METHOD AND DEVICE FOR THE RAMAN SPECTROSCOPIC, IN OVO SEX DETERMINATION OF FERTILISED AND INCUBATED BIRDS' EGGS

Abstract

The invention relates to a method for the Raman spectroscopic, in ovo sex determination of fertilised and hatched birds' eggs (1), wherein the embryo, including the extra-embryonic structures, can move in the egg, and is not yet attached to the shell at the time of measuring. In addition, the following steps are carried out: monitoring the time course of the hatched egg until forming at least one recognisable blood vessel (21); creating a hole (2) in the shell in the region near to the attached bloody vessel, using a hole-generating unit; finding the blood vessel forming in the egg, using a vision system (19, 13) and a coaxial or lateral illumination with light (10a) in the visible wavelength range; positioning at least one blood vessel in the laser focus of a laser source (3), either by moving the egg or moving a lens (6) of a device (5) for introducing the laser light (3a), and detecting the Raman scattered radiation (7); registering the Raman scattered radiation of the irradiated blood vessel using the device for introducing the laser light, and for detecting the Raman scattered radiation, wherein, during the measuring process, a movement of the blood vessel out of the focus can be avoided by tracking using the vision system; evaluating the Raman scattered radiation in an evaluation unit; determining and displaying the sex of the embryo in the bird's egg.

Claims

1. A method for the Raman spectroscopic in ovo sex determination of fertilized and incubated birds' eggs (1), wherein the embryo (23), including the extra-embryonic structures, can move in the egg (1) and is not yet attached to the shell (28) at the point in time of a measurement of the Raman scattered radiation (7), characterized by the following steps: monitoring the chronological progression of the incubation until at least one identifiable blood vessel (21, 27) develops; creating a hole (2) in the shell (28) in the proximate region of the attached blood vessel (21, 27) by means of a hole-creating unit (29); finding the blood vessels (21, 27) developing in the egg (1) using a vision system (34; 19, 13) and a coaxial or lateral illumination with light (10a) in the visible wavelength range; positioning at least one blood vessel (21, 27) into the laser focus of a laser source (3), either by moving the egg (1) or by moving an objective lens (6) of a device (5) for introducing the laser light (3a) and detecting the Raman scattered radiation (7); recording the Raman scattered radiation (7) of the irradiated blood vessel (21, 27) by means of the device (5) for introducing the laser light (3a) and detecting the Raman scattered radiation (7), wherein during the measurement a movement of the blood vessel (21, 27) out of the focus can be prevented by means of tracking using the vision system (34; 19, 13); evaluating the Raman scattered radiation (7) in an evaluation unit (9); determining and displaying the sex of the embryo (23) in the bird egg (1).

2. The method according to claim 1, characterized in that a hole (2) at the upwards-oriented pointed end of the egg (1) a diameter up to 18 mm, preferably between 8 mm and 15 mm, is formed by means of a hole-opening device (29).

3. The method according to claim 1, characterized in that for the monitoring of the chronological progression of the processes during the incubation of the bird egg (1), white or blue and/or green light from the light source (10) of the radiation device (19) of the vision system (34) is used a light (10a) in the visible wavelength range.

4. The method according to claim 1 through 3, characterized in that the method for the Raman spectroscopic in ovo sex determination of fertilized and incubated birds' eggs (1) is carried out starting from the third incubation day.

5. A method for the Raman spectroscopic in ovo sex determination of fertilized and incubated birds' eggs (1), wherein the extra-embryonic structures can no longer move freely, and wherein an attachment of the chorioallantoic membrane (CAM) to the shell (28) begins, characterized by the following steps: monitoring the chronological progression of the incubation until an attachment of the chorioallantoic membrane (CAM) to the shell (28) begins and blood vessels (21, 27) are already present; identifying the blood vessel position (24, 25, 26) by means of illumination using light (10a) in the visible wavelength range from a light source (10); creating a hole (2) in the shell (28) by means of a hole-creating unit (29) in the form of a laser or by means of mechanical perforation in the proximate region of the attached blood vessel (21, 27); positioning the blood vessels (21, 27) into the laser focus of a laser source (3), either by moving the egg (1) or an objective lens (6) of a device (5) for introducing the laser light (3a) and detecting the Raman scattered radiation (7); recording the Raman scattered radiation (7) of at least one irradiated blood vessel (21, 27) and measuring the blood in the capillaries or in larger vessels inside or beneath the chorioallantoic membrane (CAM); evaluating the Raman scattered radiation (7) in an evaluation unit (9); determining and displaying the sex of the embryo (23) in the bird egg (1).

