DE102007013107A1 - Bird sex determination involves testing DNA relevant cell material of bird with light, and measuring molecule oscillations, where spectrum of molecule oscillations resulting from light is detected, and is compared - Google Patents

Abstract

Bird sex determination involves testing DNA relevant cell material of the bird with light, and measuring molecule oscillations. Spectrum of the molecule oscillations resulting from the light is detected, and is compared with provided and sex specific DNA structures of the reference spectrum. A sex classification of the bird, based on the spectral comparison, is crossed on the basis of DNA content of cell material. The molecule oscillations is measured by using UV resonance Raman spectroscopy.

Description

The The invention relates to a method for determining sex of birds from the study of DNA-containing cell material, preferably from incubated eggs or from the shaft or the scabbard of bird feathers.

It is well known, a distinction of the sex of birds based on external characteristics, such as height and morphological differences, such as frontal bumps, Plumage or feather length to hit. In not uncommon but this presents difficulties or such a distinction can be made (especially in young birds, certain bird species or even at the embryonic stage) or at least not not be done reliably.

However, sexing options are also based on the study of the DNA of birds. So it is known (for example KBC Appa Rao, D. Mohan and SM Totey: Polymerase chain reaction and its applications: Special emphasis on its role in embryo sexing, Biotechnology Advances 12, 1994 341-55 ) to amplify certain DNA sequences by PCR (polymerase chain reaction) and to differentiate by gel electrophoresis.

There are numerous publications in this regard for the study on birds (for example KK De Mattos, SN Del Lama, ARC Salles, MR Francisco, A. Medaglia, AM Tomasulo, FMS Carmo, JMC Pereira and MA De Oliveira: Sex determination of parrots Amazona a. aestiva and Amazona amazonica using the polymerase chain reaction, Journal of Experimental Zoology, 281, 1998, 217-219 ; T. Minematsu, M. Sugiyama, Y. Tohma, A. Tajima and Y. Kanai: Simplified DNA extraction methods for sexing chick embryos, Journal of Poultry Science, 41, 2004, 147-154 ; L. Huynen, CD Millar and DM Lambert: A DNA test to sex ratite birds, Molecular Ecology, 11, 2002, 851-856 ; LG Bermudez-Humaran, A. Garcia-Garcia, CH Leal-Garza, VM Riojas-Valdes, G. Jaramillo-Rangel and R. Montes-de-Oca-Luna: Molecular sexing of monomorphic endangered macaw birds, Journal of Experimental Zoology, 292, 2002 677-680 ; Y. Itoh, M. Suzuki, A. Ogawa, I. Munechika, K. Murata and S. Mizuno: Identification of the wide range of Carinatae birds by PCR using primer sets EE0.6 and its related sequences , Journal of Heredity, 92, 2001, 315-321 ; US 5,876,942 ) and mammals (such as US 5,876,942 ; K. Makondo, GS Amiridis, IA Jeffcoate, PJ O'Shaughnessy, JS Boyd, C. Paterson and L. Robertson: Use of the polymerase chain reaction to sex the bovine fetus using cells recovered by ultrasound-guided fetal fluid aspiration, Animal Reproduction Science, 49, 1997, 125-133 ) known.

The DNA amplification succeeds with different primers and must be adapted to the species of bird.

There is also known a method ( WO 9749806 ), which uses sex-specific markers to differentiate the sex of birds regardless of their species.

The precision and verification of the PCR method itself are criticized in recent studies ( BC Robertson and NJ Gemmell: PCR-based sexing in conservation biology: wrong answers from an accurate methodology ?, Conservation Genetics, 7, 2006, 267-271 ).

Also known are optical methods for sex discrimination of birds by differentiation of the spring structure with UV light ( US 6,396,938 ) or tissue with NIR light ( US Pat. No. 6,493,566 B1 ). As this method involves the thickness measurement of the dermis and the subcutaneous fat layer, the results can be influenced by the nutritional status of the birds.

