Patent Application
20120318981
The invention relates to a method and apparatus for determining the sex of fertilized and not embryonated bird eggs, wherein an egg exhibits a solid egg shell, an egg yolk surrounded by one of the egg shells and further fetal membranes, a germinal disc having associated with the egg yolk and wherein a probe for measuring a spectrum is led through a hole in the egg shell guided toward the germinal disc with the germ disc cells,
comprising the steps of
It is important here for example with birds and, also reaching into economic areas, especially with poultry the determination whether to develop as individuals for example from previously fertilized eggs either males or females.
There is already described a method for sex determination of chicks by means of infrared Fourier transform spectroscopy in the publication Steiner et al. Sexing of turkey poults by Fourier transform infrared spectroscopy, Analytical and Bio-analytical Chemistry, Vol 396 (1) 2010 pages 465-470 which allows to determine the sex of six-week-old chicks.
The use of infrared spectroscopy for the determination of the sex of young birds shows that the method is fast and precise, and can be potentially used by the poultry industry to identify and select eggs with female embryonic stage or male embryonic stage for breeding purposes.
A multitude of birds, especially nestlings and farmers, veterinarians, aviculturists and ornithologists, however, the exact sex embryonic stages of birds show no external sexual characteristics. For poultry-determination is a fundamental aspect. But for the poultry industry, the knowledge is of paramount importance: fast, objective and inexpensive procedures for possible early sex determination, for example, in chickens and turkeys, are for the poultry industry important because in the preferred gender for incubation can be identified.
The Fourier transform infrared spectroscopy is used for this purpose. The method is based on that cells extracted from the growing contour springs of six weeks old, male and female turkeys are employed. The technique exploits the cellular genetic information and allows a direct determination of the sex of the bird.
One problem comprises that the effort to determine the specific gender involves many steps and therefore the time expenditure is very high.
A method for determining the sex of birds is described in the German Patent Application publication DE 10 2007 013 107 A1, wherein DNA-relevant cellular material of the sexually to be determined bird was studied with light and the molecular oscillations are measured, wherein the light generated spectrum of molecular vibrations is recorded and compared with predetermined reference spectra of gender-specific DNA structures of the to be investigated bird species. A sex assignment of the bird is rendered based on the basis of the DNA content of the cell material.
The molecular oscillations are measured here by means of application of Raman spectroscopy, or IR spectroscopy, it being possible for example, the relevant DNA cellular material can be removed from the stem of a young spring of a bird. The cell material is prepared on a support and is scanned with light.
In another sub-method the light is focused for measuring the molecular vibrations of the DNA relevant cell material from unhatched birds through the egg shell and onto the embryo or germ-disc, wherein the spectrum of the radiation generated by molecular oscillations in the egg is measured with a probe guided through the shell of the egg.
At least a microscopically small hole is drilled through the eggshell for the passage of the probe for measuring the spectrum. The light through the small opening through the eggshell is immediately focused on the germinal disc as cell material. The probe is inserted by the same or other access of small opening size, by means of the reflected spectrum and is measured by the probe recorded spectrum of the afore mentioned molecular motion in the interior of the egg.
The resulting spectral information are compared with reference data in a second step and are fed to a classification algorithm. These represent preferably statistically derived data on the bird species to be examined. The assignment of gender of the DNA material to be investigated is made from this comparison
One problem comprises that a high time expenditure is required for the insertion of a probe into pre-made holes for a very large number of bird eggs to be examined. In addition, with a focusing of the light from the probe to the germinal disc, a considerable adjustment has to be furnished for an optical imaging, wherein the focusing plane can exhibit a different position from egg to egg and no determination of the sex can be carried out therewith.
No arrangement is given how the IR spectrum of the germinal disc cells under in-ovo conditions can be taken quickly and reliably.
A spectroscopy device is described in the U.S. Pat. No. 4,835,389 A, which contains a total reflection element which is disposed at the bottom of a long probe tube that is together with the total reflection element deeply immersed in a container with liquid in order to capture with the complete surface of the crystal and immersed in the liquid and many edge side total reflections within the crystal and to receive analytical information on an optical path received and forward them to the spectroscopic device.
An immersion probe is described in U.S. Pat. No. 5,459,316 A, which probe is immersed in powder or liquid and an infrared spectroscopy is performed with it. The immersion probe consists of at least one tube, which tube exhibits a built-in attenuated total reflection ATR element operating on the basis of attenuated total reflection (ATR) at the bottom. The ATR element furnished serves as a back reflector.
