Method for in-ovo sex determination in an unopened avian egg

Abstract

The invention concerns the field of process engineering and relates to a method for in-ovo sex determination in an unopened avian egg, the method for example being used in commercial chicken breeding. The problem addressed by the invention is that of specifying a method by means of which in-ovo sex determination in an avian egg is made possible with a high prediction accuracy. The problem is solved by a method in which the following method steps are carried out: providing a fertilized avian egg incubated for a maximum of 9 days, detecting the position of the blood-carrying cardiovascular system in the avian egg, irradiating the avian egg, acquiring and storing absorption spectra using at least one detector and storage device, detecting and selecting the blood-specific absorption spectra and the non-blood absorption spectra and subsequently compensating the absorption spectra, performing an evaluation by determining at least one value of the oxygenation of the haemoglobin, classifying the blood-specific absorption spectra, determining the sex of the embryo.

Description

[0001] Methods for in-ovo sex determination in unopened bird eggs

[0002] Description

[0003] The invention relates to the fields of process engineering and agriculture and concerns a method for in-ovo sex determination in unopened bird eggs. The method can be used for in-ovo sex determination, for example, in commercial chicken breeding.

[0004] A strong negative correlation between egg-laying and meat production prevents the simultaneous use of domestic chickens as both egg producers and meat suppliers. As a result, specialized lines of commercial chickens have been developed through selective breeding in recent decades. For hens, the focus is on the production of high-quality eggs, while both roosters and hens are suitable for efficient, industrial meat production.

[0005] While hens from broiler lines are fattened, roosters from laying lines can currently only be raised and marketed economically to a very limited extent. This problem affects not only conventional egg production but also organic laying hen farming.

[0006] In recent years, particularly due to the efforts of legislators and animal welfare organizations, initial non-invasive methods have been developed to identify male offspring of laying hen lines at an early embryonic stage, in order to avoid, in particular, the killing of male chicks during egg production.

[0007] To ensure healthy embryonic development and to implement an economically viable solution, sex determination has been pursued for years in closed, i.e., unopened, bird eggs at the earliest possible stage of development, but also in unincubated eggs. From WO 2014 086 335 A1, a method and an arrangement for the non-invasive, non-destructive identification of molecule-specific and / or biological properties of an internal structure of a biological object of investigation through an optically opaque barrier are known, in which the object of investigation, such as an egg, is not altered, i.e., the eggshell is not opened. The egg is positioned under an array of pulse transmitters and receivers, with the electromagnetic pulses emitted by the pulse transmitters being in the spectral range of 0.01 to 1 THz.The radiation emitted by the object under investigation is captured by the receivers and analyzed by computer. This analysis is performed by comparing the recorded THz absorption spectra with reference spectra. To extract the relevant individual spectrum of the genetic material from the germinal disc, spectra of the individual components of the bird egg are calculated and subtracted from the measured integral spectrum. The model is refined within a batch using adaptive software algorithms. At the end of the measurement period for a batch, the data is evaluated and analyzed using statistical methods, taking into account the expected sex-specific characteristics.

[0008] The disadvantage is that the proportion of absorption from the germinal disc with the DNA-containing cells is very small compared to the sum of absorptions from the other egg components (yolk, egg white, eggshell).

[0009] From WO 2016 131 124 A1, a method and a device for characterizing unhatched birds are known, the characteristics obtained including the fertilization rate and sex. Data acquisition is performed using an imaging system, and data evaluation is carried out with a feature extractor and subsequent classification. However, it is known that on day 0 after fertilization, the difference between male and female eggs is based solely on the different sizes of the sex chromosomes W and Z. DNA shows no absorption in the spectral range of 900 to 1700 nm, so the classification disclosed in this method is based on undescribed differences in the egg components yolk, albumen, and eggshell. For this reason, the robustness of such a classification on day 0 is questionable due to the high natural variability of the eggs, e.g.,They are considered very susceptible to seasonal changes, changes in feed, the age of the animals, or varying shell thickness.

[0010] Furthermore, a method for in-ovo fertilization and sex determination in the closed egg is known from DE 10 2018 105 512 A1. In this method, a closed egg is positioned, illuminated, and / or scanned. An image of the closed egg is then taken, the acquired data is evaluated, and the position of the cardiovascular system within the egg is calculated. A detection unit is positioned over the calculated position of the cardiovascular system using a positioning unit, and subsequently, the blood is stimulated. The blood-specific and extraneous absorption spectra are then detected and selected, fertilization is determined, and the spectra containing extraneous information are compensated using a compensation procedure. The resulting spectra are then classified for sex determination.

