Isotopic marking and identification of animals and plants
11561214 · 2023-01-24
Assignee
Inventors
Cpc classification
International classification
Abstract
The invention relates to an isotopic identification method making it possible, where appropriate, to link a livestock animal or animal product to a specific farm, or a plant or a plant product to a farm, by analyzing the concentration of ratios or stable isotopes, and comparing with isotopic codes previously generated in a unique fashion for a set of frames. The invention also relates to a method which makes it possible to impose a unique code on the animals of a farm or on the plants of a farm, a computer making it possible to store the unique codes generated in memory, to generate unique codes for new farms and to perform comparisons.
Claims
1. A method of imposing a unique code specific to animals of a farm or a subset of a farm, or to plants of a farm or a field, or to products from said animals or plants, said method comprising: i- determinin the concentration of chemical elements and stable isotopes of several chemical elements in water and/or in food used in the farm and/or in flesh, or in skin and/or bones of said farm animals of at least one farming cycle in advance of 2i below, or in the spray water for the growing and/or in the plant from at least one growing cycle in advance of 2i below, said concentration of chemical elements and stable isotopes of several elements constituting a basal geochemical signature (BGS) of the farm or the farm subset, or of the farm or the field; and 2i- selecting several elements having stable isotopes among those present in the BGS providing said animals or plants from this cycle, or animals or plants of another farming or growing cycle with an isotopic food comprising a determined concentration of stable isotopes of the selected chemical elements, said concentration being calculated while taking account of the accumulation rate of said isotopes in said animals or plants in order to impart, at the moment of the slaughter of the animal or harvest of the plant and in light of the BGS of the farm or the crop, a unique code to said animals or plants from this farm, or said crop.
2. The method according to claim 1, wherein i further comprises analyzing in advance of 2i the ratios of stable isotopes of several chemical elements the flesh, skin, and/or bones of said animals from the farm, or in the tissues of plants, and/or analyzing the ratios of stable isotopes of several chemical elements in the water and food used for consumption by the animals or plants, and obtaining based on said analysis the BGS of the farm or the farm subset.
3. The method according to claim 1, wherein 2i further comprises providing the animals with the isotopic food, during a determined period so as to obtain, at the time of slaughter, animals having acquired the unique code specific to the farm or the farm subset.
4. The method according to claim 1, further comprising after i and 2i, during a farming or growing cycle, analyzing at least once the water and/or food in order to detect a potential variation in the concentration of the stable isotopes of the selected chemical elements.
5. The method according to claim 1, futher comprising using an electronic computer in which are stored (a) the unique codes specific to animal farms or farm subsets, or to other farms or fields, these unique codes having been previously determined and recorded, and/or (b) said BGS of the farm or farm subset, or the farm or field.
6. The method according to claim 5, wherein the computer stores the data of said animals of the farm or subset, or said plants, as a function of the farming and resepctive growing conditions, and said computer comprises a computer program or an algorithm so as to establish a correlation between a variation in concertation of isotope elements and the feeding diet with the isotopic food, in order to obtain a unique code of the time of slaughter or harvest.
7. The method according to claim 5, wherein the computer is equipped with a computer program or an algorithm so as to allow, from said BGS of the farm or its subset or of the farm or field, and knowledge of the unique codes specific to said other animal farms or farm subsets, or said other farms or fields, to compute and propose to a farmer which isotopes and which amounts must be added in order to define the isotropic food and which diet with aid isotropic food in order to deliver said food to said animals from the farm or farm subset so that said animals have the unique code specific to the moment of the slaughter, and to respectively feed the plants so that said plants also have the unique code specific to the moment of the harvest.
8. The method according to claim 1, wherein the unique code integrates: an isotopic signature of several rare elements, say at least 5 of the following elements: La, Ce, Pr, Nd, Pm, Sm, Eu, Dg, Tb, Dy, Ho, Er, Tm, Yb, Lu, these rare elements being associated with the geographical location of the farm or of the farm or field, at the continent, country or region level, an isotopic signature of at least 5 of the elements Li, Be, B, F, Na, Mg, Al, P, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, As, Se, Rb, Sr, Y, Zr, Nb, Mo, Rh, Pd, Ag, Cd, Sn, Sb, Te, I, Ba, Hf, Ta, W, Re, Ir, Hg, Ti, Pb, Si, the latter being related to the identity of the animal farm or its subset, or the farm or field, and an isotropic signature of the elements C, O, N, H, S.
