of reasons, including egg and meat production. On an industrial scale, farmers are responsible for hundreds or even thousands of animals: the higher the number of animals, the more efficient mass production. Management of animal health, welfare, and performance is of paramount importance in industrial animal production.

[0003] One approach to increasing the profitability of industrial animal production is precision animal nutrition. To optimize profitability, farmers attempt to influence animal performance by adapting nutrition to the specific needs of the animals.

[0004] In order to benefit from accurate animal nutrition, it is necessary to accurately measure analyte status and relate the status to the health and performance of the animal. It is generally known that analytes in feed do not always correlate with the bioavailability and blood levels of said analytes. What is really important is how much of the analyte is bioavailable to the animal. Therefore, there is a need for practical tools that allow easy measurement of the analyte status of animals at any time.

[0005] [Summary of the Invention] The present invention relates to industrial animal production. In particular, the present invention relates to a method for multiplying a herd of animals and a method for determining analytes in the blood and / or egg yolk of animals.

[0006] The present invention relates to a method for determining at least one analyte in the blood of an animal, comprising: i) providing a drop of blood from the animal; ii) collecting the blood provided in step i) on a carrier; iii) drying the blood on the carrier; iv) analyzing the blood on the carrier provided in step iii) to determine the value of at least one analyte parameter; The present invention relates to a method comprising the steps of:

[0007] The present invention further comprises: v) comparing the value of at least one analyte measured in step iv) with a level of at least one analyte recommended in nutritional guidelines; vi) if the value of at least one analyte measured in step iv) falls outside the range recommended in the nutritional guidelines, adapting the feed given to the animal to ensure the animal's nutritional level recommended in accordance with the nutritional guidelines; may include.

[0008] In a preferred embodiment, the blood is venous blood.

[0009] In a preferred embodiment, the animal is a pig or poultry, preferably a broiler chicken.

[0010] In a preferred embodiment, the analysis in step iv) is carried out by liquid chromatography coupled with mass spectrometry (LC-MS / MS).

[0011] The method may be enhanced by the simultaneous measurement of multiple analytes. In a preferred embodiment, the values ​​of at least two, preferably at least three, more preferably at least four analytes are measured in step iv).

[0012] In another embodiment, the present invention provides a method for determining at least one analyte in egg yolk, comprising the steps of: i) providing a drop of egg yolk; ii) collecting the egg yolk provided in step i) on a carrier; iii) drying the egg yolk on the carrier; iv) analyzing the egg yolk on the carrier provided in step ii) to determine the value of at least one analyte in the egg yolk; The present invention relates to a method comprising the steps of:

[0013] The method further comprises: v) comparing the value of at least one analyte measured in step iv) with a level of at least one analyte recommended in nutritional guidelines; vi) if the value of at least one analyte measured in step iv) falls outside the range recommended in the nutritional guidelines, adapting the feed given to the animal to ensure the animal's nutritional level recommended in accordance with the nutritional guidelines; may include.

[0014] In a preferred embodiment, the animal is a pig or poultry, preferably a broiler chicken.

[0015] In a preferred embodiment, the analysis in step iv) is carried out by liquid chromatography coupled with mass spectrometry (LC-MS / MS).

[0016] The method may be enhanced by the simultaneous measurement of multiple analytes. In a preferred embodiment, the values ​​of at least two, preferably at least three, more preferably at least four analytes are measured in step iv).

[0017] The present invention further provides a method for propagating a herd of animals of the same species and breed, comprising: i) providing a drop of blood from at least one member of the group; ii) collecting the blood provided in step i) on a carrier; iii) drying the blood on the carrier; iv) analyzing the blood on the carrier provided in step iii) to determine the value of at least one analyte; v) comparing the value of at least one analyte measured in step iv) with a level of at least one analyte recommended in nutritional guidelines; vi) if the value of at least one analyte measured in step iv) falls outside the range recommended in the nutritional guidelines, adapting the feed given to the animal to ensure the animal's nutritional level recommended in accordance with the nutritional guidelines; The present invention relates to a method comprising the steps of:

[0018] In a preferred embodiment, the blood is venous blood.

[0019] In a preferred embodiment, the animal is a pig or poultry, preferably a broiler chicken.

[0020] In a preferred embodiment, the analysis in step iv) is carried out by liquid chromatography coupled with mass spectrometry (LC-MS / MS).

[0021] The method of the invention allows one to determine the analyte status of the animal from which the blood sample was taken, and the subsequent adaptation of the feed fed to the animal.

[0022] The method may be enhanced by the simultaneous measurement of multiple analytes. In a preferred embodiment, the values ​​of at least two, preferably at least three, more preferably at least four analytes are measured in step iv).

[0023] In another embodiment, the present invention provides a method for propagating a herd of animals of the same species and breed, comprising: i) providing a drop of egg yolk from an egg of at least one member of the clutch; ii) collecting the egg yolk provided in step i) on a carrier; iii) drying the egg yolk on the carrier; iv) analyzing the egg yolk on the carrier provided in step iii) to determine the value of at least one analyte; v) comparing the value of at least one analyte measured in step iv) with a level of at least one analyte recommended in nutritional guidelines; vi) if the value of at least one analyte measured in step iv) falls outside the range recommended in the nutritional guidelines, adapting the feed given to the animal to ensure the animal's nutritional level recommended in accordance with the nutritional guidelines; The present invention relates to a method comprising the steps of:

[0024] In a preferred embodiment, the animal is a pig or poultry, preferably a broiler chicken.

[0025] In a preferred embodiment, the analysis in step iv) is carried out by liquid chromatography coupled with mass spectrometry (LC-MS / MS).

[0026] The method of the invention allows one to determine the analyte status of the animal from which the blood sample was taken, and the subsequent adaptation of the feed fed to the animal.

[0027] The method may be enhanced by the simultaneous measurement of multiple analytes. In a preferred embodiment, the values ​​of at least two, preferably at least three, more preferably at least four analytes are measured in step iv).

[0028] In a preferred embodiment, the animal is a pig or poultry, preferably a broiler chicken.

[0029] Due to the accuracy of the measurements, appropriate measures can be taken to mitigate potential losses. Such measures include adaptation of the animal's nutrition. In a preferred embodiment, the feed in step vi) of the method of the invention is adapted by adjusting the amount of at least one feed additive.

[0030] [Detailed Description of the Invention] Method for determining an analyte parameter in the blood of an animal A preferred embodiment of the method of the present invention relates to a method for determining an analyte in the blood of an animal.

[0031] In a preferred embodiment of the invention, the animal is an avian, such as a chicken or a duck. More preferably, the animal is a broiler. Well-known breeds are Ross (e.g., Ross 708) and Cobb (e.g., Cobb 500).

[0032] In another preferred embodiment of the invention, the animal is a pig.

[0033] In step iv) of the method of the invention, a blood sample of an animal is analyzed ex vivo. The blood sample analyzed in step iv) preferably comprises venous blood. Thus, the preceding step i) of the method of the invention preferably comprises the provision of venous blood of an animal, and the method of the invention is preferably not a diagnostic method performed on an animal body.

[0034] In step iv) of the method of the present invention, the venous blood prepared in step i), collected on a carrier in step ii) and dried in step iii) is analyzed ex vivo. The blood sample is preferably taken at the site where the animals are bred. The method of the present invention preferably includes a means for collecting venous blood from the animals. An example of the sampling procedure is described in Example 2. The sample can be sent directly to the laboratory using a carrier card, which eliminates biological risks. Compared to conventional blood samples, which are difficult to process and require refrigeration, and cannot be sent across borders or by mail, for the method of the present invention, refrigeration is not required for transportation and subsequent storage. It is safe to send worldwide, does not pose biological risks, and does not require an import license.