[0011] Also known from DE 10 2022 121 790 A1 is a measuring system and a method for the non-invasive, preferably automated, sex determination of embryos in an egg during early embryonic development, and in particular before the seventh day of incubation, in which the egg is irradiated with electromagnetic radiation by means of at least one irradiation unit. An evaluation unit with at least one spectrometer receives the radiation transmitted by the egg and generates a spectrum of the radiation transmitted by the egg. Furthermore, the measuring system includes a data processing unit with which the spectra generated by a spectrometer are received and stored as a reference spectrum or measurement spectrum. The measurement spectrum is then evaluated using the stored reference spectrum belonging to the egg, and the sex is determined by means of an evaluation unit.

[0012] A disadvantage of the previously described methods is that the scattering of light and the influence of the shell and the non-cardiovascular components of the bird's egg make determining the sex of the egg difficult and increase the uncertainties in accurately predicting the sex of the fertilized egg. The object of the invention is to provide a method for in-ovo sex determination in the unopened bird's egg during early embryonic development, which facilitates and simplifies in-ovo sex determination, is also time- and cost-saving, and enables a high degree of predictive accuracy regarding the sex of the bird's egg.

[0013] The problem is solved by the invention specified in the claims. Advantageous embodiments of the invention are the subject of the dependent claims.

[0014] The problem according to the invention is solved with a novel method for in-ovo sex determination on the unopened bird egg, which facilitates and simplifies the in-ovo sex determination of the bird egg, which is also time- and cost-saving and enables a high prediction accuracy of the sex of the bird egg.

[0015] The technical advantages and effects are achieved through a method for in-ovo sex determination on the unopened bird egg, in which the following procedural steps are carried out:

[0016] - Providing a fertilized bird egg that has been incubated for a maximum of 9 days,

[0017] - Determining the components and location of the blood-conducting cardiovascular system in the bird egg,

[0018] - Irradiation of the bird's egg with at least one radiation device emitting electromagnetic waves,

[0019] - Acquisition and storage of absorption spectra with at least one detector and storage device,

[0020] - Detecting and selecting the blood-specific absorption spectra and the absorption spectra of foreign substances, and subsequently compensating the absorption spectra with information from foreign substances using a compensation method.

[0021] Evaluate the blood-specific absorption spectra by determining at least one hemoglobin oxygenation value; - Classify the blood-specific absorption spectra using the analyzed hemoglobin oxygenation values ​​by means of a multiple classification procedure by determining a classification value.

[0022] - Determining the sex of the embryo by comparing the classification value with determined threshold values ​​for oxygenated and deoxygenated hemoglobin.

[0023] Advantageously, the procedure is carried out between the 3rd and 6th day of incubation.

[0024] In an advantageous embodiment of the process, at least one physical and / or chemical stimulation of at least the cardiovascular system is carried out before and / or during the execution of the process, wherein the stimulation is particularly advantageously carried out by changing the ambient temperature, the air pressure and / or the oxygen content outside the bird's egg.

[0025] Advantageously, the temperature of the bird's egg is set to between 16°C and 35°C before and during the procedure.

[0026] It is advantageous if, based on the position of the pointed and blunt poles relative to each other, the closed egg is positioned essentially horizontally.

[0027] It is also advantageous if at least one broadband LED light source, a fluorescent lamp, a broadband supercontinuum white light laser, a tunable laser and / or a halogen / xenon lamp is used as the radiation device.

[0028] Advantageously, a radiation device is used that emits electromagnetic waves in the spectral range of 350nm to 900nm.

[0029] It is also advantageous if the components and location of the blood-conducting cardiovascular system in the bird egg are determined by segmenting a generated camera image. In an advantageous embodiment of the method, the detection and selection of blood-specific and / or extraneous absorption spectra is carried out with at least one spectrometer, one photodiode, one HSI camera, one MSI camera, and / or one FPA detector, together with at least one lens.