6. The method according to claim 5, characterized in that a hole (2) with a diameter up to 5 mm, preferably between 0.1 mm and 3 mm, is formed by means of a hole-creating unit (29).

7. The method according to claim 5, characterized in that for the monitoring of the chronological progression of the processes during the incubation of the bird egg (1), white or blue and/or green or blue and green light from the light source (10) of the radiation device (19) of the vision system (34) is used as light (10a) in the visible wavelength range.

8. The method according to claim 5, characterized in that the method for the Raman spectroscopic in ovo sex determination of fertilized and incubated birds' eggs (1) is carried out approximately or starting from the fifth incubation day.

9. The method according to claim 1, characterized in that after the respective measuring procedures, a classification takes place to detect the sex of each measured incubated bird egg (1) before the sex is displayed.

10. The method according to claim 1, characterized in that the Raman scattered radiation (7) from blood of extra-embryonic blood vessels (21) and/or from blood of embryonic blood vessels (27) is used for the non-invasive, in ovo sex determination in the incubated bird egg (1).

11. The method according to claim 1, characterized in that the following parameters are used for the Raman spectroscopy: excitation wavelengths of the laser light (3a) from the laser source (3): >600 nm (e.g., HeNe laser 633 nm, solid-state laser (Nd-based, e.g. Nd:YAG laser 1064 nm; VIS NIR diode laser, e.g. 785 nm); coupling of the laser excitation beam (3a) directly with mirrors and/or with optical fibers (4a); Raman scattered radiation measurements are conducted using optical devices (5) having a large numerical aperture, such as microscope objective lenses (6) or a Raman fiber probe; direct decoupling of the collected Raman scattered radiation (7) with mirrors to the spectrometer (8) or by means of transport with optical fibers (4b); use of a spectrometer (8) in the form of a dispersive Raman spectrometer and Fourier transform Raman spectrometer.

12. The method according to claim 1, characterized in that during the positioning process for the bird egg (1), continuously back-guided Raman spectra are recorded and supplied to an evaluation, wherein an automatic classification of the Raman scattered radiation (7) takes place based on the spectral fingerprint of the blood.

13. The method according to claim 1, characterized in that the egg (1) is positioned on end, and a hole (2) is created in the region of the upwards-oriented pointed end with a hole size of 10 mm at three incubating days or with a smaller hole, also in a possible horizontal position, at incubation days lasting longer than five days.

14. The method according to claim 1, characterized in that the hole (2) in the shell (28) of the egg (1) ensures optical access to a blood vessel (21, 27) through which blood is flowing.

15. The method according to claim 1, characterized in that the laser beam (3a) is automatically focused onto the blood vessel (21, 27).

16. The method according to claim 1, characterized in that the output introduced from the laser source (3) does not result in a local or global heating of the egg (1) above 40 Celsius.

17. The method according to claim 1, characterized in that the Raman scattered radiation (7) is recorded by an objective lens (6) having a large numerical aperture (NA>0.3).

18. The method according to claim 1, characterized in that the Raman scattered radiation (7) is recorded by a Raman fiber probe.

19. The method according to claim 1, characterized in that the detected Raman scattered radiation (7) is supplied to the spectrometer (8) via fiber lines (4b).

20. The method according to claim 1, characterized in that for the evaluation of the Raman bands in the evaluation unit (9), the range from 500 cm1 to 4000 cm1 (Raman shift) is used.