9. The method according to claim 1, comprising (a) feeding the animals of the farm or subset, with (solid and/or liquid) isotopic food determined for this farm or subset, or (b) spraying the plants of the farm or field with an isotopic water determined for this farm or field, including determined concentrations of stable isotopes of the following chemical elements: at least 5 of the following elements: Li, Be, B, F, Na, Mg, Al, P, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, As, Se, Rb, Sr, Y, Zr, Nb, Mo, Rh, Pd, Ag, Cd, Sn, Sb, Te, I, Ba, Hf, Ta, W, Re, Ir, Hg, Ti, Pb, Si, at least of the following elements: La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu and, C, O, N, H, S, said unique code being achieved at the moment of the slaughter of the animal, respectively of the harvest of the plants.
10. The method according to claim 1, wherein i further comprises measuring C, H, O, N, S elements or their isotopes in the food and/or water for each farming or growing cycle.
11. The method according to claim 10, wherein, the unique code comprises natural ratios of elements or isotopes of elements from the farm or crop, coming from the soil, water and food, and imposes concentrations or ratios of other elements or their isotopes, which imposed concentrations or ratios are the result of the elements or isotopes brought by the water and/or food over a sub-period of the farming or growing period.
12. The method according to claim 11, wherein the unique code comprises natural ratios of isotopes of at least 5 of the elements Sr, B, Li, Ca, Na, Mg, K, F, P, Cl, As, Pb, Cd and all of the elements C, H, O, N, S.
13. The method according to claim 11, wherein the unique code comprises imposed isotope concentrations or ratios for at least 3 elements selected from the group consisting of Be, Al, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, Se, Rb, Y, Zr, Nb, Mo, Rh, Pd, Ag, Sn, Sb, Te, I, Ba, Hf, Ta, W, Re, Ir, Hg, Ti, Si.
Description
(1) The invention will now be described in more detail using embodiments taken as non-limiting examples and in reference to the appended drawing.
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EXAMPLES
Example 1: Description of a Computer Assembly for Managing and Defining Isotopic Codes
(7) The assembly 1 includes a programmable electronic computer 2, provided with a programmable logic unit 3, an information recording medium 4 and a data exchange interface 5 connected between them by an internal data bus. The electronic computer 2 here also includes a man-machine interface 6.
(8) The unit 3 for example includes a microprocessor or a programmable microcontroller. The medium 4 here includes a memory module, for example using FLASH or EEPROM technology, or a magnetic hard drive. The support 4 contains software instructions suitable for carrying out steps of the method of
(9) The man-machine interface 6 here includes a display screen, a data entry tool such as a keyboard and a speaker. In a variant, the man-machine interface 6 can be made differently.
(10) For example, the electronic computer 2 is a microcomputer or a mobile communication device, such as a tablet or telephone. It can also be a remote computer server, accessible through the Internet or a dedicated computer network. In this case, the interface 6 can be omitted and replaced by a dedicated communication interface, for example a computer, a communication device such as a tablet or a television, which performs the same functions as this interface 6 but which is physically separate from the electronic computer 2.
(11) The computer 2 is in particular programmed to implement a predefined model M, for example owing to executable instructions stored in the medium 4.
(12) The model M in particular makes it possible to impose, on the animals or plants, a unique isotopic code specific to a farm or farm subset, respectively farm or field, and optionally even finer granularity levels (e.g., species or varieties, production type, optionally lot), this code being based on the nature, the concentrations or the ratios of stable isotopes of chemical elements. The model (M) further makes it possible, if applicable, to couple a farmed animal or an animal product with a determined farm or farm subset, or a plant or plant product with a determined farm or field, by analyzing concentrations or ratios of stable isotopes, making it possible to determine a concentration or ratio profile of these stable isotopes, in particular by mass spectrometry, and to compare with the unique codes recorded in the model (M).
(13) The data used by the model M can be stored in the medium 4 and/or be stored in a dedicated database accessible by the computer 2.
(14) For example, the interface 5 is suitable for acquiring input data, for example in the form of digital or analog signals or in the form of data structures, such as rate of accumulation values TA and/or measurements of stable isotope concentrations C2 and/or ratios R2. These data can also be transmitted to the computer 2 by means of the interface 6.
(15)
(16) The TA is known or it can be computed in step 102 by feeding the animals or the plants over a cycle with determined ratios of the stable isotopes of the selected elements, then slaughter or harvest, collecting the flesh and analysis by MS. The data are sent to the computer 2, for example by means of the interface 6.
(17) The computer has the unique codes in memory that have been generated for other farms; this knowledge is identified during a step 103 in
(18) By increasing the data obtained during steps 101, 102 and 103, the computer generates, during a step 104, a recipe for isotopic food and a dietary regimen that will make it possible to obtain, on this farm, animals or plants having the unique code at the time of slaughter or harvest.