[0030] Advantageously, the compensation of the extraneous absorption spectra is realized by forming differences or quotients, whereby the difference or quotient is formed from the spectra with blood-specific and extraneous absorption spectra and from spectra with extraneous absorption spectra.

[0031] It is also advantageous to record the absorption spectra from the blood of the extraembryonic blood vessels.

[0032] In an advantageous embodiment of the method, the absorption spectra are recorded, evaluated and classified separately according to spectra of the extraembryonic blood vessels, the heart and / or the blood-conducting embryonic organs.

[0033] Advantageously, the absorption spectra of oxygenated hemoglobin and deoxygenated hemoglobin are referenced to the isosbestic point of the spectra by normalization.

[0034] In an advantageous embodiment of the method, the gradient of the oxygenation of hemoglobin and of at least one additional physiological parameter and / or biochemical substance or compound of the cardiovascular system is evaluated on different incubation days, wherein the heart rate, the temperature and / or the flow velocity of the blood, the size and / or shape of the vessels and / or the embryo and / or the blood pressure are particularly advantageously evaluated as additional physiological parameters.

[0035] The procedure can also advantageously provide for the detection, classification and / or reference of the absorption of biochemical substances or compounds that are a precursor of hemoglobin.

[0036] According to the invention, a novel method for in-ovo sex determination on the unopened and thus closed bird egg is provided, which is based in particular on the surprisingly discovered findings on the sex-specific oxygenation of hemoglobin in blood-carrying embryonic organs, tissue structures and extraembryonic blood vessels.

[0037] With the method according to the invention, spectroscopic sex determination is carried out at a very early stage of development of the unopened bird egg by analyzing absorption spectra of the embryonic blood, wherein the subsequent evaluation of the optical signals obtained from the analysis is carried out using multiple classification algorithms, taking into account and evaluating the oxygenated and deoxygenated values ​​of the hemoglobin.

[0038] Spectra obtained through absorption spectroscopic analysis, also known as absorption spectra, encompass all recorded signals reflected, non-elastically scattered, or transmitted by the bird's egg. The interconnected processes of transmission, absorption, and extinction, each of which can be associated with a specific spectrum, are collectively referred to here as the absorption spectrum.

[0039] To carry out this novel method, at least one fertilized bird egg, incubated for a maximum of 9 days, is provided, with the procedure advantageously being performed between the 3rd and 6th day of incubation. It was surprisingly discovered that, particularly between the 3rd and 6th day of incubation, the sex-specific differences in hemoglobin oxygenation become significantly more pronounced, resulting in a high degree of predictive accuracy for sex determination when analyzing and evaluating the absorption spectra.

[0040] Although differences in the degree of hemoglobin oxygenation between male and female embryos persist even after the sixth day of incubation, it has been observed that the differences in absorption spectra relating to sex-specific hemoglobin oxygenation decrease with increasing incubation time. This can lead to a decrease in the predictive accuracy of sex determination. Furthermore, it has been found that the blood cells of male and female embryos differ not only in number but also in their potential to respond to physical and / or chemical stimuli that influence sex-specific cell metabolism.For stimulation purposes, it may advantageously be provided that at least one physical and / or chemical stimulation of the bird's egg is carried out before and / or during the execution of the process, whereby the stimulation can be effected by changing the temperature, air pressure and / or oxygen content outside the bird's egg.

[0041] A particular advantage of physical and / or chemical stimulation is that the sex-specific difference in embryonic blood, which is advantageously reflected in the oxygenation level, is highlighted by variations in ambient temperature, incubation period, air pressure or composition of the ambient air, thus making the subsequent classification more robust and significantly improving the sex-specific prediction accuracy.

[0042] It has proven advantageous to adjust the temperature of the unopened bird's egg to between 16°C and 35°C shortly before and / or during the procedure. It has been observed that lowering the temperature of the unopened bird's egg compared to the usual incubation temperature of 38°C results in more pronounced differences in the oxygen saturation of the hemoglobin of male and female embryos. This leads to greater differences in oxygenated and deoxygenated hemoglobin when evaluating the absorption spectra, resulting in a further improved and more accurate method for determining the sex of the bird's egg.

[0043] In a subsequent procedural step, the location and components of the cardiovascular system in the bird egg are recorded.