21. The method according to claim 1, characterized in that the sex-specific featuresgenetic fingerprintare contained in the Raman bands of the nucleic acids, carbohydrates, lipids and proteins, which bands are supplied to a mathematical analysis.

22. The method according to claim 21, characterized in that for the mathematical analysis, methods of supervised and unsupervised classification are used, wherein the sex-specific characteristics are stored in a table of characteristics of an evaluation unit (9).

23. The method according to claim 1, characterized in that the form of the detected Raman scattered radiation (7) is corrected such that background signals due to fluorescence or other scattering processes are eliminated and the spectra are normalized in a predetermined form.

24. The method according to claim 1, characterized in that non-linear coherent anti-Stokes Raman scattering spectroscopy (CARS) is used as Raman spectroscopy, wherein the CARS scattered radiation from the blood of the blood vessel (21, 27) is recorded using the same device (20) as for spontaneous linear Raman spectroscopy, with or without fiber optics (5).

25. The method according to claim 24, characterized in that the CARS signal is generated by a wide-band femtosecond laser as a laser source (3), which femtosecond laser serves as a pump laser and as a Stokes laser.

26. The method according to claim 25, characterized in that the CARS signal is generated by two lasers, by one wide-band laser and one narrow-band laser as laser sources (3) for using and evaluating a multiplex CARS spectroscopy.

27. The method according to claim 24, characterized in that the CARS spectra are used for the classification.

28. The method according to claim 24, characterized in that the resonant portion of the CARS spectrum [Im(Chi(3))] is separated from the non-resonant portion [Re(Chi(3))], and only the resonant portion is used for the classification.

29. The method according to claim 24, characterized in that for the CARS spectroscopy, a similar classification strategy is carried out as for the linear Raman spectra.

30. A device (20) for determining the for the Raman spectroscopic in ovo sex determination of fertilized and incubated birds' eggs (1) using the methods according to claim 1, comprising at least one egg-mounting unit (16); one blood vessel-positioning evaluation device (14) which is connected to the egg-mounting unit (16); one radiation device (19) with light (10a) from a light source (10) for detecting at least one blood vessel (21, 27), wherein the radiation device (19) irradiates at least one part of the egg (1); one detector (13) for the light (10b) for detecting blood vessels (21, 27), wherein the detector (13) is connected to the blood vessel-positioning evaluation device (14), wherein the radiation device (19) and the detector (13) form at least one vision system (34, 19, 13); one hole-creating unit (29) for creating a hole (2) in the shell (28) in the proximate region of the attached blood vessel (21, 27); one device (5) for introducing laser light (3a) into the created hole (2) in the egg (1) and for detecting the Raman scattered radiation (7), which device is connected to at least one laser source (3) emitting the laser light (3a), wherein the laser light (3a) is directed in a focused manner onto at least one blood vessel (21, 27), one spectrometer (8) for recording the Raman scattered radiation (7) from the blood of the blood vessel (21, 27) irradiated by the laser light (3a) via at least one line (4b), and one control unit (18) for XYZ-positioning of the device (5) onto a hole (2) created in the egg (1); one sex-determination evaluation unit (9) which is connected to the spectrometer (8) and the blood vessel-positioning evaluation device (14) and indicates the sex of the incubated bird egg (1) from the processed detected Raman scattered radiation (7) of the spectrometer (8).

31. The device according to claim 30, characterized in that the hole-creating unit (29) in the form of a laser or a device for mechanical perforation is connected to the blood vessel-positioning evaluation device (14) via line (35).

32. The device according to claim 30, characterized in that the vision system (34) is a device for identifying the position of the blood vessels (21, 27) with a detector (13).

33. The device according to claim 32, characterized in that the device (34) for identifying the position of a blood vessel (21, 27) is connected via at least one supply and signaling line (17) to the control unit (18).

34. The device according to claims 32 and 33 claim 32, characterized in that the device (34) for identifying the position of a blood vessel (21, 27) is connected to a height-adjustment device and the egg-mounting unit (16).