Nowadays, endoscopic sex determinations are usually only carried out if, in any case, an evaluation of internal organs of birds is carried out on the basis of a veterinary indication. This method has the disadvantages of being an operative (though minimally invasive) procedure. For this purpose, the animals must first be anesthetized, which on the one hand requires additional expenses; On the other hand, the animals are exposed to an extreme stress situation during such treatment ( G. TH. Kaal, sexual characteristics in birds, Verlag M. & H. Scharper Hannover 1982 ).

Furthermore, methods are used for in ovo sex determination, whereby the sex determination is so far only under experimental conditions at stages of development of different ages practicable:
For in ovo sex determination of the non-incubated egg by molecular genetic methods, the Keim slice is located by magnetic resonance tomography, some blastoderm cells are removed from the germinal disc by biopsy and sex-specific sequences of the W chromosome are determined by PCR or in situ hybridization. However, the development of practicable methods are questioned for the mere reason of the relatively high technical and financial expense for the localization of the germinal disc.

For an in ovo sex differentiation based on the hormone content in the allantoic fluid (eg. DV Gill, HA Robertson and TW Betz; In vivo estrogen synthesis by the developing chicken (Gallus gallus) embryo, General and Comparative Endocrinology, 49, 1983, 176-86 ; WO 9814781 ) are taken, for example, on the 15th to 17th day of incubation 20 .mu.l allantoic fluid. Estrogen determination may be performed using a so-called "LiveSensor ^™ " assay, however, the method is not available for commercial use, and the relatively late sampling time (15-17th day of incubation) is considered problematic from an animal welfare point of view.

In addition, in ovo sex differentiation is known from the different DNA content in cells of male and female domestic chickens ( A. Herzog: Clinical Cytogenetics of Pets, JF Lehmann's Fachbuchhandlung, 1995, 200-205 ). The DNA content in cells of male individuals (ZZ) is about 2% higher at about 2.50 pg than in cells of female individuals (about 2.45 pg), whereby racial or line-specific differences must obviously be taken into account , The determination of the DNA content is carried out by means of known flow cytometry. Due to a hitherto unsatisfactory precision in sex determination and the high equipment costs, this method has not yet been further developed to practical maturity.

• – unzureichende Präzision der DNA-Vervielfältigung
• – hohe Verbrauchskosten in der Routineanalytik (PCR-Primer, Elektrophorese-Gel)
• – späte Diagnose bei bisherigen optischen Verfahren erst nach der Geburt
• – Stresssituation für das Tier bei endoskopischen Untersuchungen
• – hohe Gerätekosten bei der NMR-Analyse
• – Flüssigkeitsentnahme in ovo für die Hormonbestimmung unter Tierschutzgesichtspunkten zu einem problematischen Zeitpunkt

The disadvantages of known methods for sexing birds can be summarized as follows:

• - insufficient precision of DNA duplication
• - high consumption costs in routine analysis (PCR primer, electrophoresis gel)
• - late diagnosis in previous optical procedures only after birth
• - Stress situation for the animal during endoscopic examinations
• - high equipment costs in the NMR analysis
• - Taking liquid in ovo for the purpose of animal hormone determination at a problematic time

Furthermore, Raman spectroscopy has been known as a method of investigation for the characterization of DNA per se for decades (for example JM Benevides and GJ Thomas Jr .: Raman spectral studies of nucleic acids. Part XXV. Characterization of DNA structures by Raman spectroscopy: high-salt and low-salt forms of double helical poly (dG-dC) in water and D2O solutions and application to B, Z and A-DNA, Nucleic Acids Research, 11, 1983, 5747 -61 ).

It is also used to investigate sex transpositions in the succession using isolated SRY-HMG: DNA complex in the human domain (for example JM Benevides, G. Chan, X.-J. Lu, WK Olson, MA Weiss and GJ Thomas: Protein-Directed DNA Structure, I. Raman Spectroscopy of a High Mobility Group Box with Application to Human Sex Reversal, Biochemistry, 39, 2000, 537-547 ). This examines specific genes for the presence of gender-specific markers of inheritance. Another application involves determination of protein / DNA content in B and T cells (e.g. WO 2006130728 ).