[0035] The analysis is preferably carried out in the laboratory to which the sample was mailed. Analysis of the sample on the carrier is carried out by conventional methods, such as mass spectrometry by liquid chromatography (HPLC) or high performance liquid chromatography coupled with mass spectrometry (LC-MS / MS), to determine the level of the analyte in the animal. This procedure is highly correlated with conventional HPLC testing of plasma for the analyte.

[0036] Preferably, the decision as to which blood parameters are to be analysed in step vi) has been made prior to carrying out the analysis in step iv).

[0037] The optional correlation carried out in step v) can correlate analytes with levels of said analytes to improve health and performance factors.

[0038] The optional adaptation of the feed to the analyte values ​​carried out in step vi) may be facilitated by ordering the feed or feed additives from an external source.

[0039] The procedure of the method of the present invention, included in steps i) to iv), is summarized for the purposes of the present invention as dried blood spot test (DBS). DBS allows for easier and less invasive animal sampling, since it requires only one drop of blood from the animal. Considering the problem of sending test material, DBS is a safer and less complicated technique, since the carrier card preserves the material for a longer period, inhibiting the presence of any infectious agents, and allows the sample to be easily transported overseas or stored at room temperature. All this kind of helps to improve the quality and promptness of sending samples.

[0040] Method for determining analyte parameters in egg yolk A preferred embodiment of the method of the present invention relates to a method for determining an analyte in egg yolk.

[0041] In a preferred embodiment of the invention, the animal is an avian, such as a chicken or a duck. More preferably, the animal is a broiler. Well-known breeds are Ross (e.g., Ross 708) and Cobb (e.g., Cobb 500).

[0042] In step iv) of the method of the present invention, the egg yolk prepared in step i), collected on a carrier in step ii) and dried in step iii) is analyzed ex vivo. The egg yolk sample is preferably taken at the place where the animals are bred. An example of the sampling procedure is described in Example 3. The sample can be sent directly to the laboratory using a carrier card, which eliminates biological risks. Compared to conventional biological samples, which are difficult to process and require refrigeration, and cannot be sent across borders or by mail, for the method of the present invention, refrigeration is not required for transportation and subsequent storage. It is safe to send worldwide, does not pose biological risks, and does not require an import license.

[0043] The analysis is preferably carried out in the laboratory to which the sample was mailed. Analysis of the sample on the carrier is carried out by conventional methods, such as mass spectrometry by liquid chromatography (HPLC) or high performance liquid chromatography coupled with mass spectrometry (LC-MS / MS), to determine the level of the analyte in the egg yolk.

[0044] Preferably, the decision as to which blood parameters are to be analysed in step vi) has been made prior to carrying out the analysis in step iv).

[0045] The optional correlation carried out in step v) can correlate analytes with levels of said analytes to improve health and performance factors.

[0046] The optional adaptation of the feed to the analyte values ​​carried out in step vi) may be facilitated by ordering the feed or feed additives from an external source.

[0047] The procedure of the method of the present invention, included in steps i) to iv), is summarized for the purposes of the present invention as the dried egg yolk spot test (DYS). DYS allows for easier and less invasive animal sampling, since only one drop of egg yolk is required. Considering the problem of sending test material, DYS is a safer and less complicated technique, since the carrier card preserves the material for a longer period, inhibiting the presence of any infectious agents, and allows the sample to be easily transported overseas or stored at room temperature. All this kind of helps to improve the quality and promptness of sending samples.

[0048] [Animal blood is analyzed, a method of breeding a group of animals] A preferred embodiment of the method of the present invention relates to a method for multiplying a group of animals of the same species and breed.

[0049] The number of animals in a flock of animals may vary and depends on the species. In a preferred embodiment of the invention, the animals are birds, such as chickens or ducks. More preferably, the animals are broilers. Well-known breeds are Ross (e.g. Ross 708) and Cobb (e.g. Cobb 500). Thus, a preferred embodiment of the method of the invention relates to a method for multiplying flocks of birds, preferably chickens, of the same species and breed.

[0050] In another preferred embodiment of the invention, the animal is a pig.

[0051] Preferably, the group of animals is grown in the same compartment, for example in the same cage or in the same house. If the animals are chickens, the cage typically contains up to 100 chickens, while a chicken house can contain more than 1,000 chickens. Thus, one embodiment of the present invention relates to a method for growing a group of animals of the same species and breed, said group being grown in the same compartment and / or said group containing more than 10 animals, preferably more than 50 animals, even more preferably more than 100 animals, most preferably at least 1,000 animals.

[0052] In step iv) of the method of the invention, a blood sample of at least one member of the group of animals is analyzed ex vivo. Although it is possible to analyze blood samples of multiple members of the group, typically it is sufficient to analyze a blood sample of only one member of the group. Thereby, at least one member of the group is preferably randomly selected from the group of animals. The blood sample analyzed in step iv) preferably comprises venous blood. Thus, the preceding step i) of the method of the invention preferably comprises the provision of venous blood from at least one member of the group, and the method of the invention is preferably not a diagnostic method performed on an animal body.

[0053] In step iv) of the method of the present invention, the venous blood prepared in step i), collected on a carrier in step ii) and dried in step iii) is analyzed ex vivo. The blood sample is preferably taken at the site where the animals are bred. The method of the present invention preferably includes a means for collecting venous blood from the animals. An example of the sampling procedure is described in Example 2. The sample can be sent directly to the laboratory using a carrier card, which eliminates biological risks. Compared to conventional blood samples, which are difficult to process and require refrigeration, and cannot be sent across borders or by mail, for the method of the present invention, refrigeration is not required for transportation and subsequent storage. It is safe to send worldwide, does not pose biological risks, and does not require an import license.

[0054] The analysis is preferably carried out in the laboratory to which the sample was mailed. Analysis of the sample on the carrier is carried out by conventional methods, such as mass spectrometry by liquid chromatography (HPLC) or high performance liquid chromatography coupled with mass spectrometry (LC-MS / MS), to determine the level of the analyte in the animal. This procedure is highly correlated with conventional HPLC testing of plasma for the analyte.

[0055] Preferably, the decision as to which blood parameters are to be analysed in step vi) has been made prior to carrying out the analysis in step iv).

[0056] The correlation carried out in step v) may correlate analytes with levels of said analytes to improve health and performance factors.

[0057] The adaptation of the feed to the analyte values ​​carried out in step vi) can be facilitated by ordering feed or feed additives from an external supplier.Thus, a preferred embodiment of the present invention relates to a set-up comprising the method described herein for raising a group of animals and at least one supplier of feed additives, premixes and / or feed, wherein the results of the analysis step iv) or the correlations of step v) are communicated to at least one supplier of feed additives, premixes and / or feed.

[0058] The procedure of the method of the present invention, included in steps i) to iv), is summarized for the purposes of the present invention as dried blood spot test (DBS). DBS allows for easier and less invasive animal sampling, since it requires only one drop of blood from the animal. Considering the problem of sending test material, DBS is a safer and less complicated technique, since the carrier card preserves the material for a longer period, inhibiting the presence of any infectious agents, and allows the sample to be easily transported overseas or stored at room temperature. All this kind of helps to improve the quality and promptness of sending samples.