35. The device according to claim 32, characterized in that the device (34) for identifying the position of the blood vessel (21, 27), the height-adjustment device and the egg-mounting unit (16) are connected in the blood vessel-positioning evaluation device (14) using programming means for performing a coordination of the position of the blood vessel (21, 27).

36. The device according to claim 30, characterized in that the optical device (5) is a flexible optical fiber.

37. The device according to claim 30, characterized in that the radiation device (19) emits light (10a) in the visible wavelength range, at least green light (10a) for detecting at least one blood vessel (21, 27).

38. The device according to claim 30, characterized in that the evaluation unit (9) comprises programming means for the sex-determination evaluation of the detected Raman scattered radiation (7) and the detected CARS scattered radiation.

39. The device according to claim 30, characterized in that the device (5) for introducing the laser light (3a) and detecting the Raman scattered radiation (7) is connected to the control unit (18) which helps the laser light (3a) to focus on the identified detectable blood vessel (21, 27) in the case of a movable embryo (23).

Description

[0206] The invention is explained in greater detail by means of an exemplary embodiment with at least one drawing.

[0207] The following show:

[0208] FIG. 1 An illustration of the difference between the determination levels (compound, compound classes) from the prior art and the invention level (molecular bonds including functional groups);

[0209] FIG. 2 A schematic illustration of the device according to the invention for determining the sex of fertilized and incubated birds' eggs;

[0210] FIG. 3 A schematic illustration of a part of the device for introducing laser light into the hole of the shell, wherein

[0211] FIG. 3a shows the final part, facing the egg, for the irradiation of at least one identified blood vessel and for the detection of the Raman scattered radiation from the irradiated blood vessel, and

[0212] FIG. 3b shows an enlarged section with a view into the hole of the shell and with the possible measurement points on at least one blood vessel of the movable embryo;

[0213] FIG. 4 A spectroscopic illustration of the evaluated Raman scattered radiation intensities in a predefined wavelength range for identifying the difference between female birds' eggs and male birds' eggs, wherein the continuous line shows the mean spectrum for the female condition and the dotted line shows the mean spectrum for the male condition; and

[0214] FIG. 5 A block diagram explaining the functional principle for classifying the back-guided and detected Raman spectra, and thus for the sex determination of the bird egg embryo.

[0215] In FIG. 2, a device 20 for the Raman spectroscopic in ovo sex determination of a fertilized and incubated bird egg 1 is shown in a schematic illustration,

wherein the device 20 comprises at least [0216] one egg-mounting unit 16, on which the egg 1 is mounted; [0217] one blood vessel-positioning evaluation device 14 which is connected to the egg-mounting unit 16; [0218] one radiation device 19 with visible or green light 10a from a light source 10 for the detection of at least one blood vessel 21; [0219] one detector 13 for visible or green light 10b for the detection of blood vessels 21, wherein the detector 13 is connected to the blood vessel-positioning evaluation device 14; [0220] one hole-creating unit 29 for a hole 2 in the shell 28 at least in the proximate region of the attached blood vessel 21; [0221] one device 5 for introducing laser light 3a into the egg 1, which is connected to at least [0222] one laser source 3 emitting laser light 3a and [0223] one spectrometer 8 for recording the Raman scattered radiation 7 and [0224] one control unit 18 for XYZ-positioning of the device 5 onto the hole 2 created in the egg 1; [0225] one sex-determination evaluation unit 9 which is connected to the spectrometer 8 and the blood vessel-positioning evaluation device 14 via the control unit 18.

[0226] The device 20 comprises a device 34 for identifying the position of the blood vessels 21 inside the egg 1a vision systemin connection with the radiation device 19.

[0227] Furthermore, the device 34 includes a detector 13 in the form of a camera.

[0228] The device 34 for identifying the position of a blood vessel 21 is connected via at least one supply and signaling line 17 to the control unit 18 in the form of a coordinative positioning unit.

[0229] The device 34 for identifying the position of a blood vessel 21 is connected to a height-adjustment device (not drawn) and the egg-mounting unit 16.