(19) The dietary regimen can be tested and the data kept in the computer 2, for a correlation between this regimen and the obtainment of a ratio of stable isotopes of an element at the time of the slaughter or harvest. Adjustments (in particular in terms of content) can be made in order to obtain usable ratios of isotopes, that is to say, with significant differences measurable by MS at the time of slaughter or harvest.
(20) The computer generates the composition of the isotopic food and/or the dietary regimen, the user being able to access it for example from the interface 6.
(21) In a variant, the dietary regimen can have been determined in advance, and the computer indicates the composition of the isotopic food to the user.
Example 2: Application to a Chicken Farm
(22) Lifecycle of a chicken on the farm: 10 day old chick, 1.sup.st and second growth phases 21 days, maturity as of the 32.sup.nd day and slaughter at 45 days, weight between 1.8 and 2.3 kg. The food is specific to each of the 4 phases. The chicks consume an average of 3.5 liters of water over their entire lifetime. The farming cycles follow one another during farming.
(23) Reference will be made to
(24) The BGS of the farm is determined in step 101, and the data are sent to the computer 2, for example by means of the interface 6. Drinking water and solid foods are collected, then analyzed. The ratios of stable isotopes of the following elements (it was possible to determine their presence on the farm, for example by analysis by mass spectrometry (MS) on the water, food, soil, flesh, feathers, bones and/or feet): these 26 elements: Li, Be, B, F, Na, Mg, Al, Ca, Cr, Mn, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Mo, Rh, Pd, Ag, Cd, Te, Ba, Ti, Pb, Si, these 15 rare earths: La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu.
(25) The MS measurements in the invention in general, and in this example in particular, can be done using the available methods, in particular: Inductively Coupled Plasma Mass Spectrometry (ICP-MS), Multicollector-Inductively Coupled Plasma Mass Spectrometer (MC-ICPMS), Isotope-Ratio Mass Spectrometry (IRMS), Laser Ablation (LA-ICP/MS), Laser Induced Breakdown Spectroscopy (LIBS).
(26) The TA is known or it can be computed in step 102 by feeding the animals over a cycle with determined ratios of the stable isotopes of the indicated elements, then slaughter, collecting the flesh and analysis by MS. The data are sent to the computer 2, for example by means of the interface 6.
(27) The computer has the unique codes in memory that have been generated for other farms; this knowledge is identified during a step 103 in
(28) By increasing the data obtained during steps 101, 102 and 103, the computer generates, during a step 104, a recipe for isotopic food and a dietary regimen that will make it possible to obtain, on this farm, hens having the unique code at the time of slaughter.
(29) The dietary regimen can be tested and the data kept in the computer 2, for a correlation between this regimen and the obtainment of a ratio of stable isotopes of an element at the time of the slaughter at 45 days. Adjustments (in particular in terms of content) can be made in order to obtain usable ratios of isotopes, that is to say, with significant differences measurable by MS at the time of slaughter at about 35 days.
(30) The computer generates the composition of the isotopic food and/or the dietary regimen, the user being able to access it for example from the interface 6.
(31) In a variant, the dietary regimen can have been determined in advance, and the computer indicates the composition of the isotopic food to the user.
(32) For the hens, it is thus possible to define a diet in which the isotopic food constitutes the solid and liquid food for the week leading up to slaughter, step 105.
Example 3: As an Example, on a Chicken, One has
(33) Added .sup.86Sr to the drinking water over the entire cycle of the animal Added .sup.66Zn during the 14 days leading up to slaughter
(34) The MS measurements yielded the following:
(35) TABLE-US-00001 Isotopes .sup.84Sr .sup.86Sr .sup.87Sr .sup.88Sr Natural 0.5574 9.8566 7.0015 82.5845 abundance in % Isotopes .sup.64Zn .sup.66Zn .sup.67Zn .sup.68Zn .sup.70Zn Natural 48.63 27.90 4.10 18.75 0.62 abundance in %
Example 4: Model Marking on Several Farms Using Different Isotope Ratios
(36) There are 6 farms, farms A, B, C and X with 90,000 hens per cycle, farm D with 300,000 hens per cycle, farm E with 120,000 hens per cycle.
(37) It has been chosen to vary the isotope content levels according to the following table.