[0059] [Egg yolks are analyzed, a method of multiplying a group of animals] A preferred embodiment of the method of the present invention relates to a method for multiplying a group of animals of the same species and breed.

[0060] The number of animals in a flock of animals may vary and depends on the species. In a preferred embodiment of the invention, the animals are birds, such as chickens or ducks. More preferably, the animals are broilers. Well-known breeds are Ross (e.g. Ross 708) and Cobb (e.g. Cobb 500). Thus, a preferred embodiment of the method of the invention relates to a method for multiplying flocks of birds, preferably chickens, of the same species and breed.

[0061] Preferably, the group of animals is grown in the same compartment, for example in the same cage or in the same house. If the animals are chickens, the cage typically contains up to 100 chickens, while a chicken house can contain more than 1,000 chickens. Thus, one embodiment of the present invention relates to a method for growing a group of animals of the same species and breed, said group being grown in the same compartment and / or said group containing more than 10 animals, preferably more than 50 animals, even more preferably more than 100 animals, most preferably at least 1,000 animals.

[0062] In step iv) of the method of the invention, an egg yolk sample of an egg of at least one member of the group of animals is analyzed. Although it is possible to analyze egg yolk samples of eggs of several members of the group, typically it is sufficient to analyze an egg yolk sample of only one member of the group. Thereby, the at least one member of the group is preferably randomly selected from the group of animals.

[0063] The egg yolk sample is preferably taken at the site where the animals are bred. The method of the present invention preferably includes a means for obtaining the sample from the egg. An example of the sampling procedure is described in Example 3. The sample can be sent directly to the laboratory using a carrier card, which eliminates biological risks. Compared to conventional biological samples, which are difficult to process and require refrigeration, and cannot be sent across borders or by mail, for the method of the present invention, no refrigeration is required for transportation and subsequent storage. It is safe to send worldwide, has no biological risks, and does not require an import license.

[0064] The analysis is preferably carried out in the laboratory to which the sample was mailed. Analysis of the sample on the carrier is carried out by conventional methods, such as mass spectrometry by liquid chromatography (HPLC) or high performance liquid chromatography coupled with mass spectrometry (LC-MS / MS), to determine the level of the analyte in the animal.

[0065] Preferably, the decision as to which egg yolk parameters are analysed in step vi) has been made prior to carrying out the analysis in step iv).

[0066] The correlation carried out in step v may correlate analytes with levels of said analytes to improve health and performance factors.

[0067] The adaptation of the feed to the analyte values ​​carried out in step vi) can be facilitated by ordering feed or feed additives from an external supplier.Thus, a preferred embodiment of the present invention relates to a set-up comprising the method described herein for raising a group of animals and at least one supplier of feed additives, premixes and / or feed, wherein the results of the analysis step iv) or the correlations of step v) are communicated to at least one supplier of feed additives, premixes and / or feed.

[0068] The procedure of the method of the present invention, included in steps i) to iv), is summarized for the purposes of the present invention as the dried egg yolk spot test (DYS). DYS allows for simpler and less invasive animal sampling, since it requires only one drop of blood from the animal. Considering the problem of sending test material, DYS is a safer and less complicated technique, since the carrier card preserves the material for a longer period, inhibiting the presence of any infectious agents, and allows the sample to be easily transported overseas or stored at room temperature. All this kind of helps to improve the quality and promptness of sending samples.

[0069] [Definition] Analyte: In the context of the present invention, the term "analyte" refers to a component or chemical species of interest in an analytical procedure. In a preferred embodiment of the present invention, the analyte may be a nutrient or a biomarker.

[0070] Animals: In the context of the present invention, the term "animals" includes all non-human members of the kingdom Animalia. Preferably, animals are monogastric animals, such as pigs (including but not limited to piglets, breeding pigs, and sows), poultry (including but not limited to poultry, turkeys, ducks, quails, guinea fowl, geese, pigeons, chicks, chickens, broilers, layers, pullets, and chicks); pet animals, such as cats and dogs, fish (amberjack, arapaima, barb, bass, bluefish, boca chick, bream, bullhead, cachamas, carp, catfish, cattla, milkfish, char, cichlid, cobia, cod, crappie, gilthead, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, lai, loach, mackerel, milkfish, black mullet, pako, pearl spot, pejerey, perch, pike, trevally, roach, salmon, sampa, sauger, grouper, sea bream, shiner, sleeper, snakehead, snapper, scallop, sole, rabbitfish, sturgeon, sunfish, sweetfish, tench, teller, tilapia, trout, tuna, flounder, vendace, walleye, and whitefish); and crustaceans (including, but not limited to, shrimp and prawns). Preferably, the animal is selected from the group of pigs (including but not limited to piglets, growers, and sows) or poultry (including but not limited to poultry, turkeys, ducks, quails, guinea fowl, geese, pigeons, chicks, chickens, broilers, layers, pullets, and chicks).

[0071] Anticoagulants: Sometimes anticoagulants are added to blood samples. Well-known anticoagulants are heparin and EDTA. In the context of the present invention, blood containing heparin is referred to as "heparinized blood".

[0072] Biomarker: A biomarker (short for biological marker) for the purposes of the present invention is an objective, quantifiable characteristic of a biological process. A biomarker is an objective measure that captures what is happening in a cell or organism at a given time. Biomarkers can, for example, help improve the ability to understand the relationship between environmental factors and disease and to diagnose, monitor, or predict disease risk. Biomarkers for the purposes of the present invention can be selected from, but are not limited to, albumin, calcium, carotenoids, creatine kinase, globulin, glucose, hemoglobin, phosphorus, potassium, sodium, total carbon dioxide, and uric acid.

[0073] Albumin: In the context of the present invention, the term "albumin" refers to a family of globular proteins. All proteins of the albumin family are water-soluble, moderately soluble in concentrated salt solutions, and undergo thermal denaturation. Albumin is generally found in plasma and differs from other blood proteins in that it is not glycosylated. Substances that contain albumin are called albuminoids. The concentration of albumin in serum (serum albumin, SA) can be influenced by several factors, including its synthesis rate, catabolic rate, extravascular distribution, and exogenous loss. In addition, both nutritional status and systemic inflammation affect the synthesis of SA. Determination of SA concentration aids risk prediction in various clinical situations. An increased risk of all-cause mortality and cardiovascular (CV) mortality can be associated with low SA concentration.

[0074] Calcium: Calcium deficiency can lead to metabolic abnormalities or potential pathological changes. Thus, calcium can act as a biomarker for metabolic problems and / or pathological changes.

[0075] Carotenoids: In the context of the present invention, the term "carotenoid" refers to organic pigments produced by plants and algae, as well as some bacteria and fungi. Carotenoids are obtained from the diet as brightly colored pigments. They may serve as, but are not limited to, biomarkers of fruit and vegetable intake.

[0076] Creatine kinase: In the context of the present invention, the term "creatine kinase" (CK) refers to an enzyme present in tissues and energy-requiring cells, such as skeletal and cardiac muscles, and is considered the best marker for the detection and monitoring of skeletal muscle diseases. Creatine kinase activity is typically measured in serum or plasma.