[0044] In principle, spectroscopic sex determination can be performed in any egg position. However, the preferred position is horizontal, with the pointed and blunt ends of the egg positioned as horizontally as possible. In this horizontal position, the cardiovascular system is located directly beneath the eggshell, as metabolic processes create density differences between the various egg components during the first days of incubation. These differences ensure that, if the egg rotates, the embryo attached to the yolk, including the extraembryonic vessels, is always located at the highest point within the egg. This simplifies the identification of the position and components of the cardiovascular system within the egg and makes the procedure more accurate.

[0045] If the bird egg were left in the vertical incubation position, as is used in regular incubation management in a commercial hatchery, there would be an air chamber between the embryo, including the embryonic cardiovascular system, and the eggshell. This air chamber would affect the X-raying of the bird egg and would have to be taken into account and additionally compensated for as extraneous absorption spectra in the subsequent evaluation.

[0046] After the cardiovascular system has been scanned, the bird's egg is irradiated with at least one electromagnetic wave-emitting radiation device. The irradiation of the bird's egg is carried out using a radiation device with light in the visible and / or near-infrared spectrum, whereby irradiation is also understood to include X-raying the bird's egg.

[0047] Within the scope of the invention, the irradiation of the bird's egg is understood to mean the registration of the absorption spectra, preferably in transflexion mode.

[0048] In the context of the invention, the recording of the absorption spectra when the egg is X-rayed is preferably understood to be in transmission mode.

[0049] The procedure step of irradiating the bird's egg serves to locate the cardiovascular system, for example by means of an optical camera system, in particular by means of a camera.

[0050] The radiation device used according to the invention can emit light in the visible spectral range. If the optical recording systems are equipped accordingly, the radiation device can also emit light in the ultraviolet and / or infrared spectral range. Crucially, the optical camera system enables the detection of the cardiovascular system within the unopened bird egg. The radiation device can, in principle, be positioned and arranged from any side of the egg, for example, laterally or above the egg, but advantageously below the horizontally positioned egg.

[0051] Advantageously, a broadband radiation device is used, for example a thermal radiator, a broadband LED light source, fluorescent lamps, gas discharge lamps, broadband supercontinuum white light lasers or halogen lamps with filters, wherein the filters serve to avoid strong heat radiation on the positioned egg.

[0052] In principle, the cardiovascular system in a bird egg can be located directly using a high-sensitivity imaging (HSI) camera or a focal-plane array (FPA) detector. For this purpose, it is advantageous to use an HSI camera with a wide-angle lens and / or a large array detector to capture the entire egg surface where the embryo and the cardiovascular system are located. However, processing the three-dimensional data set from the HSI camera or FPA detector in the evaluation unit is more time-consuming than acquiring an RGB or monochrome image. Therefore, coupling such a vision system with an HSI camera, an MSI camera, or an FPA detector represents a technically advantageous solution.

[0053] The radiation device for irradiating the bird's egg can simultaneously be used to excite blood fluorescence or to measure transmission in the cardiovascular system. This is the case when the transmission method is used, in which the bird's egg is illuminated from below and / or laterally by the radiation device to acquire positional data of the cardiovascular system, and is thus positioned opposite or at a specific angle to the optical camera system and the detection unit. In this arrangement, the radiation device positioned below and / or to the side of the bird's egg simultaneously serves as the excitation unit.

[0054] Fluorescence or transmission light is detected using at least one detector and storage device via transmission spectroscopy and stored as a spectrum or individual photometric value. However, the detected and stored absorption spectra contain not only blood-specific information but also signals from other parts of the blood, such as those resulting from the influence of the shell (including the outer and inner shell membranes), the yolk, the air bubble, or the egg white.

[0055] For this reason, data pretreatment is required in which the blood-specific absorption spectra and the extraneous absorption spectra are detected and selected in order to subsequently eliminate the absorption spectra with extraneous information by means of a compensation procedure.

[0056] The detected absorption spectra can be divided into two groups: spectra that consist of blood-specific information and information foreign to the blood, and spectra that contain exclusively information foreign to the blood.

[0057] The spectra of the excited blood can be recorded in the spectral ranges from UV to non-IR light, but preferably in the spectral range of 350 nm to 900 nm, on the unopened bird egg. In the subsequent evaluation of the absorption spectra, the optical signals of all egg components surrounding the cardiovascular system and thus foreign to the blood, including the eggshell, are removed by optical, biochemical, and / or mathematical compensation.