[0230] The device 34 for identifying the position of the blood vessel 21, the height-adjustment device, for example inside the coordinative positioning unit 18, and the egg-mounting unit 16 can be connected in the blood vessel-positioning evaluation device 14 by programming means for a coordination and identification of the position of the blood vessel 21.

[0231] The radiation device 19 for monitoring the development of blood vessels 21, 27 can comprise the light source 10, a filter 11 and a lens 12 and can be directed at the top part of the egg 1.

[0232] The optical device 5 can be a flexible optical fiber.

[0233] Additionally, the following measures are taken such that [0234] the egg 1 preferably stands upright and the hole 2 is created in the region of the upwards-oriented pointed end by the hole-creating unit 29, wherein measurements are primarily possible at three incubation days with a hole size of 10 mm, and also horizontally at incubation days >5 days with a smaller hole; [0235] the hole 2 is created in the shell 28 using a laser 29 or using a mechanical means; [0236] the hole 2 in the shell 28 ensures optical access to the previously detected blood vessel 21 through which blood is flowing; [0237] the laser beam 3a is focused onto the blood vessel 21 inside the hole 2 and, if necessary, automatic tracking takes place; [0238] the output introduced from the laser source 3 does not result in local or global thermal damage to the egg 1 (temperature increase of <1 C. during measurement); [0239] the Raman scattered radiation 7 is recorded by an objective lens 6 having a large numerical aperture (NA>0.3); [0240] the Raman scattered radiation 7 is recorded by a Raman fiber probe 5; [0241] the recorded Raman scattered radiation 7 is supplied to a spectrometer 8 via a fiber line 4b; [0242] preferably the range from 500 cm.sup.1 to 4000 cm.sup.1 (Raman shift) is used for the evaluation of the Raman bands; [0243] the sex-specific characteristics in the Raman bands are fed to a mathematical analysis; [0244] for the mathematical analysis, methods of supervised and unsupervised classification are used, wherein the sex-specific characteristics are stored in a table of characteristics; [0245] the form of the Raman spectra is corrected such that background signals due to fluorescence or other scattering processes are eliminated and the spectra are normalized in a predetermined form; [0246] membranes and fluids of the egg can also be used for enhanced sex determination; [0247] the sex determination can be performed at any day between the start of incubation and the hatching.

[0248] In FIG. 3, schematic illustrations of a part of the device 5 for introducing laser light 3a into the hole 2 in the shell 28 are shown, wherein in FIG. 3a the final part, which faces the egg, for the irradiation of the at least one identified blood vessel 21 and for the detection of the Raman scattered radiation 7 originating from the irradiated blood vessel 21 is shown, and in FIG. 3b an enlarged section with a view into the hole 2 in the shell 28 and the possible measurement points on the at least one blood vessel 21 of the movable embryo 23 are shown.

[0249] In FIG. 3a, a laser light 3a focused on the blood vessel 21 is directed by the device 5. The blood vessel 21 is located outside the embryo 23. The Raman scattered radiation 7 produced is detected by the device 5 via the objective lens 6 and is transmitted. The embryo 23 also has blood vessels 27, which in FIG. 3b can likewise be the point of origin for measuring a Raman scattered radiation 7. In FIG. 3b, an enlarged section 22 of the hole 2 created in the egg 1 from FIG. 3a is shown. The embryo 23 (gray area in the hole 2), along with the embryonic blood vessels 27 thereof (shown in black), is located beneath the hole 2 in the shell 28 of the egg 1, as are multiple extra-embryonic blood vessels 21. Both blood vessels 21 and 27 can be points for the measurements and detection of the Raman scattered radiation 7, wherein the possible measurement points 24, 25, 26 can be detected depending on a chronological development of the blood vessels 21 and/or 27 and can be defined in the control and/or evaluation units 14, 9 by the programming means at least for comparison with stored Raman spectra.