[0077] Globulin: In the context of the present invention, the term "globulin" refers to a family of globular proteins with a molecular weight higher than albumin, insoluble in pure water, but soluble in dilute salt solutions. Some globulins are produced in the liver, while others are produced by the immune system. Globulins, albumin, and fibrinogen are major blood proteins. Higher globulin levels are strongly associated with the risk of periprosthetic joint infection (PJI) and may serve as a biomarker in the diagnosis of PJI. The serum albumin / globulin ratio is further recognized as a valuable prognostic biomarker in various cancers.

[0078] Glucose: In the context of the present invention, the term "glucose" has the molecular formula CH 12 Glucose refers to a simple sugar with O6. Glucose is the most abundant monosaccharide, a subcategory of carbohydrates. Glucose is primarily made by plants and most algae during photosynthesis from water and carbon dioxide, using energy from sunlight, and is used to make cellulose in cell walls, the most abundant carbohydrate in the world. Continuous glucose monitoring provides detailed real-time data that is of value in clinical decision-making, assessing response to new diabetes drugs and the development of closed-loop artificial pancreas technology.

[0079] Hemoglobin: In the context of the present invention, the term "hemoglobin" or hemoglobin (abbreviated Hb or Hgb) refers to the iron-containing oxygen-transporting metalloprotein in the red blood cells of almost all vertebrates and in the tissues of some invertebrates. Hemoglobin in the blood carries oxygen from the lungs or gills to other parts of the body (i.e., tissues). There, hemoglobin releases oxygen so that aerobic respiration can provide the energy that powers the functions of the organism in a process called metabolism. High hemoglobin levels (Hb) may be associated with an increased risk of total and breast cancer incidence.

[0080] Phosphorus: In the context of the present invention, the term "phosphorus" refers to the element with the symbol P and atomic number 15. Elemental phosphorus exists in two major forms, yellow phosphorus and red phosphorus, but because it is highly reactive, phosphorus is never found as a free element on Earth. In minerals, phosphorus generally exists as phosphate salts. 24-hour urinary phosphorus is commonly used as a surrogate measure for phosphorus intake and absorption.

[0081] Potassium: In the context of the present invention, the term "potassium" refers to the element with the symbol K and atomic number 19. Potassium occurs in nature only as an ionic salt. Potassium is the most abundant intracellular cation that plays an important role in nerve impulse transmission, cardiac activity, membrane transport, acid-base balance, and neuromuscular function. A potassium-rich diet, such as DASH [Dietary Approaches to Stop Hypertension], may lower blood pressure and reduce kidney disease progression.

[0082] Sodium: In the context of the present invention, the term "sodium" refers to the element with the symbol Na and atomic number 11. Free metal does not occur in nature. Excessive sodium intake increases the risk of hypertension, which is a major risk factor for cardiovascular disease.

[0083] Total Carbon Dioxide: Carbon dioxide is produced in the body from cellular respiration. As a biomarker, carbon dioxide in serum is actually a measure of bicarbonate in the blood. Most CO2 produced from cellular respiration (about 75%) is carried in the blood as bicarbonate ions. 5% remains in solution as dissolved CO2, and the remaining 20% ​​remains combined with hemoglobin and other plasma proteins. Dissolved CO2 formed in the lungs contributes very little to the CO2 value. In blood tests, CO2 or bicarbonate is a common measure of tissue acidity or alkalinity. CO2 content refers to bicarbonate, which is an alkaline or base molecule. It is in solution and is primarily regulated by the kidneys and is related to CO2 in blood chemistry panels. CO2 gas, on the other hand, refers to dissolved CO2, which is primarily an acid. It is regulated by the lungs. Both have a powerful effect on acid-base regulation and are regulated by various organ systems. Carbon dioxide serum as bicarbonate serves as one of the reserve alkaline elements in the blood. Bicarbonate neutralizes metabolic acids such as hydrochloric acid and lactic acid. Serum CO2 can be looked at to aid in the assessment of the tendency towards alkalosis or acidosis in the body. Elevated levels of serum CO2, or bicarbonate, can be associated with a tendency towards metabolic alkalosis, while decreased levels are associated with a tendency towards metabolic acidosis. In this situation, bicarbonate is being used up in the body to buffer the increasing levels of acidity or H+. Serum total carbon dioxide can also be a prognostic factor for 28-day mortality in septic patients.

[0084] Uric acid: In the context of the present invention, the term "uric acid" refers to a heterocyclic compound of carbon, nitrogen, oxygen, and hydrogen with the formula C5H4N4O3. It forms ions and salts known as urates and acid urates, such as ammonium acid urate. Uric acid is a product of the metabolic breakdown of purine nucleotides, and it is a normal component of urine. High blood concentrations of uric acid can cause gout and are associated with other medical conditions, including diabetes and the formation of ammonium acid urate kidney stones. Uric acid (UA) has recently emerged as an inflammatory factor that increases oxidative stress and promotes the activation of the renin-angiotensin-aldosterone system. As a result, higher UA levels are associated with various stages of the development and progression of diabetic nephropathy, including metabolic, cardiovascular, and renal function abnormalities. Serum UA levels may serve as a biomarker of renal and cardiovascular risk, and a potential additional therapeutic target in diabetes.

[0085] Blood parameters: values ​​of "blood parameters" can be measured. As an example, the blood parameter Na (electrolyte) can be 140 mmol of ionic sodium per liter of blood. The value of a blood parameter can depend on several contributing variables, such as age and breed.

[0086] Breed: In the context of the present invention, the term "breed" refers to a lineage of animals within a species that has a distinctive appearance and that has typically been developed by careful selection. Thus, the animals are likely related homoeologously from a common ancestor. As an example, broiler chickens from breeds Ross 708 and Cobb 500 may be commercially sourced from a local commercial hatchery.

[0087] Carrier: In the context of the present invention, the term "carrier" refers to a sample collection card. Said sample collection card can be made of various substrates, e.g. cardboard, wood, glass, plastic. Preferably, the sample collection card is a filter paper with collection circles printed on its surface, where a drop of blood and / or egg yolk can be deposited for further analysis. Each collection circle can be used to deposit a drop of sample. Multiple collection circles allow the placement of multiple samples and separate analysis of the samples.

[0088] More preferably, the sample collection card is a chemically treated filter paper designed for the collection, storage, and shipping of dry biological samples for subsequent DNA and RNA analysis, where special chemicals dissolve and inactivate bacteria and / or viruses and preserve the DNA and RNA for detection by analytical methods such as PCR. Such sample collection cards are commercially available as Whatman® 903 (GE Healthcare, Piscataway, NJ, EUA) and are disclosed in WO 2000062023 A1.

[0089] Samples placed and dried on such carriers are preserved so they can be taken virtually anywhere. Dried samples on carriers do not require refrigerated transport.

[0090] Calcidiol: The terms "25-OH D3", "25-hydroxyvitamin D3", "HyD", and "calcidiol" are used interchangeably.

[0091] Cholecalciferol: The terms "cholecalciferol" and "vitamin D3" are used interchangeably.

[0092] Dietary guidelines: The terms "dietary guidelines", "feed-based dietary guidelines", or "nutritional guidelines" are used interchangeably and refer to guidelines intended to ensure recommended animal nutritional levels and / or establish the basis for providing nutritionally complete feed rations needed to improve health and performance factors.

[0093] Group of animals: The term "group of animals" refers to a group of animals, preferably at least 10, more preferably at least 100, and most preferably at least 1000 animals, kept in the same compartment (e.g., in the same cage or in the same house).