A refractometric method for a long-term stable measurement of concentrations of dissolved substances and a device for performing the process are described in the publication German patent DE 100 30 927 C1, wherein a linearly polarized electromagnetic measuring radiation beam is guided by total reflection in a prism. Here the longitudinal sides and a narrow side of the prism are in contact with the solution to be measured.
Edge side multiple total reflections of the measuring radiation beam are fed to a detector after they emerge from the prism via phase shifter and analyzer to a detector, wherein the concentration is determined from the signals and from a calibration curve.
An ATR-probe for measurement of whole blood is described in the document U.S. Pat. No. 5,170,056 A, wherein measurement solutions can be measured remote from the spectrometer through a light conductor. Here, the ATR probe is a portion of a fiber optic coupled device for carrying out IR-spectroscopy. The long cylindrical ATR probe crossing the solution exhibits coupled connectors of the light guides at the two front sides, wherein the infrared radiation is passed through the ATR probe over the coupled connectors. The detected IR radiation is measured and used for parameter determination of the blood from the many attenuated edge side total reflections within the ATR probe during the passage of the radiation bundle through the ATR probe.
There are problems that can occur in that falsifications can occur in the detected spectra based on the configurations of the recited probes with the multitude of total reflections on the edge sides and that with the total reflection bundles detected no precise positioning of the crystal tip on blastoderm cells is possible. Thus, no precise determination of the sex of bird eggs can be performed.
It is therefore the object of the present invention to provide a method and an apparatus for determining the sex of fertilized and non-incubated bird's eggs, which are so designed to be suitable, that sex can be unequivocally determined quickly and reliably already in the eggs.
The object is solved by a method having the features of claim 1 and an apparatus having the features of claim 8.
The procedure for determining the sex of fertilized and non-incubated bird's eggs, wherein one egg comprises a solid egg shell, an egg yolk surrounded by the eggshell and other egg cases and an embryonic disc coordinated to the egg yolk, with a probe for the measurement of a spectrum through a hole in the egg shell through is guided toward the germinal disc with germ disc cells,
includes the following steps
The advantages include that no material must be removed in the conduct of the processing of the egg. Also, there are no foreign substances introduced for signal detection in the egg. In addition, no entry is made of energy, such as heat or light.
The automatic positioning of the optical crystal with respect to the germinal disc cells with the crystal tip or output surface is carried out until the evanescent field of the total reflection at the crystal tip or output face captures the germinal disc and the crystal is finally brought into a final disc coordinating position, in which the evanescent field enters into interaction with the germ disc cells and thus an extinction becomes registrable.
The positioning of the optical disk mapping crystal can be oriented in all directions in space possible in order to hit the germinal disc with the germinal disc cells as precisely as possible.
Continuously recirculated IR- and/or NIR spectra are recorded and fed to an evaluation during the positioning operation, wherein an automatic classification of the spectra using the spectral fingerprint, of for example, proteins, lipids and nucleic acids takes place.
In the gender-absorption of the incident IR and/or NIR light, the germ disc cells are identified based on absorption bands of nucleic acids (DNA and RNA) and other biochemical compounds such that the sex of the tested egg is determined and displayed.
The measurement can be performed with a conventional infrared spectroscopy.
Simultaneously, a plurality of bird eggs can be measured, wherein several optical crystals are disposed preferably equally spaced (array like) from each other and a plurality of optical crystals are arranged and coupled in each case with a separate optical fiber, or wherein the reflected light without fibers directly radiates onto an IR- and/or NIR-sensitive detector. In the latter case can also mirror/mirror systems can be employed for beam steering.
The inventive method is a procedure that is sufficiently precise for the poultry industry for the selection of eggs with female embryonic or male embryonic stages for breeding, egg production or fattening, wherein the killing of millions of chicks of the unwanted sex can be avoided shortly after hatching.
The apparatus for determining the sex of fertilized and non-incubated bird's eggs, wherein the egg includes a solid egg shell, a yolk surrounded by the eggshell and other egg envelopes, and germinal disc germ coordinated to the egg yolk, wherein a probe for measuring a spectrum is led through a hole of the eggshell and is guided toward the germinal disc in which the germinal disc cells, according to the procedure above,
contains according to the characterizing part of patent claim 8
The disk mapping position of the optical crystal within the egg and in the region of the germinal disc can be obtained by a hole placed into the egg shell, wherein the hole is either already present or is placed during the installation of the optical crystal into the egg shell by the optical crystal itself.