(38) TABLE-US-00002 Farm isotopes A .sup.68Zn/ B .sup.68Zn/ C .sup.68Zn/ D .sup.68Zn/ E .sup.68Zn/ X .sup.68Zn/ .sup.57Fe/.sup.95Mo .sup.58Fe/.sup.95Mo .sup.57Fe/.sup.95Zr .sup.58Fe/.sup.87Rb .sup.58Fe/.sup.95Mo .sup.57Fe/.sup.46Ti Content mg/1 0.344/0.049/ 0.344/0.0065/ 0.344/0.0049/ 0.344/0.0065/ 0.344/0.0065/ 0.344/0.0049/ production 0.0044 0.0044 0.00138 0.072 0.00462 0.00528 cycle, isotopic beverage for 10 days
(39) This amounts to producing, for 8 farming cycles per year, in kg:
(40) TABLE-US-00003 .sup.68Zn .sup.57Fe .sup.58Fe .sup.95Mo .sup.91Zr .sup.87Rb .sup.46Ti 2.41488 0.11907 0.029835 0.012118 0.001125 0.1944 01004277
Example 5: Isotopic Identification of Animals or Plants
(41) This example illustrates the method making it possible to determine whether an animal or plant X comes from a farm belonging to the tracking and identification method according to the invention, and determining its precise origin, namely which farm in the world.
(42) Reference will be made to
(43) A sample of the animal or plant is collected in 201, and subject to MS analyses of all of the elements and their stable isotopes in 202. In 203, the user enters the data from the profile obtained in the computer 2 (or an interface with the mass spectrometer does so automatically, with or without a wired connection), the latter looking in its information recording medium 4 for whether a recorded profile (unique code) is found in the obtained profile, step 204. If there is no correlation (205), the user is informed in 206 that the tested animal or plant does not come from a farm monitored and conditioned according to the invention. If there is a correlation (207), in 208 the computer supplies the user with the precise identity of the farm from which the animal or plant comes.
Example 6: Isotropic Identification of Hens
(44) This example illustrates the method making it possible to determine whether a hen X comes from a farm belonging to the tracking and identification method according to the invention, and determining its precise origin, namely which farm in the world.
(45) Reference will be made to
(46) A sample of flesh is collected in 201, and subject to MS analyses of all of the elements and their stable isotopes in 202. In 203, the user enters the data from the profile obtained in the computer 2 (or an interface with the mass spectrometer does so automatically, with or without a wired connection), the latter looking in its information recording medium 4 for whether a recorded profile (unique code) is found in the obtained profile, step 204. If there is no correlation (205), the user is informed in 206 that the tested animal does not come from a farm monitored and conditioned according to the invention. If there is a correlation (207), in 208 the computer supplies the user with the precise identity of the farm from which the animal comes.
(47) The example relates to a whole animal from which a sample has been collected. This animal can for example have been collected from a stall, or a piece of packaged meat sold in pieces can also have been collected. The method also applies in the same way to another animal or to a plant.
Example 7: Internal Marketing of a Unique Code Through the Use of Stable Isotopes of Industrial Broil Chickens
(48) The internal unique code corresponds to the ingestion of isotopic markers by industrial broil chickens. This unique code thus corresponds to a controlled variation of the markers in the entire body of the chicken.
(49) These variations are imposed by the quantity of markers ingested by the chicken. These quantities, for a same unique code, must be adjusted as a function of certain environmental criteria: The farming conditions of the industrial broil chickens The ingestion route of the markers The metabolization of these markers by the body of the industrial broil chickens
(50) Under industrial conditions, the broil chickens are raised in a closed building, heated to between 30 and 20° C., and under artificial light for 23 to 18 hours. The chicks arriving in the building are raised until the age of 35 to 42 days to an average weight between 1.8 and 2.2 kg. The information is compiled in Table 1:
(51) TABLE-US-00004 TABLE 1 Farming conditions of industrial broil chickens of strain ROSS 308 (Manual management of ROSS broil chickens, Aviagen, 2010) Average Cumulative Cumulative live Brightness food water weight/ Cycle Temperature (hours/ consumption consumption chicken (days) (° C.) day) (g) (mL) (g) 1 30 23 13 28 57 6 27 23 131 275 160 15 24 20-18 613 1124 535 21 22 20-18 1183 2055 929 27 20 20-18 1968 3357 1414 35 20 20-18 3322 5716 2144 42 20 20-18 4741 8213 2809
(52) The ingestion route of the markers can be through water or food. In each of the cases, the quantity of markers to be contributed is computed as a function of the concentration of the markers initially present in the water and food. Another important factor to compute the quantity of markers to be ingested is the metabolization/absorption by the body of the chicken.