[0058] The compensation for extraneous information is achieved by selecting blood spectral signals from all detected absorption spectra of the unopened bird egg, by detecting at least one, but preferably several, blood-specific absorption spectra and at least one, but preferably several, extraneous absorption spectra. From the total number of detected absorption spectra, those spectra are selected which, in addition to the extraneous absorptions of the components of the bird egg (i.e., yolk, albumen, egg membranes, and shell), also exhibit the absorption spectra of embryonic blood.

[0059] The group of unselected absorption spectra contains only spectral signals from all other components of the bird's egg, but no information about the embryonic blood and therefore, in particular, no information about the oxygenation of hemoglobin. Mathematical operations between the extraneous and blood-specific absorption spectra, such as calculating differences or ratios, enable data preprocessing and compensation for the absorption signals of the other components of the bird's egg surrounding the blood vessels, in order to obtain only unambiguous blood-specific absorption spectra for further analysis.

[0060] According to the invention, sex determination is achieved for the first time by evaluating blood-specific absorption spectra using the sex-specific oxygenation of hemoglobin. The observed higher cell division rate of male blood cells leads to a higher hemoglobin content compared to female blood cells, but also to a higher oxygen demand due to increased cell metabolism. This increased cell metabolism, through the oxygen diffusion barrier of the inner shell membrane and in the protein layer between the yolk sac and the inner shell membrane, leads to a sex-specific difference in the ratio of oxygenated to deoxygenated hemoglobin during the early embryonic phase, and particularly up to the 9th day of incubation.Only as development progresses and the subembryonic fluid under the yolk vascular network increases, does the buoyancy of the yolk ensure that the blood vessels are positioned under the inner shell membrane, thereby enabling a higher oxygen supply in particular.

[0061] The differences in the oxygenation of hemoglobin are particularly pronounced in the extraembryonic blood vessels, so that in an advantageous embodiment of the procedure, sex-specific absorption spectra can be obtained from the blood-carrying vessels.

[0062] According to the invention, blood-specific absorption spectra are classified using the analyzed oxygenation of hemoglobin by means of a multiple classification method by determining a classification value. The classification of the blood-specific, and thus also sex-specific, absorption spectra is carried out using a multiple classification method, wherein, advantageously, after the acquisition of the sex-specific absorption spectra and the data pretreatment of the blood-specific absorption spectra, a multiple classification method is carried out with at least two different methods for data pretreatment of the spectra and the method for classification associated with the respective data pretreatment.

[0063] Advantageously, supervised methods can be used for the classification of absorption spectra, in which the absorption spectra of blood from embryonic organs, extraembryonic blood vessels and the heart are recorded and classified separately.

[0064] According to the invention, the sex of the embryo is determined after classification by comparing the respective classification value with determined threshold values ​​for the oxygenated and deoxygenated hemoglobin of the blood.

[0065] The inventive method for sex determination can be further improved in its accuracy if, in addition to the hemoglobin oxygenation gradient, at least one additional physiological parameter and / or biochemical substances and / or compounds of the cardiovascular system are evaluated on different incubation days. Advantageously, the heart rate, temperature and / or blood flow velocity, the size and / or shape of the vessels and / or the embryo, and / or blood pressure can be evaluated as additional physiological parameters.

[0066] In an advantageous embodiment of the method, to further improve the predictive accuracy in sex determination, it may be provided that, in addition to the oxygenation level of the hemoglobin during absorption, at least one biochemical substance and / or compound, for example precursors of hemoglobin, is detected, classified and / or used for referencing.Such hemoglobin precursors, or other compounds not directly associated with hemoglobin or hemoglobin synthesis, such as biochemical substances or compounds that influence the oxygen affinity of hemoglobin or biochemical substances or compounds that affect cell metabolism with regard to oxygen demand (also referred to as markers in technical terminology), provide additional sex-specific information and can further improve the interpretability of the recorded and classified absorption spectra of oxygenated and deoxygenated hemoglobin. The inventive method achieves various technical effects and advantages compared to the prior art.

[0067] Surprisingly, it was found that the oxygen saturation of embryonic blood differs between the sexes, and this difference is particularly pronounced in the early stages of embryonic development. This makes it possible, for the first time, to determine the sex of an unopened bird's egg using blood-specific spectra, taking into account the sex-specific oxygenation of hemoglobin, after compensating for the other components of the egg. The method according to the invention uses not only the absolute hemoglobin content for sex determination, but also clearly evident sex-specific differences in hemoglobin oxygenation.

