Method and device for the raman spectroscopic, in ovo sex determination of fertilised and incubated birds' eggs

Patent metadata

Field	Value
Publication number	US-9835560-B2
Application number	US-201515317802-A
Country	US
Kind code	B2
Filing date	Jul 3, 2015
Priority date	Jul 4, 2014
Publication date	Dec 5, 2017
Grant date	Dec 5, 2017

Abstract

The invention relates to a method for the Raman spectroscopic, in ovo sex determination of fertilized 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 recognizable 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 ( 10 a ) 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 ( 3 a ), 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.

First claim

The invention claimed is:

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

2. The method according to claim 1 ,
wherein
a hole at an upwards-oriented pointed end of the egg a diameter up to 18 mm, preferably between 8 mm and 15 mm, is formed with a hole-opening device.

3. The method according to claim 1 ,
wherein
for the monitoring of the chronological progression of the incubation, white or blue and/or green light from a light source of a radiation device of the vision system is used as the light in the visible wavelength range.

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

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

6. The method according to claim 5 ,
wherein
a hole with a diameter up to 5 mm, preferably between 0.1 mm and 3 mm, is formed with a hole-creating unit.

7. The method according to claim 5 ,
wherein
for the monitoring of the chronological progression of the incubation, white or blue and/or green or blue and green light from the light source of a radiation device of a vision system is used as the light in the visible wavelength range.

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

9. The method according to claim 1 ,
wherein
after the the evaluating, a classification takes place to detect the sex of each incubated bird egg before the sex is displayed.

10. The method according to claim 1 ,
wherein
the Raman scattered radiation from blood in the blood vessels of the extra-embryonic structures and/or from blood in the blood vessels of the embryo is used for non-invasive, in ovo sex determination in the incubated bird egg.

11. The method according to claim 1 ,
wherein
the Raman spectroscopy comprises:
excitation wavelengths of the laser light from a laser source: >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 light directly with mirrors and/or with optical fibers ( 4 a );
Raman scattered radiation measurements are conducted using optical devices having a large numerical aperture, such as microscope objective lenses or a Raman fiber probe;
direct decoupling of collected Raman scattered radiation with mirrors to a spectrometer or by transport with optical fibers;
use of a spectrometer in a form of a dispersive Raman spectrometer and Fourier transform Raman spectrometer.

12. The method according to claim 1 ,
wherein
during the positioning, continuously back-guided Raman spectra are recorded and supplied to an evaluation, wherein an automatic classification of the Raman scattered radiation takes place based on a spectral fingerprint of the blood.

13. The method according to claim 1 ,
wherein
the egg is positioned on end, and a hole is created in a region of a upwards-oriented pointed end of the shell 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 13 ,
wherein
the hole in the shell of the egg ensures optical access to a blood vessel through which blood is flowing.

15. The method according to claim 1 ,
wherein
the laser beam is automatically focused onto the blood vessel.

16. The method according to claim 1 ,
wherein
an output introduced from a laser light source does not result in a local or global heating of the egg above 40° Celsius.

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

18. The method according to claim 1 ,
wherein
the Raman scattered radiation is recorded by a Raman fiber probe.

19. The method according to claim 1 ,
wherein
the recorded Raman scattered radiation is supplied to a spectrometer via fiber lines.

20. The method according to claim 1 ,
wherein
for evaluating Raman bands in the evaluation unit, a range from 500 cm −1 to 4000 cm −1 (Raman shift) is used.

21. The method according to claim 1 ,
wherein
sex-specific features—genetic fingerprint—are contained in Raman bands of nucleic acids, carbohydrates, lipids and proteins, which bands are supplied to a mathematical analysis.

22. The method according to claim 21 ,
wherein
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 the evaluation unit.

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

Assignees

• Univ Dresden Tech
• Univ Leipzig

Inventors

• Galli Roberta
• Preusse Grit
• Koch Edmund
• Steiner Gerald
• Krautwald-Junghanns Maria-Elisabeth
• Bartels Thomas

Classifications

• G01N2201/06113

  Coherent sources; lasers · CPC title

• G01N33/08

  Eggs, e.g. by candling · CPC title

• G01N2021/653

  Coherent methods [CARS] · CPC title

• G01N21/65 Primary

  Raman scattering · CPC title

• G01N33/4833

  of solid biological material, e.g. tissue samples, cell cultures (tissue in vivo A61B5/00; cell suspensions G01N33/48735) · CPC title