[0094] Health and performance factors: In the context of the present invention, the term "health and performance factors" refers to objective, quantifiable characteristics of animal performance and health. Preferably, the health and performance factors are selected from the group of, but not limited to, bone strength, meat yield and eggshell strength, milk fever potential, hatchability, embryo viability, gut health (microflora), nutrient absorption, skeletal health, electrolyte balance, liver health, oxidative stress, and inflammation.

[0095] By correlating biomarker levels in blood and / or egg yolk with performance gains, it is possible to estimate the impact on economically important parameters. For example, comparing performance gains in different broiler trials, it could be concluded that, on average, a 1% increase in plasma levels of 25-OH-D3 leads to 0.029%, 0.173%, 0.008%, and 0.296% gains in ADG, bone strength, breast meat yield, and ambulatory score, respectively (Sakas et al., 2019; Bray et al., 2012; Sauders et al. 2004; Vignale et al., 2015).

[0096] Bone strength: Bone strength is determined by bone geometry, cortical thickness and porosity, trabecular bone morphology, and the intrinsic properties of bone tissue. Bone strength can be indirectly estimated by bone mineral density (BMD) using analytical methods such as dual energy X-ray absorptiometry (DXA). Osteoporosis is a disease defined by reduced bone mass and altered microarchitecture that leads to increased bone fragility and increased risk of fracture. The main complication of osteoporosis, fractures, is due to low bone strength. Therefore, any treatment of osteoporosis implies an improvement in bone strength.

[0097] Meat Yield: In the context of the present invention, the term "meat yield" or "lean meat yield" (LMY%) refers to the proportion of the carcass that is lean (muscle), expressed as a percentage. The LMY% of a carcass is a standard way of assessing the composition of the carcass and does not vary according to the cut specifications used to market the carcass. LMY% is calculated differently for sheep and beef. For sheep, LMY% is predicted using Hot Standard Carcass Weight (HSCW) and knife GR tissue depth. Hot carcass weight is the weight of the carcass before freezing. Beef carcasses consist of 70-75 percent water. As the carcass cools and ages, water is lost by evaporation. Within just the first 24 hours, the carcass can lose up to 2-5 percent of its initial weight. The higher the HSCW and the lower the GR value, the higher the LMY%. The algorithm for beef LMY% is calculated using HSCW, rib fat depth, and sometimes eye muscle area (EMA). The higher the HSCW and the lower the rib fat depth (and the greater the EMA, if EMA is used), the higher the LMY%. Processors can use a variety of systems to determine LMY%, but these systems are all calibrated against CT scan, the gold standard system for measuring LMY%. This allows processors and producers to compare carcasses in a standard way. Producers can manage LMY% through key on-farm practices, such as nutritional supplementation and genetic selection. Armed with information about LMY% in relation to LDL measures, producers can make better informed management decisions to maximize carcass value in the future.

[0098] Eggshell Strength: Shell breakage is, and always has been, a financial drain on the poultry industry. Methods for measuring eggshell strength can involve quasi-static compression. In this destructive process, an egg is compressed between two parallel plates under a steadily increasing load until failure results. The force and deformation are continuously recorded and the strength of the eggshell is given in terms of the force at break. The non-destructive deformation of the shell is usually assumed to give a measure of its hardness characteristics.

[0099] Milk fever: In the context of the present invention, the terms "milk fever", "puerperal hemoglobinuria", or "parturient paresis" refer to a disease seen primarily in dairy cows, but also in beef cattle and non-bovine domesticated animals, characterized by a reduction in blood calcium levels (hypocalcemia). It occurs after parturition, at the onset of lactation, when the demand for calcium for colostrum and milk production exceeds the body's ability to mobilize calcium. "Fever" is a misnomer, since body temperature does not usually rise during the disease. Milk fever is more commonly seen in older animals (which have less ability to mobilize calcium from bones) and in certain breeds (such as the Channel Island breed).

[0100] Hatchability: As used herein, the term "hatchability" refers to the percentage of total eggs that hatch into viable chicks or poodsores (e.g., number of hatched poodsores per number of egg sets x 100). Increasing hatchability, i.e., increasing the percentage of laid eggs that hatch, is particularly desirable, since even small increases in percentage will significantly affect the number of resulting chicks. Thus, increasing hatchability is considered an important factor in large-scale breeding programs. The increase in the likelihood that an egg will hatch can be measured by calculating the "hatchability" or "fertilized hatch" of several eggs. As used herein, the term "fertilized hatch" refers to the percentage of total fertilized eggs that hatch into viable chicks or poodsores (e.g., number of hatched poodsores per number of fertilized egg sets x 100).

[0101] Embryo viability: As used herein, the term "embryo viability" refers to an estimate of the survival rate or viability of an embryo.

[0102] Gut health: The term "embryo viability" as used herein refers to improving the diversity of the microbiota and / or increasing the amount of beneficial bacteria in the intestinal tract, particularly the colon. Beneficial bacteria known to reside in the colon include Acidaminococcus spp., Akkermansia spp., Bacteroides ovatus, Bifidobacterium spp., Blautia producta, Clostridium cocleatum, Collinsella aerofaciens, Dorea longicatena, Escherichia coli, Eubacterium spp., Faecalibacterium prausnitzii, Lachnospira pectinoschiza, and others. pectinoshiza, Lactobacillus spp., Parabacteroides distasonis, Raoultella spp., Roseburia spp., Ruminococcus spp., and Streptococcus spp. Preferably, the bacteria to be increased are selected from the group consisting of Bifidobacterium spp., Akkermansia spp., Faecalibacterium spp., and Bacteriodes spp.More preferably, Bifidobacterium adolescentis, Bifidobacterium longum, Bacteroides ovatus, Bacteroides xylanisolvens, Lachnoclostridium spp., Akkermansia muciniphila, Blautia wexlerae, and / or Faecalibacterium prausnitzii are increased following administration of the antioxidant of the present invention. Increasing bacterial diversity and / or the amount of beneficial bacteria is particularly beneficial when the animal is experiencing a condition selected from the group consisting of: metabolic disorders, type 2 diabetes, obesity, Crohn's disease, ulcerative colitis, inflammatory bowel disease, irritable bowel syndrome, leaky gut, malnutrition, chronic inflammation, and cardiovascular disease.

[0103] Skeletal Health: As used herein, the term "skeletal health" may refer to various aspects of skeletal health, including, but not limited to, bone strength, good bone mineralization, high bone density, and / or resistance to fracture.

[0104] Electrolyte balance: As used herein, the term "electrolyte balance" refers to the absence of electrolyte imbalance, which is an abnormality in the concentration of electrolytes in the body, or water-electrolyte imbalance. Electrolytes play an essential role in maintaining homeostasis in the body. Electrolytes help regulate cardiac and neurological function, fluid balance, oxygen delivery, acid-base balance, and many others. Electrolyte imbalance can occur by consuming too little or too much electrolytes, and by secreting too little or too much electrolytes.

[0105] Electrolyte disorders are involved in many disease processes and are an important part of patient management in medicine. The causes, severity, treatment, and outcome of such disorders can vary greatly depending on the electrolytes involved. [3]The most serious electrolyte disorders involve abnormalities in sodium, potassium, or calcium levels. Other electrolyte imbalances are less common and often occur with major electrolyte changes. The kidneys are the most important organs in maintaining proper fluid and electrolyte balance, although other factors such as hormonal changes and physiological stresses play a role. [2] .