[0250] In FIG. 4, a spectroscopic illustration of the evaluated Raman scattered radiation intensities in a predetermined wave number range of 800 cm.sup.1 to 1800 cm.sup.1 for detecting the difference between female birds' eggs and male birds' eggs is shown, wherein the continuous line shows the mean spectrum for the female condition and the dotted line shows the mean spectrum for the male condition. Between the sex curves, there are clear differences or distances in terms of the respective wave number regions between the two intensity wave number curves.

[0251] The analyses and FIG. 4 show that, for example, three to four spectral ranges may be sufficient for the sex determination, for instance, a comparison of the intensities using the absolute values can be carried out for a sex determination: for the female sex (continuous line), the intensity curve between the predetermined wave numbers is greater or less than the intensity curve for the male sex (dotted line) between the same wave numbers, as a result of which at least one intensity comparison can take place in one of the wave number ranges.

[0252] An embodiment of the light source with optical filters for the corresponding wave numbers can also be provided. In place of a costly spectrometer, at least one or more laser diode(s) or light source(s) with a wave number-related filter, in particular an interference filter related to the predefined wave numbers, can be used, wherein the wave number is the result of multiplying the inverse of the wavelength (in micrometers) by 10000.

[0253] During the sex-specific Raman scattering of the incident IR and/or NIR light, the blood from the blood vessel 21 is identified in the spectrometer 8 such that the sex of the examined bird egg 1 can be determined and displayed.

[0254] The spectroscopic evaluation takes place in the evaluation unit 9 involving mathematic classification algorithms.

[0255] In FIG. 5, the functional principle is shown including the classification of the measured Raman spectra as part of the evaluation.

[0256] For performing the spectral classification and outputting the results thereof, a multi-stage process is specified: [0257] 1. A measuring step for the Raman spectra of the egg 1 being analyzed; [0258] 2. A quality test step for the conducted measurement in order to detect the spectra and eliminate inadequately detected spectra, with which it is not possible to perform an evaluation, wherein a check whether the detected spectra satisfy the following requirements/criteria takes place: [0259] The ratio of the integral intensities of the CH.sub.2/CH.sub.3 stretching vibrations must match that of the blood (different from the surrounding tissue or fluids), or the ratio of the intensities of CH.sub.2/CH.sub.3 is <0.3. [0260] The signal-to-noise ratio (SNR) of the hemoglobin band (750 cm.sup.1) has at least a magnitude of 5:1. [0261] 3. A measurement repetition step, in case the two criteria of the preceding step are not met, [0262] 4. A data preprocessing step with [0263] a reduction of the spectral range to a wave number range between 500 cm.sup.1 and 4000 cm.sup.1, [0264] a suppression of the noise using a Savitzky-Golay filter, [0265] a correction of the baseline, for example, using a polynomial function and the correction of the offset, [0266] a normalizing of the spectra to an integral intensity by means of surface or vector normalization. [0267] 5. A spectral classification step with [0268] the application of a supervised classification. SVMsupporting vector machine, LDAlinear discriminant analysis, KNNnearest neighbor classification or the ANNartificial neural networks method can be used as classification methods. Other methods, such as non-linear processes/methods or supervising devices or SIMCA, can also be used. LDA classifies multiple spectral ranges, that is, the intensity values of said ranges. [0269] 6. If necessary, a verification step, wherein a reference spectra set with male reference spectra and female reference spectra having a known sexual correlation is required. The algorithm compares the spectrum with other spectra of the sex class and examines the similarity of the new spectrum with the known stored spectra. [0270] 7. A step for outputting the results of the determination of the respective sex of the birds' eggs, wherein the egg 1 is separated out if the minimum certainty for male of equal to or less than 45% is reached. Otherwise a female egg 1 is present which will continue to be incubated.

[0271] The method according to the invention, and the related device, use the Raman spectra preferably of blood from extra-embryonic blood vessels for the non-invasive, in ovo sex determination in the incubated bird egg 1.