[0068] A significant advantage of the method according to the invention is that the sex determination is carried out on the unopened bird egg.

[0069] This method enables contactless sex determination in the early embryonic phase up to the 9th day of incubation, advantageously between the 3rd and 6th day. The egg's shell is neither opened nor is embryonic fluid or other sample material extracted. This eliminates the need for the complex opening and closing of the egg, a process known from prior art, making the method reproducible, cost-effective, and efficient. Pretreatment of the eggshell to increase transparency for subsequent examination is also unnecessary. In particular, sex determination performed on the unopened egg leaves embryonic development unaffected, resulting in improved hatching rates.

[0070] By utilizing absorption spectra derived solely from the oxygen saturation of hemoglobin, preferably from the extraembryonic blood vessels of the closed egg, intense, sex-specific optical signals of the embryonic blood are obtained. Compared to previously known methods and investigations based on the absolute hemoglobin content, this results in significantly improved signal quality with a low error rate in sex determination. Furthermore, according to the invention, high classification accuracy is achieved by compensating for strong absorptions from the egg components surrounding the blood vessels, which do not contribute to sex determination. The classification is therefore stable and independent of the natural variability of bird eggs and the variability during the incubation process.

[0071] Compared to other methods known from the prior art, a further significant advantage of the invention is that, in particular, the sex determination is carried out reliably and with very high accuracy at a very early stage, since the differences in the sex-specific absorption spectra due to the oxygenated and deoxygenated hemoglobin of the embryonic blood are clearly recognizable in contrast to the information from the other components of the bird egg during incubation.

[0072] A further advantage of the method according to the invention is that complementary sex-specific signals can be used for the classification of blood-specific absorption spectra. This has the advantage that sex-specific differences are highlighted by means of internal referencing, whereby the internal referencing is carried out, for example, by calculating the ratio of the intensity in the shorter-wavelength spectral range to the intensity in the longer-wavelength spectral range. A further advantage of the method is that the absolute amount of hemoglobin is not necessarily included in the evaluation, so that the method is independent of the size of the blood vessels being measured.

[0073] The invention will now be explained in more detail using an exemplary embodiment.

[0074] Example of implementation

[0075] After 5 days of incubation, a chicken egg is removed from the incubator, rotated 90° from its vertical position, and placed approximately 4 cm above a halogen lamp positioned below and to the side. This lamp has an adjustable power output of 0-50W and an emission spectrum in the spectral range between 400 nm and 900 nm. An aperture with an elliptical opening is located in the upper half of the egg's curvature. This aperture prevents direct exposure of the excitation light to the detector and the resulting saturation of the detector unit. The halogen lamp illuminates the egg from below and from the side, making the embryo and its blood vessel network visible. The egg is then positioned so that at least part of the embryo and the extraembryonic blood vessels are within the aperture opening.An RGB camera is used to capture the positions of the heart structure within the embryo and the extraembryonic blood vessel network. The resulting camera image is segmented, and the positional data is transferred to a custom-designed microrobot system. Subsequently, absorption spectra in the spectral range between 480 nm and 900 nm are recorded in the area of ​​the captured extraembryonic blood vessels, such as the anterior vitelline vein, the lateral vitelline veins and arteries, or the posterior vitelline vein, corresponding to the positional data of the embryonic heart. A second fiber optic sensor is positioned within the aperture, but outside all segmented blood vessel positions and the heart itself, and a spectrum is subsequently recorded. Finally, the oxygen content is measured in various regions of the selected extraembryonic blood vessel.The measured values ​​obtained from the veins and arteries of the yolk sac circulation and the heart in the embryo are compared to each other in such a way that the sex-specific difference becomes clearer. This comparison, or internal referencing, is performed, among other things, at the isosbestic point. For this purpose, differences are calculated between absorption values ​​from a spectral range where the absorption coefficient of oxygenated blood is greater than that of deoxygenated blood and the absorption values ​​from a spectral range where the absorption coefficient of oxygenated blood is less than that of deoxygenated blood. Subsequently, these differences are divided by the absorption values ​​at the wavelength of the isosbestic point where the absorption coefficient of oxygenated blood equals that of deoxygenated blood.In addition, the absorption values ​​for blood oxygenation are correlated or internally referenced using sex-specific parameters such as heart rate, blood flow, and / or the number and size of existing blood vessels. Finally, the blood-specific absorption spectra are classified using a multiple classification procedure based on the analyzed hemoglobin oxygenation.The classification of blood-specific and thus also sex-specific absorption spectra is carried out using a multiple classification procedure, whereby after the acquisition of the sex-specific absorption spectra and the data pretreatment of the blood-specific absorption spectra, a multiple classification procedure with at least two different data pretreatment procedures of the spectra and the classification procedure assigned to the respective data pretreatment is carried out and the sex of the embryo is determined.