[0106] Liver health: As used herein, the term "liver health" refers to the maintenance of healthy liver function.

[0107] Oxidative Stress: Oxidative stress reflects an imbalance between the systemic manifestations of reactive oxygen species and the ability of biological systems to readily detoxify reactive intermediates or repair the resulting damage. Disturbances in the normal redox state of cells can cause toxic effects through the generation of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA. Oxidative stress from oxidative metabolism causes base damage and strand breaks in DNA. Base damage is mostly indirect and occurs due to the reactive oxygen species (ROS) generated, e.g., O2 - Oxidative stress is caused by reactive oxidative species such as superoxide radical, OH (hydroxyl radical), and H2O2 (hydrogen peroxide). In addition, some reactive oxidative species act as cellular messengers in redox signaling. Thus, oxidative stress can cause disruptions in the normal mechanisms of cell signaling.

[0108] Inflammation: Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants, and is a defense reaction involving immune cells, blood vessels, and molecular mediators. The function of inflammation is to remove the initial cause of cellular damage, to clear necrotic cells and damaged tissue from the initial injury and inflammatory process, and to initiate tissue repair.

[0109] The five main signs are heat, pain, redness, swelling, and loss of function (Latin: calor, dolor, rubor, tumor, and functio laesa). Inflammation is a general response and is therefore considered a mechanism of innate immunity, as compared to adaptive immunity, which is pathogen-specific. Too little inflammation can lead to progressive tissue destruction by harmful stimuli (e.g. bacteria), compromising the survival of the organism. In contrast, too much inflammation, in the form of chronic inflammation, is associated with various diseases, e.g., hay fever, periodontal disease, atherosclerosis, and osteoarthritis.

[0110] Inflammation can be classified as either acute or chronic. Acute inflammation is the body's initial response to harmful stimuli and is accompanied by increased migration of plasma and white blood cells (particularly granulocytes) from the blood into the injured tissue. A series of biochemical events propagates and matures the inflammatory response involving the local vascular system, immune system, and various cells within the injured tissue. Inflammation over a long period of time, known as chronic inflammation, leads to a progressive shift in the types of cells present at the site of inflammation, e.g., mononuclear cells, and is characterized by the concomitant destruction and healing of tissue resulting from the inflammatory process.

[0111] High-Performance Liquid Chromatography Coupled with Mass Spectrometry: High-Performance Liquid Chromatography Coupled with Mass Spectrometry (LC-MS / MS) is a highly sensitive analytical technique. It reads the analyte, its mass / charge (m / z), and its fragments (product ions). It is a highly sensitive technique, determining parts per billion (ppb or ng / mL), but requires only a small sample volume of 150 μL (microliters), and suffers from low interference from biological matrices (plasma) and metabolite components.

[0112] Nutritional parameters: A or nutritional parameters, for the purposes of the present invention, are substances used by an organism to survive, grow, and reproduce. Nutrients can be classified to describe the nutritional requirements of an animal and can be divided into macronutrients and micronutrients. Macronutrients, such as carbohydrates, fats, proteins, and water, are consumed in relatively large amounts (grams or ounces) and are primarily used to generate energy or be incorporated into tissues for growth and repair. Micronutrients, such as vitamins and dietary minerals, are required in smaller amounts (milligrams or micrograms). Micronutrients for the purposes of the present invention can be, but are not limited to, minerals and vitamins. Nutritional parameters can be selected from the group consisting of fat-soluble vitamins, water-soluble vitamins, and trace minerals. Preferably, nutritional parameters are selected from vitamin D3, 25-OH-D3, vitamin A, and / or vitamin E.

[0113] Breeding animals: In the context of the present invention, "breeding animals" refers to the production of animals, whatever the purpose. Thus, "breeding animals" includes breeding animals for meat and / or egg production. Chickens raised for meat production are broiler chickens.

[0114] Vitamin D3: Vitamin D3 is a fat-soluble vitamin that must undergo enzymatic digestive processes (lipases) and micelle formation in the intestinal tract to be absorbed by enterocytes (Combs Jr. and McClung, 2017). As it passes through the liver, it is hydroxylated to produce the first metabolite: 25-OH-D3, which is the most abundant form in the organism and is available to be metabolized and converted to the active form of vitamin D, mainly in the kidneys.

[0115] Due to its advantages over dietary vitamin D3, the inclusion of 25-OH-D3 in the diet is a strategy currently used in animal nutrition. At the same level of inclusion, the absorption of 25-OH-D3 is higher (74.9%) than vitamin D3 (66.5%) (Bar et al., 1980). When given in the diet, 25OHD3 does not need to undergo a conversion stage in the liver (Soares et al., 1995), is more efficiently absorbed in the intestine, has greater retention and less secretion compared to vitamin D3 (Bar et al., 1980), is 2.0-2.5 times more biologically active (Soares et al., 1978; Fritts and Waldroup, 2003), and shows no toxic effects up to 10 times its recommended dose (Yarger et al., 1995).

[0116] 25-OH-D3 is the most abundant form of vitamin D in the bloodstream and its assessment is the metric for assessing vitamin D status in all animal species, including humans. Assessment of this metabolite has been used to determine ideal dietary levels and correlate with important parameters, such as calcium and phosphorus plasma levels, bone characteristics (Tizziani et al., 2019), muscle protein synthesis (Vignale et al., 2015; Prokoski et al., 2019), performance variables (Zhang et al., 2020), and establish differences between production conditions (DSM Field Trial. Lozano, 2021).

[0117] The role of vitamin D in calcium and phosphorus metabolism, physiological processes, and requirements in poultry has been well documented (Rama-Rao et al., 2006; Rama-Rao et al., 2009). In addition, its role in regulating immune responses (Chou et al., 2009; Morris et al., 2014), muscle formation, and meat yield (Hutton et al., 2014; Prokoski et al., 2019) has been widely described in the recent literature.

[0118] The inclusion of optimal levels of vitamin D3 has been the subject of research for several decades. Initial recommendations were based on studies conducted with refined or semi-refined foods under controlled experimental conditions (NRC, 1994). However, the recommendations were lower than those required under commercial production conditions or under pathogen challenges that typically increase nutritional requirements.

[0119] At commercial levels, vitamin D3 inclusions vary between 3,000-5,000 UI / kg feed (Bozkurt et al., 2017; Sakkas et al., 2019). Typically, poultry companies determine the level to be used based on available recommendations, their own production conditions, expected performance, end product, and maximum return on investment.

[0120] In general, the measurement of 25OHD3 in plasma allows the vitamin D status of animals to be known. There is a correlation between health status, immunity, and performance and vitamin D3 status. The plasma / blood levels of 25OHD3 reached by animals are always greater when HyD is supplemented in the feed due to its metabolic advantages over vitamin D and competitors. [Brief description of the drawings]

[0121] [Figure 1] Effect of inclusion of dietary 25(OH)D3 (Hy-D®) on blood concentrations of vitamin D3 in 21-day-old broiler chickens.