The optical crystal can be furnished with differently structured crystal end faces or crystal ends, e.g. with a planar, a round or oval output surface or may be also provided with a pointed tip crystal.
The needle-shaped optical crystal can be formed in the lower output area oriented toward the embryonic disc.
The apparatus for determining the sex of fertilized and not incub\eggs, can have coordinated a device for detecting the position of the germinal disc within the egg, which device can be either part of the apparatus or may be formed separately.
The device for detecting the position of the germinal disc in the egg can be formed in the egg above the support arm, wherein the support arm is height adjustable and pivotable. The disk mapping position of the optical crystal e.g. bird eggs largely can be adjustable above and preferably perpendicularly in the region facing the germinal disc.
The device for detecting the position of the germinal disc in the egg can have at least one power supply and a signal technical line connected to the control unit of the device.
The device for detecting the position of the germinal disc in the egg can further also be connected to the height adjustment device and the egg position support pad.
The device for determining the position of the germinal disc, the height adjustment device and the egg position support pad can be connected in the control unit by means of program-technical means for coordinating the position of the germinal disc and the disc mapping position of the optical crystal, whereby the precision target mapping position of the optical crystal in the region of the germinal disc can be fixed.
The respective optical element for guiding the beam or the returning beam guidance can be a flexible optical fiber for the IR- and/or NIR light beam.
The optical crystal can be formed cylindrical and can be supported in a metal sheath, wherein the metal sheath is in connection with the support arm.
A gold layer can be applied between the metal sheath and the surface of the optical crystal for improving the reflection and/or the total reflection.
The optical crystal can be directed to the germinal disc in its disk allocation position with its lower output surface, wherein the germinal disc is at least in a part in the evanescent field for the absorption of IR- and/or NIR light.
The optical crystal can be constructed such that it leads the IR and/or NIR light beam and that a total reflection occurs at the tip in the region of the output surface.
The optical crystal can be made of IR-transparent and simultaneously hard material such as germanium, silicon, calcium fluoride (CaF2), barium fluoride (BaF2), zinc selenide (ZnSe) or zinc sulfide (ZnS).
The optical crystal can comprise glass or sapphire for measurements with NIR light.
The optical crystal can also consist of a composite, wherein the optical crystal in view of the needle shape the tip can be laid out as a diamond in the region of the output area.
The optical crystal can be connected to the flexible optical fiber, wherein the light beam is fed through fiber both from the spectral light source for IR and/or NIR light to the optical crystal and from the optical crystal to the detector.
Conventional spectrometers with broadband thermal sources, laser diodes or light sources with filters, in particular interference filters, can be used as spectral light sources for IR and/or NIR light.
A detector with many detector elements/pixels (focal plane array detector) can be used as IR- and/or NIR-sensitive detector to simultaneously and in parallel detect spectra of many bird eggs to be tested.
The advantage of the inventive apparatus is the possibility of a parallel measurement of many birds' eggs, and is thus a rapid simultaneous determination of the specific gender of bird eggs.
Further developments and further embodiments of the method and device are specified in further dependent claims.
The invention is further illustrated by way of several embodiments with reference to several drawings.
There is shown in:
a infrared Fourier transform (FTIR) spectra of blastoderm cells of male bird eggs and of female bird eggs, wherein the arrows represent spectral regions for gender specific characterization/distinction,
b shows an enlarged section of the evaluated (FTIR) spectra of
In the following, the two figures
In
An egg positions support 15 with a locking of the avian egg 13,
A spectrometer can be used as a spectral light source 6 for IR and/or NIR light in
The disc mapping position 28 of the optical crystal 4 provided in the region 29 above the germinal disc 3 by the hole 17 introduced in the egg shell 14 is shown in
A device 16 for detecting the position 30 of the germinal disc 3 in the avian egg 13 can be preferably coordinated to the inventive device 1 in
The device 16 for detecting the position 30 of the germinal disc 3 in the avian egg 13 can through the support arm 8, which can be constructed height-adjustable and pivotable, the position, in particular the disc mapping position 28 of the optical crystal 4 in the avian egg 13 can be installed largely above in a region 29 and preferably be set perpendicular to the germinal disc 3.