[0272] The following parameters are used for the Raman spectroscopy: [0273] excitation wavelengths of the laser light 3a: >600 nm, e.g. HeNe laser 633 nm, solid-state laser (Nd-based, e.g. Nd:YAG laser 1064 nm; VIS NIR diode laser, e.g. 785 nm); [0274] coupling of the laser excitation beam 3a directly with minors and/or with optical fibers; [0275] Raman scattered radiation measurements are conducted using the optical devices 5 having a large numerical aperture, such as microscope objective lenses 6 or a Raman fiber probe 5; [0276] direct decoupling of the collected Raman scattered radiation 7 with mirrors to the spectrometer or for transport with optical fibers; [0277] application of spectrometers 8 in the form of dispersive Raman spectrometers and Fourier transform Raman spectrometers.

[0278] The following is specified for the formation of the blood vessels 21 used and the chorioallantoic membrane (CAM) developed:

[0279] The blood vessels 21 form starting at the second incubation day. At roughly the third incubation day, the embryonic blood circulates in the blood stream. Within this time span, the measurement for the sex determination is conducted.

[0280] The following two methods according to the invention are thus used: Depending on the incubation day, two methods for measuring the Raman scattered radiation exist: [0281] 1. a section of the period of the movement of the embryo 23, and [0282] 2. a section of the period of the development of the chorioallantoic membrane (CAM) and thus the attachment of the embryo 23.

[0283] First Variant:

[0284] Until approximately the fourth incubation day, the embryo and the extra-embryonic structures can move in the egg 1, that is, they are not attached to the shell 28. The measurement procedure is carried out as follows: [0285] Monitoring or observing the chronological progression of the incubation until maximally the fourth incubation day; [0286] Creation of a hole 2 in the shell 28 by means of a laser 29 or mechanical perforation, preferably at the pointed end of the shell 28 of the egg 1 with a laser 29, having diameters of up to 18 mm, preferably between 8 mm and 15 mm; [0287] Finding the blood vessels 21 by means of the vision system 34, preferably using an in-line vision camera 13 and a coaxial or lateral illumination with visible light 10a from a light source 10. The visible light 10a can be white light, but the contrast is improved with blue and/or green light. The vision camera 13 records the light 10b. [0288] Positioning the blood vessels 21 into the laser focus of the laser source 3, either by moving the egg 1 or the objective lens 6. [0289] Recording the Raman scattered radiation 7, wherein during the measurement a possible movement of the blood vessel 21 out of the focus can be prevented by means of real-time tracking of the blood vessels 21 through monitoring using the vision system 34.

[0290] Preferably, a measurement occurs at the third incubation day.

[0291] Second Variant:

[0292] Starting from the fifth incubation date, the extra-embryonic structures can no longer move freely, that is, the attachment of the chorioallantoic membrane (CAM) to the shell 28 is completed. From this point of incubation on, there is a variant for the measuring procedure which is carried out as follows: [0293] monitoring or observing the chronological progression of the incubation from the fifth incubation day on; [0294] identifying the position 25, 26 of the blood vessels 21 or the position 24 of the blood vessels 27 according to FIG. 3, 3a by means of illumination with white or blue or green or blue and green light 10a; [0295] creating a hole 2 in the shell 28 in the region of the identified blood vessel 21, 27 by means of a laser 29 or mechanical perforation, preferably with a laser, wherein the holes 2 are formed with diameters up to 5 mm, preferably between 0.1 mm and 3 mm; [0296] positioning the blood vessels 21 into the laser focus, either by moving the egg 1 or moving the objective lens 6; [0297] recording the Raman scattered radiation 7 in the blood vessels 21, that is, a measurement of the blood in the capillaries or in larger blood vessels inside or beneath the chorioallantoic membrane (CAM) takes place.

[0298] After the respective measuring procedures, a classification takes place in the evaluation unit 9 to detect the sex of each measured incubated bird egg.