[0122] [Embodiment of the invention] 1. A method for determining at least one analyte in the blood of an animal, comprising: vii) providing a drop of blood from the animal; viii) collecting the blood provided in step i) on a carrier; ix) drying the blood on the carrier; x) analyzing the blood on the carrier provided in step iii) to determine the value of at least one analyte; The method includes:

[0123] 2. xi) comparing the value of the at least one analyte measured in step iv) with a level of the at least one analyte recommended in a nutritional guideline; xii) if the value of at least one analyte measured in step iv) falls outside the range recommended in the nutritional guidelines, adapting the feed given to the animal so as to ensure the animal's nutritional level recommended in accordance with the nutritional guidelines; The method of claim 1, further comprising:

[0124] 3. A method for propagating a group of animals of the same species and breed, comprising: i) providing a drop of blood from at least one member of the group; ii) collecting the blood provided in step i) on a carrier; iii) drying the blood on the carrier; iv) analyzing the blood on the carrier provided in step iii) to determine the value of at least one analyte; v) comparing the value of at least one analyte measured in step iv) with a level of at least one analyte recommended in nutritional guidelines; vi) if the value of at least one analyte measured in step iv) falls outside the range recommended in the nutritional guidelines, adapting the feed given to the animal to ensure the animal's nutritional level recommended in accordance with the nutritional guidelines; The method includes:

[0125] 4. The method according to any one of claims 1 to 3, wherein the blood is venous blood.

[0126] 5. A method for determining at least one analyte in egg yolk, comprising: vii) providing a drop of egg yolk; viii) collecting the egg yolk provided in step i) on a carrier; ix) drying the egg yolk on the carrier; x) analyzing the egg yolk on the carrier provided in step iii) to determine the value of at least one analyte in the egg yolk; The method includes:

[0127] 6. xi) comparing the value of the at least one analyte measured in step iv) with a level of the at least one analyte recommended in a nutritional guideline; xii) if the value of at least one analyte measured in step iv) falls outside the range recommended in the nutritional guidelines, adapting the feed given to the animal so as to ensure the animal's nutritional level recommended in accordance with the nutritional guidelines; 6. The method of claim 5, further comprising:

[0128] 7. A method for propagating a group of animals of the same species and breed, comprising: vii) providing a drop of egg yolk from an egg of at least one member of the flock; viii) collecting the egg yolk provided in step i) on a carrier; ix) drying the egg yolk on the carrier; x) analyzing the egg yolk on the carrier provided in step iii) to determine the value of at least one analyte; xi) comparing the value of the at least one analyte measured in step iv) with a level of the at least one analyte recommended in a nutritional guideline; xii) if the value of at least one analyte measured in step iv) falls outside the range recommended in the nutritional guidelines, adapting the feed given to the animal so as to ensure the animal's nutritional level recommended in accordance with the nutritional guidelines; The method includes:

[0129] 8. The method of any one of claims 1 to 7, wherein the carrier is a sample collection card.

[0130] 9. The method of claim 8, wherein the sample collection card is a cardboard covered with a substrate for lysing cells and purifying nucleic acids.

[0131] 10. The method of claim 9, wherein the sample collection card is a Whatman® 903 (GE Healthcare, Piscataway, NJ, EUA).

[0132] 11. The method according to any one of claims 1 to 10, wherein the analysis in step iv) is carried out by liquid chromatography coupled with mass spectrometry (LC-MS / MS).

[0133] 12. The method of any one of claims 1 to 11, wherein the analyte is a nutrient.

[0134] 13. The method of any one of claims 1 to 12, wherein the analyte is selected from the group consisting of fat-soluble vitamins, water-soluble vitamins, and trace minerals.

[0135] 14. The method of any one of claims 1 to 13, wherein the analyte is selected from 25-OH-D3, vitamin A, and / or vitamin E.

[0136] 15. The method of any one of claims 1 to 11, wherein the analyte is a biomarker.

[0137] 16. The method of any one of claims 1 to 11 and 15, wherein the analyte is selected from the group consisting of albumin, calcium, carotenoids, creatine kinase, globulin, glucose, hemoglobin, phosphorus, potassium, sodium, total carbon dioxide, and uric acid.

[0138] 17. The method of any one of claims 1 to 13, 15 and 16, wherein the health and performance factors are selected from the group of bone strength, meat yield and eggshell strength, milk fever potential, hatchability, embryo viability, gut health, nutrient absorption, skeletal health, electrolyte balance, liver health, oxidative stress and inflammation.

[0139] 18. Animals include monogastric animals such as pigs (including but not limited to piglets, growers, and sows), poultry (including but not limited to poultry, turkeys, ducks, quails, guinea fowl, geese, pigeons, chicks, chickens, broilers, layers, pullets, and chicks); pet animals such as cats and dogs, fish (amberjack, arapaima, barbu, bass, bluefish, boca chick, bream, bullhead, cachamas, carp, catfish, cattla, milkfish, char, cichlid, cobia, cod, crappie, sea bream, drum, eel, goby, goldfish, gourami, grouper, guapote, 18. The method of any one of claims 1 to 17, wherein the fish is a fish such as halibut, java, rabeo, rae, loach, mackerel, milkfish, black mullet, pako, pearl spot, pejeray, perch, pike, koban trevally, roach, salmon, sampah, sauger, grouper, red sea bream, shiner, sleeper, snakehead, snapper, akame, sole, rabbitfish, sturgeon, sunfish, sweetfish, tench, teller, tilapia, trout, tuna, flounder, vendace, walleye, and whitefish; and crustaceans (including but not limited to shrimp and prawn).

[0140] 19. The method of any one of claims 1 to 18, wherein the animal is selected from the group of pigs (including but not limited to piglets, growers, and sows) or poultry (including but not limited to poultry, turkeys, ducks, quail, guinea fowl, geese, pigeons, chicks, chickens, broilers, layers, pullets, and chicks).

[0141] [Example] Example 1 Poultry Sampling [1. Birds and Containment Unit] Five hundred and sixty one-day-old broiler chickens were used from 1 to 21 days of age in the Metabolism Room facility of the Laboratory of Studies and Research on Production and Nutrition of Non-Ruminant Animals (LEPNAN) (Federal University of Parana, Curitiba, Brazil). Birds were housed in metabolism cages (two cages per floor) with four floors and a section of 0.98 x 0.90 x 0.50 m (length x width x height). All cages were equipped with trough feeders and nipple drinkers. A maximum / minimum thermometer was used for temperature verification.

[0142] Birds received food and water ad libitum. Lighting programs and appropriate room temperature were set according to breeder guidelines by using a digital timer and an electric portable heater, respectively. Rooms were allowed to breathe naturally, controlled by opening and closing windows. Daily tasks included checking temperature, providing food, and inspecting cages for dead birds, which were removed and their weights noted to calculate mortality rates.

[0143] 2. Experimental Design A completely randomized design was performed with or without Hy-D® (Table 1), with a total of 2 treatments, 7 replicates of 40 birds each.

[0144] [Table 1]

[0145] 3. Preparation of experimental foods The diet is provided in mash form, based on corn and soybean meal, and is formulated with attention to the nutritional requirements of starter phase broilers. The diet varies according to the inclusion of Hy-D® (with or without 250 g / ton of feed).

[0146] 4. Analyzed variables On day 20, blood samples were collected from 75 randomly selected birds per group (control, HyD) for analysis of vitamin D metabolite levels measured by the dried blood spot (DBS) method.