(53) Let us take the example of the case of zinc as marker for fixed conditions. It will be added for 10 days, from the 24.sup.th to the 33.sup.rd days, through the drinking water or the food. The chicken will eat about 1.5 kg of food, the zinc concentration of which is 85 mg/kg. It will drink about 2.6 L of water, the zinc concentration of which is about 9 μg/L. The total quantity of zinc ingested during this period will be 127.73 mg.
(54) Zinc has 5 stable isotopes, the natural abundances of which are listed in Table 2. Let us choose as marker to add zinc 68, the natural abundance of which is 18.75%. The total quantity of zinc 68 contributed through the food and water is therefore 23.95 mg (18.75%*127.7 mg). The quantity to be added depends on the selected target value: in this example, it is 10% of the ratio .sup.68Zn/.sup.64Zn. It is computed according to equation (1) as a function of the abundances of each isotope.
(55) In the case of a complete homogenization of the natural zinc and the marker, a modified average abundance of zinc 68 of 18.907% will then be necessary for an addition of 0.25 mg spread out over ten days. In the case where the marker is more or less well metabolized by the chicken than the natural zinc, the quantity of markers to be added will be recalculated downward or upward, respectively.
(56) TABLE-US-00005 TABLE 2 Natural abundances of the isotopes of zinc. Isotopes of zinc 64 66 67 68 70 Natural abundances 48.63% 27.90% 4.10% 18.75% 0.62%
(57)
Example 8: Test on a Chicken Farm
(58) The isotopic marking of a quantity of chickens was tested, grouped into different lots. Our experiment consisted of adding isotopic markers into the drinking water and/or the food of these chickens so that they are marked in vivo. The concentrations and the marking periods (cycles) varied for the different lots as a function of the applied test scenarios. The chickens were next killed, cut into pieces and analyzed for compositions in terms of concentrations and isotopes done in order to quantify and evaluate the marking. This made it possible to show the metabolization phase of the markers in the body of the chickens. The presence and the durability of the markers were also studied by analyzing organic samples. Identical farming conditions for the different lots (excluding markers) Closed environment (battery on hard soil type) Chickens of fast-growing breed (ROSS 308) Food produced all at once for the different lots (homogeneity) Lifetime of the chickens from 35 to 42 days (1.8 to 2.2 kg)
(59) 1. Farming Conditions
(60) a. The Hen House
(61) The henhouse is made in a garden shed, in order to simulate intensive farming conditions. Four enclosures were built in order to be able to conduct four tests at once. Each enclosure measures 0.72 m.sup.2 (0.8 m*0.9 m). According to the laws on intensive farming, it is possible to place 12 to 15 chickens/m.sup.2, weighing 2.2 to 1.8 kg each, respectively. It is therefore possible to place up to 8 chickens per enclosure. Let us consider a maximum of 5 chickens per half-enclosure in order to account for the well-being of the animals.
(62) b. Management of the Chicks and Chickens
(63) The fast-growing ROSS 308 chicks come from Couvoir Duc, Quartier des Blaches, Crest, 26400. They are available with chick sexing.
(64) The chicks are raised in an enclosure on the ground. Boxes are placed on the ground and the bedding is made up of wood shavings. It is replaced every other day for sanitary reasons. The enclosure is equipped with a water trough suitable for chicks (and chickens) and a feed trough. The necessary heat is produced by an infrared bulb suitable for poultry. The chicks need a temperature around 35° C. for the first few days, which should decrease to around 20° C. (Table 3).
(65) The chickens are next distributed into their respective enclosures several days before the beginning of the tests. Each enclosure is equipped with a feed trough and a water trough with nipples. The ground is covered with a PVC floor and bedding made up of wood shavings. It is replaced every other day for sanitary reasons.