Raman spectroscopy is also used for food control of meat ( DI Ellis, D. Broadhurst, SJ Clarke and R. Goodacre: Rapid identification of closely related muscle foods by vibrational spectroscopy and machine learning, Analyst (Cambridge, United Kingdom), 130, 2005, 1648-1654 ) or also pigment studies of birds ( M. Veronelli, G. Zerbi and R. Stradi: Raman spectra of carotenoids in situ in bird's feathers, Journal of Raman Spectroscopy, 26, 1995, 683-92 ).

about Studies on the DNA content of the cell material of birds with Raman spectroscopy to determine gender is nothing has become known in the professional world.

Of the Invention is based on the object, the basis of external Features not or little recognizable gender of birds in the simplest possible way, fast, reliable as well as without appreciable and also for animal welfare reasons problematic loading of the animals (for example by stunning) to determine.

According to the invention the sex of birds is determined by DNA-related Cellular material of the bird (not individual chromosomes) by exposure to light, For example, a laser beam, examined and the molecular movements be measured. The spectrum of the molecule movements resulting radiation is detected and given as well as gender-specific Representing DNA structures of the bird species to be examined Reference spectra compared. These reference spectra, for example in a database, include in particular a statistical recorded gender-specific assignment of DNA features of the each to be examined bird species.

Out the spectral comparison of the evaluated cell material with the said reference data is preferably a computational on Based on the DNA content of the cell material based sex allocation hit the bird.

Surprised was found to be due to the different DNA content between male and female birds the application of vibrational spectroscopic methods specific and distinguishable spectra occur between both sexes, which are of high diagnostic relevance. As a vibrational spectroscopic In particular, Raman spectroscopy, UV resonance Raman spectroscopy, IR spectroscopy and the absorption method of terahertz spectroscopy Application, wherein the DNA-relevant cell material in each case by light corresponding wavelength to molecular or skeletal vibration is stimulated.

The cell material can be removed from the shaft of a young bird's feather in a simple and animal-compatible manner and prepared on a support, preferably a quartz glass or a CaF ₂ glass. On the support surface with the said preparation, the light is focused to measure the molecular motion. Advantageously, the focusing of a laser beam appears here via the objective of a microscope. However, the focus can also be fiber-coupled, which is particularly interesting for an in-ovo sex determination.

at Unbroken birds can see the light through one small access through the eggshell directly to the germinal disc be focused as cell material. By the same or one other access small opening size a probe is guided, by means of which the spectrum of by the aforementioned molecular movement in the interior of the egg is measured.

The obtained spectral information in a second step compared with reference data of a database. These represent preferably statistically obtained data on the examined Species. From this comparison, the invention Gender assignment of the examined DNA material taken.

in the Contrary to the state of the art sexing is with high reliability, specificity, sensitivity, Speed and comparatively little effort feasible.

With The invention provides a method which is an essential improved and more specific determination of the sex of birds like ornamental birds (parrots, etc.), birds of prey or Laying or masthead even allowed in ovo and therefore an immense offers improved diagnostics option.

The present invention is based on measurement results of the DNA material, the known by application of vibration spectroscopy (preferably UV resonance Raman spectroscopy, Raman spectroscopy, IR spectroscopy and THz spectroscopy), and on a preferably computational comparison of the determined spectroscopic Data of the DNA cell material with reference values of a database.

It showed that in DNA rich cell material of birds, with high sensitivity and specificity in difference changed to the same species of the opposite sex Signals were found in the spectrum. The specific reference spectra are thus of high relevance to sexing what this procedure is for the sex distinction (even in ovo) predestined. These differences in the Raman spectrum between males and female birds of the same species, were the experts not known yet.

in the Following is the special procedure of the vibrational spectroscopic Methods exemplified by the already mentioned UV resonance Raman spectroscopy explained. Raman spectroscopy advantageously offers the Possibility of biological material non-destructive to investigate.