[0299] Instead of conventional linear Raman spectroscopy, non-linear CARS spectroscopy can be used for the sex determination according to the invention. Coherent anti-Stokes Raman scattering (CARS) is part of non-linear Raman scattering. With CARS spectroscopy, the same molecular vibrations are analyzed, though the difference from (linear) Raman spectroscopy is in the specific manner of excitation. In CARS spectroscopy, a multi-photon process is used to excite the molecular vibrations, and a coherent signal is generated. As a result, with CARS a signal is obtained which is several orders of magnitude stronger than that of spontaneous Raman emission (factor of 10.sup.5), which leads to a shorter measurement time. Furthermore, the CARS signal is blue-shifted compared to the excitation wavelength and is therefore free of fluorescence.

[0300] The excitation occurs by means of ultrafast NIR lasers, as a result of which the penetration depth is comparable to the penetration depth in NIR Raman spectroscopy and light damage is minimized.

[0301] The sensitivity is not limited by the detection of the CARS photons, but rather by the differentiation between resonant and non-resonant portions of the CARS signal. Various methods exist for separating the resonant portion by means of special configurations for exciting/collecting the light: [0302] polarization-sensitive detection, [0303] time-resolved detection, and [0304] spectral phase control and spatial phase control.

[0305] Alternatively, the Raman spectrum can be obtained from the CARS spectrum by means of a modified Kramers-Kronig transformation or with special calculation methods, based on the assumption of maximum entropy.

[0306] To carry out the method according to CARS as disclosed by the invention, the following take place: [0307] The CARS spectrum from the blood of the blood vessel 21, 27 is detected using the same recording configuration as for spontaneous Raman spectroscopy as a function of the incubation time, with or without fiber optics; [0308] the CARS signal can be generated by a wide-band femtosecond laser as a laser source 3, which femtosecond laser serves as a pump laser and Stokes laser; [0309] however, the CARS signal can also be generated by two laser sources 3, a wide-band laser and a narrow-band laser (multiplex CARS); [0310] the CARS spectra are used for the classification. [0311] The resonant portion of the CARS spectrum [Im(Chi.sup.(3))] is separated from the non-resonant portion [Re(Chi.sup.(3))], and only the resonant portion is used for the classification.

[0312] Finally, a similar classification strategy for the CARS spectroscopy takes place as for the conventional linear Raman spectra according to the prior art.

LIST OF REFERENCE NUMERALS

[0313] 1 Egg [0314] 2 Hole in the shell [0315] 3 Laser source/laser sources [0316] 3a Laser light introduced into the hole in the shell [0317] 4a Optical glass fiber conducting laser light to the egg [0318] 4b Optical glass fiber conducting Raman scattered radiation to the spectrometer/detector [0319] 5 Device for introducing the laser light into the egg/fiber probe [0320] 6 Lens for focusing the laser light and collecting the Raman scattered radiation [0321] 7 Raman scattered radiation/Raman scattered light [0322] 8 Spectrometer with detector [0323] 9 Evaluation unit [0324] 10 Light source [0325] 10a Light with which the egg is irradiated [0326] 10b Transmitted or scattered visible light [0327] 11 Green filter [0328] 12 Lens [0329] 13 Camera for the detection of light 10b [0330] 14 Blood vessel-positioning evaluation device [0331] 15 Control line [0332] 16 XYZ-positioning unit for egg position control [0333] 17 Control lines [0334] 18 Coordinative XYZ-positioning unit/control unit [0335] 19 Radiation device [0336] 20 Device according to the invention [0337] 21 Extra-embryonic blood vessel [0338] 22 Section of a shell [0339] 23 Embryo [0340] 24 Location for detecting the Raman scattered radiation of an embryonic blood vessel 27 [0341] 25 Locations for detecting the Raman scattered radiation of an extra-embryonic blood vessel 21 [0342] 26 Locations for detecting the Raman scattered radiation of an extra-embryonic blood vessel 21 [0343] 27 Embryonic blood vessel [0344] 28 Shell [0345] 29 Hole-creating unit/laser/mechanical perforation [0346] 30 Compound field [0347] 31 Compound class field [0348] 32 Molecular bonds field [0349] 33 Detection level diagram [0350] 34 Device for identifying the position of blood vessels/vision system [0351] 35 Line