(66) TABLE-US-00006 TABLE 3 Evolution of the ambient temperature during cycle 1 Ideal Cycle 1-20 chicks Cycle temperature T (° C.) T (° C.) IR bulb (days) (° C.) Date morning Afternoon Mortality (day) 1 30 Aug. 29, 2017 40 yes 2 Aug. 30, 2017 28 1 yes 3 28 Aug. 31, 2017 26 yes 4 Sep. 1, 2017 27 yes 5 Sep. 2, 2017 27 yes 6 27 Sep. 3, 2017 30 1 yes 7 Sep. 4, 2017 16 1 yes 8 Sep. 5, 2017 16 yes 9 26 Sep. 6, 2017 16 1 yes 10 Sep. 7, 2017 14.5 yes 11 Sep. 8, 2017 14 yes 12 25 Sep. 9, 2017 14 yes 13 Sep. 10, 2017 13 yes 14 Sep. 11, 2017 15 yes 15 24 Sep. 12, 2017 15 yes 16 Sep. 13, 2017 14 yes 17 Sep. 14, 2017 15 yes 18 23 Sep. 15, 2017 17 no 19 Sep. 16, 2017 14 No 20 Sep. 17, 2017 14 No 21 22 Sep. 18, 2017 13 no 22 Sep. 19, 2017 No heating 23 Sep. 20, 2017 No heating 24 21 Sep. 21, 2017 No heating 25 Sep. 22, 2017 No heating 26 Sep. 23, 2017 No heating 27 20 Sep. 24, 2017 No heating 28 Sep. 25, 2017 No heating 29 Sep. 26, 2017 No heating 30 Sep. 27, 2017 No heating 31 Sep. 28, 2017 No heating 32 Sep. 29, 2017 No heating 33 Sep. 30, 2017 No heating 34 Oct. 1, 2017 No heating 35 20 Oct. 2, 2017 No heating 36 Oct. 3, 2017 No heating 37 Oct. 4, 2017 8 (marked) No heating 42 Oct. 9, 2017 4 (controls) No heating
(67) 2. Experimental Conditions
(68) During a cycle, up to 3 different marking tests and 1 control test without marking can be done. The lots can be 5 chickens each.
(69) The test parameters to be varied are: The marking duration: 10, 20, 35, 42 days, etc. The mode of administration: drinking water or food The concentration and the intensity of the markers Different breeds of chickens: fast and slow growth, etc. The choice of markers
(70) 3. Cycle 1
(71) a. Experimental Conditions of Cycle 1:
(72) First, the tests of cycle 1 are done on ROSS 308 fast-growing chickens, through the drinking water and for 10 days (24 to 34 days, Lot 1) and 15 days (19 to 34 days, Lot 2). The marked chickens were ultimately killed early in the morning on the 37.sup.th day. The “control” chickens were killed on day 42.
(73) Among the 20 chicks in the experiment, a first lot is considered to be the “control” and is not marked. A second and third lot are marked at 30 per mil (Table 4). The quantity of chickens per lot will depend on the mortality rate. The mortality is four chicks. Four chickens are distributed for each test.
(74) TABLE-US-00007 TABLE 4 Experimental conditions for Cycle 1. By default, lot 1 of each cycle will be the control lot without marking. Period Marking Chickens Nb (days) Markers Marking vector Cycle 1 - ROSS 4 24-34 .sup.57Fe, .sup.68Zn 30/1000 Drinking Lot 1 308 water Cycle 1 - ROSS 4 19-34 .sup.57Fe, .sup.68Zn 30/1000 Drinking Lot 2 308 water Cycle 1 - ROSS 4 — — None — Lot 3 308
(75) The chicken food consists of corn, soy extract feedstock cake, wheat, hulled sunflower extract feed cake, bran, soybean oil, calcium carbonate, salt, magnesium oxide, monocalcium phosphate and a premix of additives.
(76) TABLE-US-00008 TABLE 5 Composition of the food for chicks and chickens in cycle 1. Concentration Concentration Concentration Composition Formulation (photo) (cycle 1, bag 1) (cycle 1, bag 2) Guarantee Aug. 29, 2017 Sep. 25, 2017 Crude protein 19.70% 19.60% 19.60% Crude fat 2.70% 2.70% 2.70% Raw ash (M. Miner.) 5.40% 5.70% 5.70% Crude cellulose 4.20% 4.20% 4.20% Calcium 0.90% 1.05% 1.05% Phosphorus 0.55% 0.55% 0.55% Sodium 0.13% 0.15% 0.15% Methionine 4.60 g/kg 4.80 g/kg 4.80 g/kg Lysine 10.70 g/kg 10.80 g/kg 10.80 g/kg Additives (not Ratio 1.15-1.2 Ratio 1.15-1.2 guaranteed, minimum) Vitamins A 8600 UI/kg 10000 UI/kg 10000 UI/kg D3 2600 UI/kg 3000 UI/kg 3000 UI/kg E Alpha- 30 UI/kg 35 UI/kg 35 UI/kg tocopheryl acetate C 5 mg/kg 6 mg/kg 6 mg/kg Oligo-elements Iron E1 FeSO.sub.4•H.sub.2O 50 mg/kg 58 mg/kg 58 mg/kg Copper E4 CuSO.sub.4•5H.sub.2O 12 mg/kg 14 mg/kg 14 mg/kg Manganese E5 MnO 78 mg/kg 91 mg/kg 91 mg/kg Zinc ZnO 75 mg/kg 87 mg/kg 87 mg/kg Iodine KI 0.90 mg/kg 1 mg/kg 1 mg/kg Selenium Na.sub.2SeO.sub.3 0.30 mg/kg 0.35 mg/kg 0.35 mg/kg Enzymes 6-Phytase EC 3, 1, 3, 375 OTU/kg 440 OTU/kg 440 OTU/kg 26 Endo-1, 3 (4) - beta- 1500 UV/kg 1750 UV/kg 1750 UV/kg glucanase EC 3, 2, 1, 6 Endo-1, 4 - beta- 1100 UV/kg 1285 UV/kg 1285 UV/kg xylanase EC 3, 2, 1, 8 Antioxidants Propyl gallate E310 4 mg/kg 4 mg/kg 4 mg/kg BHT E321 9 mg/kg 11 mg/kg 11 mg/kg
(77) b. Formulation of the Marking of Cycle 1
(78) We have chosen iron and zinc as marking elements, with their associated isotopes: .sup.57Fe and .sup.68Zn. These elements are two oligo-elements whose content levels in food are generally between 80 and 150 μg/g and 40-120 μg/g, respectively. In the food that is given to the chickens, the minimum concentration of iron is 50 μg/g and of zinc is 75 μg/g.