The support arm 8 can also be controlled through a control line 10 led to a height adjustment device 9 for installing a hole in the egg shell 14 with jerky or abrupt executable mechanical impulses, possibly accompanied by a short rotary turn.
The device 16 for detecting the position 30 of the embryonic disc 3 in the bird egg 13 may have at least one power supply and signal technology line 25 be connected to the control unit 11, which control unit 11 is technically connected through the line 35 to the spectral light source 6 for IR and/or NIR light energy supply and signal-line.
Here, the device 16 for detecting the position 30 of the germinal disc 3 in the avian egg 13 can be connected to the height adjustment device 9 and the egg position support 15 through the control line 10.
The device 16 for detecting the position 30 of the embryonic disc 3, the height adjustment device 9 and the egg position pad 15 can be connected by way of program-technical means to a joint coordination in the control unit 11, with the precision target mapping position 28 of the optical crystal 4 above the germinal disc 3 of the avian egg 13.
The practical process of measuring bird eggs at 13 is that the egg yolks 2 through the existing chalazae (chalaze cords) can rotate so that the germinal disc 3 is located in the upper part of egg yolk 2.
It is appropriate to determine the position 30 of the germinal disk 3 before the determination of gender by means of the device 16 for detecting the position 30 of the germinal disc 3. Only then can a hole installation and a disc mapping position 28 of the optical crystal 4 with respect to the germinal disc 3 can be performed by the controllable height adjustment device 9.
The jacket framed optical crystal 4 in its disc mapping position 28 is directed in
The IR and/or NIR light guided out of the spectrometer 6 to the lower output area 27 of the crystal 4 oppositely positioned to the germinal disc 3 through the incoming guided beam 20 within the frame of beam guidance, wherein there through the beam set total reflection 31 the evanescent field 21 is formed and which evanescent field 21 depending on the absorption of IR and/or NIR light at the blastoderm cells 23 by absorption spectrally changes at the output area 27 crystalline side total-reflection optical beam path, 22 wherein the attenuated IR and/or NIR light of beam path 22 passes through the optical crystal 4 passes via the optical element 5 for guiding the beam to the detector 32 and following to the evaluation unit 7 for the spectral analysis.
The crystal tip 33 of the optical crystal 4 can for example also formed needle shaped.
An essential point during the execution of the method is the cleaning/sterilization of the crystal tip and/or of the output area 27. For authentic recordings of the spectra neither foreign substances nor organisms/germs can be entered in the following bird eggs 13. Therefore, the inventive device 1 can be used to determine the gender of a fertilized not incubated avian egg 13 and a device for cleaning and sterilization (not shown) can be coordinated, which device for cleaning and sterilization after each measurement cleans and sterilizes the crystal tip 33. The device for cleaning and sterilization can perform on the basis of a steam sterilization and/or by chemical means. It may also penetrate any liquids into the intermediate space between the optical crystal 4 and the metal shell 19 or the reflecting gold layer 18. The device for cleaning and sterilization can be supported at the height adjustment device 9, and can be supported in the way the supporting arm 8 is supported and can be activated depending on the termination of the measuring process for cleaning and sterilization of the optical crystal 4 or the crystal tip 33.
The needle-shaped or cone-shaped crystal tip 33 may also consist of a synthetic or natural diamond. Diamond material, for example, sp3 carbon does not absorb the infrared and/or NIR light, is extremely hard and very insensitive to contamination with lipids, proteins, etc. In addition, the diamond material can be cleaned quickly.
a and 4b (higher magnification) are FTIR spectra 45, 46 of embryonic disc cells 23 male birds' eggs (45) and female bird eggs (46) is shown with the arrows 41, 42, 43, 44 representative for spectral ranges representing the gender characterization. The studies show that, for example three or four spectral regions 41, 42, 43, 44 for sex determination are sufficient, for example can be performed for a determination, a comparison of the absorbances in the absolute amounts: for the female sex is the absorbance between the wave numbers vi, v 2, v3, v 4 is greater than the extinction of the male running over the same wave numbers, so that a comparison in a wave number range can at least take place.
Another possibility is to determine the difference
of the extinction E 4−E 2 (female)>E 4′−E 2′ (male), as shown in
The sex determination is based here as the vibrations of the phosphate of DNA or RNA strands.