By exploiting the resonance effect under excitation in the UV wavelength range, important signals of the DNA and proteins can be selectively amplified and the signal-to-noise ratio improved by about 10 ⁶ due to an increase in intensity. Thus, even low concentrations of DNA-containing cell components are sufficient to investigate these UV resonance Raman spectroscopy. Accordingly, this method is suitable for characterizing Raman spectra of DNA-containing cell material of the birds to be examined. Although the Raman spectra of the investigated DNA-containing cell material are very similar, the complex vibrational structure contains important information that varies between the sexes. The main vibrational bands for gender determination can be found in the wavenumber range below 1800 cm ^-1 (fingerprint area).

It could be shown that the components are mainly contribute to the Raman spectrum of DNA-containing cell material, from DNA bases such as guanine, adenine, cytosine and thymine result. There In addition, proteins are present in the investigated cell material are, other important bands in the Raman spectrum of Signals of aromatic amino acids, such as tryptophan, Tyrosine and phenylalanine, as well as amide I, II and III vibrations.

by virtue of the complexity of the spectra are differences in the spectrum difficult to visualize between the sexes, which is why in particular chemometric methods have been used. chemometrics involves the application of statistical and mathematical methods for data analysis. Chemometric methods can be applied will develop to plan chemical processes and experiments or to evaluate with maximum information. Especially at very large or very similar records chemometric methods are helpful. Such are the methods for the classification of samples using analytical data, such as the detection of diseases using the Raman spectra of the tissue, used. This can be both monitored and unattended classification methods are used. Unmonitored classification methods are the hierarchical ones Cluster Analysis (HCA) and Principal Component Analysis (PCA Principal Component Analysis). For supervised classification methods in contrast to the unsupervised methods, the class membership considered. These procedures are usually used if unknown samples are identified after classification should be. For this the model must first with known samples created and then validated. It could preferably the known methods K Nearest Neighbor Classification (KNN), Nearest Mean Classifier (NMC), linear discriminant analysis (LDA), artificial neural networks (ANN - Artificial Neural Networks) and Support Vector Machines (SVM) come.

For a classification for the evaluation according to the invention It is necessary to have a large record of DNA-containing Cell material of bird species of different sex UV resonance Raman spectroscopy to examine these spectra as reference values in a Database to be used. This will be a chemometric model placed to distinguish between these samples. After completion the collection of spectra in the database and the creation of a classification model, this is used as a basis after recording a UV resonance Raman spectrum an unknown sample using the elaborated model the spectrum of this sample directly as male or to classify female.

The The present invention thus provides a fast and reliable Method for sexing of birds also in ovo.

The Molecular or skeletal vibrations of the examined DNA materials can be detected in particular by UV resonance Raman spectroscopy, Raman spectroscopy, IR spectroscopy and THz spectroscopy be recorded.

As IR and THz spectroscopy is Raman spectroscopy, a vibrational spectroscopic investigation method. However, during IR spectroscopy, the light absorption when passing through a sample is measured in Raman spectroscopy analyzed the scattered light. When the stray light is the same frequency and that it has the same energy as the excitation light, is included elastic light scattering process, Rayleigh scattering before. Beyond that a few light quanta of the laser light are inelastically scattered. Here, the light quanta undergo an interaction with the molecules a sample in such a way that the scattered radiation is higher or lower energy than the excitation light. You then watch next to the elastically scattered light of Rayleigh scattering on both Sides of the excitation line a frequency-shifted inelastic Scattered radiation, the Raman scattering.

The Energy of the THz radiation or the far infrared corresponds to the Energy of weak intramolecular interactions, such as the skeletal vibrations of proteins that are essential for whose folding and function are. Differentiates in THz spectroscopy one the absorption spectroscopy and the emission spectroscopy. Emission spectroscopy measures the radiation that everyone has Body radiates. There is no need for further stimulation occur. The states are already thermally excited and must be measured with a very sensitive detector become. This would be a passive measurement. The whole thing can also take place actively in the form of an absorption experiment with help a radiation source (preferably with germanium lasers and quantum cascade lasers) and detector. It is also possible through the shell of a hatched egg.

The Invention will be described below with reference to an illustrated in the drawing Embodiment with UV resonance Raman spectroscopic Investigation will be explained in more detail.

It demonstrate:

1 : 1. Derivation of UV resonance Raman spectra from spring pulp material of male (bright) and female (dark) leybridges.