Claims

Patent claims 1. Method for in-ovo sex determination on the unopened bird egg, in which the following procedural steps are carried out: - Providing a fertilized bird egg that has been incubated for a maximum of 9 days, - Determining the components and location of the blood-conducting cardiovascular system in the bird egg, - Irradiation of the bird's egg with at least one radiation device emitting electromagnetic waves, - Acquisition and storage of absorption spectra with at least one detector and storage device, - Detecting and selecting the blood-specific absorption spectra and the absorption spectra of foreign substances, and subsequently compensating the absorption spectra with information from foreign substances using a compensation method. - Evaluate the blood-specific absorption spectra by determining at least one value of hemoglobin oxygenation, - Classifying blood-specific absorption spectra using the analyzed hemoglobin oxygenation values ​​by means of a multiple classification procedure by determining a classification value, - Determining the sex of the embryo by comparing the classification value with determined threshold values ​​for oxygenated and deoxygenated hemoglobin.

2. The method according to claim 1, which is carried out between the 3rd and 6th day of incubation.

3. The method according to claim 1, wherein at least one physical and / or chemical stimulation of at least the cardiovascular system is performed before and / or during the execution of the method.

4. Method according to claim 3, wherein the stimulation is carried out by changing the ambient temperature, air pressure and / or oxygen content.

5. The method according to claim 1, wherein the temperature of the bird's egg is set to 16°C to 35°C before and during the execution of the method.

6. Method according to claim 1, wherein, starting from the position of the pointed and blunt poles relative to each other, the closed egg is positioned substantially horizontally.

7. Method according to claim 1, wherein at least one broadband LED light source, a fluorescent lamp, a broadband supercontinuum white light laser, a tunable laser and / or a halogen / xenon lamp is used as the radiation device.

8. Method according to claim 1, wherein a radiation device is used which emits electromagnetic waves in the spectral range from 350nm to 900nm.

9. Method according to claim 1, wherein the detection of the components and location of the blood-conducting cardiovascular system in the bird egg is carried out by segmenting a generated camera image.

10. Method according to claim 1, wherein the detection and selection of the blood-specific and / or foreign absorption spectra is carried out with at least one spectrometer, one photodiode, one HSI camera, one MSI camera and / or one FPA detector together with at least one lens.

11. Method according to claim 1, wherein the compensation of the foreign absorption spectra is realized by forming differences or quotients, wherein the difference or quotient is derived from the spectra with blood-specific and absorption spectra foreign to the blood and is formed from spectra with absorption spectra foreign to the blood.

12. Method according to claim 1, wherein absorption spectra from the blood of the extraembryonic blood vessels are recorded.

13. Method according to claim 1, wherein the absorption spectra are recorded, evaluated and classified separately according to spectra of the extraembryonic blood vessels, the heart and / or the blood-conducting embryonic organs.

14. Method according to claim 1, wherein the absorption spectra of oxygenated hemoglobin and deoxygenated hemoglobin are referenced to the isosbestic point of the spectra by normalization.

15. Method according to claim 1, wherein the gradient of oxygenation of hemoglobin and of at least one additional physiological parameter and / or biochemical substance or compound of the cardiovascular system is evaluated on different incubation days.

16. Method according to claim 15, wherein the heart rate, temperature and / or blood flow velocity, size and / or shape of the vessels and / or embryo and / or blood pressure are evaluated as additional physiological parameters.

17. Method according to claim 15, wherein the absorption of biochemical substances or compounds that are a precursor of hemoglobin is detected, classified and / or referenced.