[0147] [5. Table 3. Composition of experimental diets]

[0148] [Table 2]

[0149] [Table 3]

[0150] [6. Dried Blood Spot Analysis]

[0151] [Table 4]

[0152] Example 2 Blood Sampling Instructions 1A (poultry) - Hold the bird and either puncture a wing vein or take a sample with a syringe 1B (Pig) - The pig is restrained and the marginal ear vein located on the back of the ear is punctured and a sample is taken with a syringe. 2- Using the FTA card, carefully collect a drop of blood. Touch the blood to the FTA and place the sample within each circle. 3- Repeat the process with another animal in another field (circle). 4- Allow the sample to dry until the coloration is brown (characteristic of dried blood). 5- Accurately identify the card with animal data, for example, identify each sample with the corresponding project (customer) and animal information: age, sex, date of collection, farm, and treatment. 6- Place the samples in a plastic bag, removing as much air as possible to avoid samples not suitable for analysis.

[0153] Sampling does not require any form of processing, such as drying or homogenization, prior to sample collection using the FTA card.

[0154] Example 3 Egg Yolk Sampling Instructions 1- Crack the egg and take a sample of the yolk with a syringe or pipette. 2- Carefully collect one drop of egg yolk using the FTA card. Place a sample in each circle by touching the egg yolk to the FTA. One circle per sample. 3- Repeat the process with another sample in another field (circle) 4- Allow the sample to dry. 5- Accurately identify the card with animal data, for example, identify each sample with the corresponding project (customer) and animal information: age, sex, date of collection, farm, and treatment. 6- Place the samples in a plastic bag, removing as much air as possible to avoid samples not suitable for analysis.

[0155] Sampling does not require any form of processing, such as drying or homogenization, prior to sample collection using the FTA card.

Claims

1. A method for determining at least one analyte in the blood of an animal, comprising: i) providing a drop of blood from the animal; ii) collecting the blood provided in step i) onto a carrier; iii) drying the blood on the carrier; iv) analyzing the blood on the carrier prepared in step iii) to measure the value of at least one analyte and a method comprising the steps of:

2. v) comparing the value of the at least one analyte measured in step iv) with the level of the at least one analyte recommended in the nutritional guidelines; vi) if the value of the at least one analyte measured in step iv) is outside the range recommended in the nutritional guidelines, adapting the feed given to the animal to ensure the animal nutrition level recommended according to the nutritional guidelines The method according to claim 1, further comprising the steps of:

3. A method for increasing a group of animals of the same species and the same breed, comprising: i) providing a drop of blood from at least one member of the group; ii) collecting the blood provided in step i) onto a carrier; iii) drying the blood on the carrier; iv) analyzing the blood on the carrier prepared in step iii) to measure the value of at least one analyte; v) comparing the value of the at least one analyte measured in step iv) with the level of the at least one analyte recommended in the nutritional guidelines; vi) If the value of the at least one analyte measured in step iv) is outside the range recommended in the nutrition guidelines, adapting the feed given to the animal to ensure an animal nutrition level recommended according to the nutrition guidelines A method comprising.

4. The method according to any one of claims 1 to 3, wherein the blood is venous blood.

5. A method for determining at least one analyte in egg yolk, comprising: i) providing one drop of egg yolk; ii) collecting the egg yolk provided in step i) on a carrier; iii) drying the egg yolk on the carrier; iv) analyzing the egg yolk on the carrier prepared in step iii) to measure the value of at least one analyte in the egg yolk A method comprising.

6. v) comparing the value of the at least one analyte measured in step iv) with the level of the at least one analyte recommended in the nutrition guidelines; vi) if the value of the at least one analyte measured in step iv) is outside the range recommended in the nutrition guidelines, adapting the feed given to the animal to ensure an animal nutrition level recommended according to the nutrition guidelines The method according to claim 5, further comprising.

7. A method for increasing a group of animals of the same species and the same breed, comprising: i) providing one drop of egg yolk from the eggs of at least one member of the group; ii) collecting the egg yolk provided in step i) on a carrier; iii) drying the egg yolk on the carrier; iv) analyzing the yolk on the carrier prepared in step iii) to measure the value of at least one analyte; v) comparing the value of the at least one analyte measured in step iv) with the level of the at least one analyte recommended in the nutritional guidelines; vi) if the value of the at least one analyte measured in step iv) is outside the range recommended in the nutritional guidelines, adapting the feed given to the animal to ensure the animal nutrition level recommended according to the nutritional guidelines A method comprising.

8. The method according to any one of claims 1 to 3 and 5 to 7, wherein the carrier is a sample collection card.

9. The method according to claim 8, wherein the sample collection card is blotting paper covered with a substrate for lysing cells and purifying nucleic acids.

10. The method according to claim 9, wherein the sample collection card is Whatman (registered trademark) 903 (GE Healthcare, Piscataway, NJ, USA).

11. The method according to any one of claims 1 to 3 and 5 to 7, wherein the analysis in step iv) is performed by liquid chromatography coupled with mass spectrometry (LC-MS / MS).

12. The method according to any one of claims 1 to 3 and 5 to 7, wherein the analyte is a nutrient.

13. The method according to any one of claims 1 to 3 and 5 to 7, wherein the analyte is selected from the group consisting of fat-soluble vitamins, water-soluble vitamins, and trace minerals.

14. The method according to any one of claims 1 to 3 and 5 to 7, wherein the analyte is selected from 25-OH-D3, vitamin A, and / or vitamin E.

15. The method according to any one of claims 1 to 3 and 5 to 7, wherein the analyte is a biomarker.

16. The method according to any one of claims 1 to 3 and 5 to 7, wherein the analyte is selected from the group consisting of albumin, calcium, carotenoid, creatine kinase, globulin, glucose, hemoglobin, phosphorus, potassium, sodium, total carbon dioxide, and uric acid.

17. The method according to any one of claims 1 to 3 and 5 to 7, wherein the health factor and the performance factor are selected from the group consisting of bone strength, meat yield and eggshell strength, milk fever potential, hatchability, embryo viability, intestinal health, nutrient absorption, skeletal health, electrolyte balance, liver health, oxidative stress, and inflammation.

18. The method according to any one of claims 1 to 3, 6 and 7, wherein the animal is a monogastric animal.

19. The method according to any one of claims 1 to 3, 6 and 7, wherein the animal is selected from the group consisting of pigs, poultry, pet animals, fish, and crustaceans.

20. The method according to any one of claims 1 to 3, 6 and 7, wherein the animal is selected from the group consisting of pigs or poultry.

21. The method according to claim 19, wherein the pig is selected from the group consisting of piglets, growing pigs, and sows.

22. The method according to claim 19, wherein the poultry is selected from the group consisting of turkeys, ducks, quails, pheasants, geese, pigeons, chicks, chickens, broilers, layers, young hens, and ducklings.

23. The method according to claim 19, wherein the pet animal is selected from the group consisting of cats and dogs. **Claim 24**: The method according to claim 19, wherein the fish is selected from the group consisting of amberjack, pirarucu, barb, bass, bluefish, bocachico, bream, bullhead, catfish, koi, loach, catla, mackerel, char, kawasuzume, sugire, cod, crappie, hadai, drum, eel, goby, goldfish, gourami, sea bass, guapote, ohyo, java, labeo, lai, dojo, mackerel, mackerel, blacktip shark, mudfish, bora, paco, pearlspot, pejerrey, perch, kawakamasu, kobanaji, roach, salmon, sampa, sawfish, sea bass, red sea bream, shiner, sleeper, snakehead, fuedai, akame, flounder, aygo, angel shark, manta ray, ayu, tench, terror, tilapia, trout, tuna, halibut, whitefish, wall eye, and koktima. **Claim 25**: The method according to claim 19, wherein the crustacean is selected from the group consisting of shrimp and crayfish.

Images