Therefore, an embodiment of the light source 6 with optical filters of the corresponding wave numbers vi, v 2, v 3, v 4 may be provided. It can be used instead of an expensive spectrometer 6 at least one or more laser diode/n or light source/n with a wave number associated filter, in particular an interference filter based on the wave numbers vi, v 2, V 3, v 4 be used, with the wave number v of the reciprocal of the wavelength λ (in microns) is multiplied-by 10000.
The method for determining the sex of fertilized and not embryonated bird eggs 13, wherein a bird's egg 13 is a solid egg shell 14, an egg yolk 2 surrounded by the egg shell and further fetal membranes and the egg yolk 2 coordinated to the germinal disc 3, wherein each for receiving one of the germinal disc 3 modified spectrum, a probe 4 is led through a microscopic hole 17 through the egg shell 14 in the direction towards the germinal disc 3,
has the following steps
According to the invention an optical crystal is used as the probe 4, with the rapid and non-reactive receiving an infrared and/or near infrared spectrum 70, as shown in
The automatic positioning of the optical crystal 4 with the crystal tip/output surface 27 in relation to the germinal disc cells 3 is carried out until the evanescent field 21 of the total reflection 31 at the crystal tip/output surface 27 captures the germinal disc 3 and the optical crystal 4 has reached its final disc mapping position 28, at which position the evanescent field 21 interacts with the embryonic disc cells 23.
The disc mapping position 28 of the optical crystal 4 can be directed in all directions in space possible in order to hit as precisely as possible the germinal disc 3 and the embryonic disc cells 23 of the germinal disc 3.
During the positioning process of the optical crystal 4, the returned totally reflected IR and/or NIR spectra 70 are permanently recorded and fed to an evaluation, wherein an automatic classification of returned spectra 70 is performed using the spectral fingerprint in proteins, lipids and nucleic acids.
In the gender-absorption of the incident IR and/or NIR light, the embryonic disc cells 23 were identified by means of absorption bands of nucleic acids (DNA and RNA) in the detector 32 so that the gender of the tested bird egg 13 can be determined and displayed.
The spectroscopic analysis is performed in the evaluation unit 11 with the IR or NIR spectroscopy or using a Fourier-transform infrared spectroscopy, with the evaluation preferably including a mathematical classification algorithm.
The inclusion of the classification of the measured FTIR spectra 70 in the context of the analysis is shown in
A six-step process is roughly indicated for the implementation of the spectral classification and its result output:
First Step 71 of the quality testing of the measurement for detecting the spectra 70 and eliminating inadequate detected spectra, with which no evaluation can be carried out, considering to check compliance if the recorded spectra 70 meet the following requirements/criteria:
Second Step 72 of the repeated measurement, if the two criteria of step 71 are not achieved,
Third Step 73 of the data pre-treatment with
Fourth Step 74 of the spectral classification with
the use of a supported (English, supervised) classification. The LDA—linear discriminance analysis—can be used as a classification method. Other methods, such as non-linear procedure and/or methods or supporting facilities or SIMCA can be used. The LDA classifies several spectral ranges, i.e., the absorbance values of these areas. In this case, the classification is based primarily on the ratio of the phosphate vibrations of DNA and RNA. Due to the differences in the genome, male germ cells and female germ cells of embryonic disc cells can be distinguished.
Fifth Step 75 of the verification, which will require a set of reference spectra with reference spectra “male” and reference spectra “female” is required with known gender association. The algorithm compares the spectrum 70 with other spectra of the sex class and analyzes the similarity of the unknown spectrum 70 with the known spectra.
Sixth Step 76 to output the results of determining the respective gender of each bird egg 13, 130.
In
The device 100 for determining the sex of fertilized and non-incubated bird eggs 130 contains
The egg position system 150 in connection with the control unit 11 can be formed into a device for automatic individual positioning of the respective egg 130
Simultaneously with the devices 100, a plurality or array-equipped arranged many bird eggs can be measured 130, with either optical crystals are arranged in several array-like crystals 40 and each coupled to a separate optical fiber 50 or the totally reflected light without fibers 50, but with mirrors 60 is fed directly to an IR and/or NIR-sensitive array detector 32.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2010 006 161.1 | Jan 2010 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/DE11/00062 | 1/14/2011 | WO | 00 | 7/23/2012 |