(79) In order to calculate the quantity of markers to be added in the drinking water and considering a metabolization of 100%, we based ourselves on the iron and zinc concentration measured in the food of a premix producer for poultry, or 144 μg/g of iron and 120 μg/g of zinc (Table 4).
(80) For lot 2, 500 mL of parent solution was prepared in a bottle. For lot 3, 750 mL of parent solution was prepared in another bottle. Each day, a volume of these parent solutions (50 mL) is withdrawn and diluted in a volume of water.
(81) The parent solution must have a pH below 2.5 in order to avoid the precipitation of the iron. To that end, nitric acid will be added: the quantity to be added depends on the initial pH of the parent solution.
(82) The daily water consumption for 4 chickens will evolve from 730 mL to 1.44 L from the 19.sup.th to the 33.sup.rd day (calculated with a 10% margin). By diluting 50 mL of the parent solution to achieve these volumes of water, the dilution rate is between 5 and 3.3% (Tables 5 and 6). The iron and zinc concentrations in the drinking water are below the recommended values of 0.3 mg/L and 5 mg/L, respectively (Tables 6 and 7).
(83) TABLE-US-00009 TABLE 6 Quantity of markers to be added in the drinking water to have a marking of 5, 10 and 30 per mil (considering 100% metabolization) over 10 days. Marking 30/1000 Marking 30/1000 Weighing precision 24.sup.th to 34.sup.th days 19.sup.th to 34.sup.th days (Without cup) .sup.57Fe 95.5% .sup.57Fe .sup.57FeCl.sub.2 .sup.57Fe .sup.57FeCl.sub.2 1 chicken/day 15.00 μg 33.68 μg 1 chicken/10 days 150.00 μg 336.80 μg 4 chickens/day 60 μg 134.72 μg 4 chickens/10 days 600.00 μg 1347.2 μg 93.1% 4 chickens/day 60 μg 134.72 μg 4 chickens/15 days 900 μg 2020.8 μg .sup.68Zn 97.8% .sup.68Zn .sup.68ZnCl.sub.2 .sup.68Zn .sup.68ZnCl.sub.2 1 chicken/day 105 μg 214.61 μg 1 chicken/10 days 1050 μg 2146.10 μg 4 chickens/day 420 μg 858.43 μg 4 chickens/10 days 4200 μg 8584.30 μg 98.7% 4 chickens/day 420 μg 858.43 μg 4 chickens/15 days 6300 μg 12876.5 μg
(84) TABLE-US-00010 TABLE 7 Theoretical chemical data on the iron and zinc concentration in the parent solutions and drinking water (diluted) of Cycle 1. Marking 30/% 1000 Solution .sup.57FeCl.sub.2 (g) 0.0017234 Parent .sup.57FeCl.sub.2 (mol) 1.34809E−05 Parent .sup.57FeCl.sub.2 (mol/L) 2.69618E−05 Parent .sup.57FeCl.sub.2 (mg/L) <0.17 Diluted .sup.68ZnCl.sub.2 (g) 0.00766 Parent .sup.68ZnCl.sub.2 (mol) 5.51751E−05 Parent .sup.68ZnCl.sub.2 (mol/L) 0.00011035 Parent .sup.68ZnCl.sub.2 (mg/L) <0.77 Diluted
(85) TABLE-US-00011 TABLE 8 Dilution of a volume of parent solution in a volume of water during the 10 days of marking of Cycle 1 Volume of Volume of water/4 parent chickens (as close Jour Date solution as possible) Lot 1 Lot 2 Notes 19 Sun. Sep. 17, 2017 50 ml 685 g no 20 Mon. Sep. 18, 2017 50 ml 731 g no 21 Tue. Sep. 19, 2017 50 ml 777 g no 22 Wed. Sep. 20, 2017 50 ml 823 g +10 g no 23 Thu. Sep. 21, 2017 50 ml 874 g no 24 Fri. Sep. 22, 2017 50 ml 925 g 25 Sat. Sep. 23, 2017 50 ml 976 g 26 Sun. Sep. 24, 2017 50 ml 1031 g 27 Mon. Sep. 25, 2017 50 ml 1087 g 28 Tue. Sep. 26, 2017 50 ml 1142 g 29 Wed. Sep. 27, 2017 50 ml 1193 g 30 Thu. Sep. 28, 2017 50 ml 1244 g 31 Fri. Sep. 29, 2017 50 ml 1295 g 32 Sat. Sep. 30, 2017 50 ml 1341 g 33 Sun. Oct. 1, 2017 50 ml 1387 g 34 Mon. Oct. 2, 2017 — — no no 35 Tue. Oct. 3, 2017 — — no no 36 Wed. Oct. 4, 2017 — — no no Slaughter 8 chickens marked lot 1 and 2 42 Mon. Oct 9, 2017 — — Slaughter 8 control chickens Each day and for both enclosures corresponding to the “marked” chickens, the appropriate dilutions are prepared.
(86) c. Results
(87) Placement in Solution of the Markers
(88) The two parent solutions have a concentration of 3.4 mg/L of .sup.57Fe, as well as of 17.6 and 19.4 mg/L of .sup.68Zn. From these data, the potential-pH diagram of the iron was calculated to check the solubilization of the iron (
C(.sup.57Fe)=((0.17/1000)*57)/(0.685+0.05)=0.01318M.
(89) The concentration in .sup.57Fe of 0.17 mg corresponds to the quantity in 50 mL of parent solution.
(90) C.sub.1 (HNO.sub.3)=(50*0.335)/(685+50)=0.023 M. The least diluted solution.
(91) pH.sub.1=1.642
(92) C2 (HNO.sub.3)=(50*0.335)/(1387+50)=0.012 M. The most diluted solution.
(93) pH.sub.1=1.933
(94) The concentration in HNO.sub.3 of 0.335 M corresponds to 250 mL of solution 1 (pH=1)+750 ml of solution 2 (pH=0.3) having a pH of 0.475.
(95) The iron must not have precipitated in the diluted solutions given to the chickens to drink because the pH is lower than 2 and stays in the range of Fe.sup.3+.
(96) TABLE-US-00012 TABLE 9 Sampling by lot of Cycle 1 Day Food Water Parent sol. Droppings Meat/bone Bedding 1-18 ✓ ✓ ✓ 19 ✓ 20 21 22 ✓ 23 24 ✓ ✓ ✓ ✓ 25 26 ✓ 27 28 ✓ 29 30 ✓ 31 32 ✓ 33 34 35 ✓ ✓ ✓ Number 2*2 = 4 2 3 6*3 = 18 3*12 = 36
(97) d. Results of Analyses of the Obtained Markers:
(98) For the doping of the Zinc: Zn.sup.68 by comparing the abundance of the stable isotopes. As a reminder, the calculated isotopic ratios are:
(99) TABLE-US-00013 Ratios Min Max 68Zn/67Zn 4.416 4.482 68Zn/66Zn 0.645 0.665 68Zn/64Zn 0.396 0.405 64Zn/66Zn 1.605 1.661
(100) We note that the samples of marked meat have an abundance value higher than the known standard.
(101) For the doping of the Iron: Fe.sup.57 by comparing the abundance of the stable isotopes. As a reminder, the calculated isotopic ratios are:
(102) TABLE-US-00014 Ratios Min Max 57Fe/56Fe 0.0224 0.2330 58Fe/56Fe 0.3664 0.00 58Fe/57Fe 16.5406 0.00
(103) We note that the samples of marked meat have an abundance value higher than the known standard.
(104) Although the doping had been done on Zn68, the variation of the abundance is not measurable with respect to the most abundant isotope. This shows that our markers are in an infintesimal quantity and that it is important the measure the correct isotopic ratio in order to detect the markers.
(105) During the experiment, we continually tried to find the minimum quantity to be ingested by the animals (the poultry). In light of the doping and the natural abundance of the isotope that will be selected, certain isotopic ratios are more indicative of the caused isotopic variation, thus determining the unique code. This is why we must look first and foremost at the Zn68/Zn67 ratio.