2 : Dendrogram as a result of Hierarchical Cluster Analysis light: male - dark: female.

3 : Principal Component Analysis 3D Rendering: x - PC1; y - PC2; z - PC3, light: male - dark: female.

The UV resonance Raman spectroscopic examinations are shown by way of example on feather-sheath-derived spring pulp of different bird species of different sex at a laser excitation wavelength of 244 nm. The sample preparation of the spring pulp is carried out by squeezing out of the spring sheath on a substrate carrier made of quartz. In 1 the 1st derivatives of various UV resonance Raman spectra of spring pulp of male and female leybrids are shown. For each spectra, an integration time of 60 s was chosen, with the laser power used being about 1 mW.

The Determination of sex based on the Raman spectrum is carried out with Help of chemometric methods. Unmonitored procedures, such as the hierarchical cluster analysis and the principal component analysis, provide information about natural groupings in the record. In addition, there are also supervised classification methods such as a support vector machine for sexing used, with which it is possible, following the Classification to identify unknown samples.

In 2 is the dendrogram resulting from a hierarchical cluster analysis. As a data pretreatment, a derivation of the 1st order as well as a vector normalization is performed. The Factorization method is used to calculate the spectral distances. The clustering is done with Ward's algorithm. The spectral range from 1140 to 1730 cm ^{-1 was} used. The smaller the spectral distances (heterogeneity) between two spectra or clusters, the more similar they are. The grouping makes it clear that a good distinction between spectra of female and male laying hybrids based on DNA material of the spring pulp is possible.

The Dendrogram shows the splitting into two clusters, with one group of spectra from female laying hybrids (shown in dark) while the other group is mainly derived from spectra of male laying hybrids. Indeed some spectra were assigned to the opposite sex. It is determine that Sample 9 and 13 are declared male, but always - also with various data pretreatments - in Cluster "female" are classified 27 declared as female but always in the "male" Clustered.

The result of the cluster analysis was confirmed by a principal component analysis (PCA). Again, the derivative of the spectra is used and there is a vector normalization. In 3 a three-dimensional score plot is shown with the first major component plotted on the x, the second on the y and the third on the z-axis. It becomes clear that feather pulp preparations differ from female laying hybrids (shown in dark) to those of the male (shown lighter). With few major components, the two sexes can be separated, with the scattering of each group high is and partially overlaps.

Furthermore, the spectra were evaluated using a support vector machine (SVM; kernel function: radial basis function (RBF); cost factor 1.5 × 10 ⁶ , γ = 1 × 10 ^-6 ). The spectral ranges of 3545-2532 cm ^-1 and 1809-913 cm ^{-1 are} included. In this classification method, attributes are assigned to the spectra, in this case the attribute "gender". The computational algorithm optimally tries to separate the spectra, which have to be subdivided by the attributes into the specified classes, by a separation plane. For this purpose, the data matrix is transferred to the higher-dimensional "hyperspace." A visual representation of the results is no longer possible because of the higher dimensions.

The result is a model that is validated by the leave-one-out method. In the process, a spectrum is left out when the model is created and an assignment is attempted again using the calculated model. The result is shown in Table 1. Table 1: Result of the SVM analysis using the leave-one-out method Domestic chicken male Domestic chicken female correct wrong Domestic chicken male 168 3 98.3 1.7 Domestic chicken female 10 145 93.6 6.4 95.9 4.1

Another type of validation of the model succeeds with a test record. The original data set is randomly divided into a model building record (215 spectra) and a model validation record (111 spectra). The result of the validation is shown in Table 2. Table 2: Result of the SVM analysis using test set method Domestic chicken male Domestic chicken female correct wrong Domestic chicken male 56 1 98.2 1.8 Domestic chicken female 4 48 92.3 7.7 95.4 4.6

The described method, which is based on the application known per se vibration spectroscopic methods and chemometric methods (here by way of example using the hierarchical cluster analysis, Principal component analysis and support vector machine of UV resonance Raman spectra), thus offers the possibility due to spectral differences of DNA material (in the present example Federpulpapräparate of laying hybrids) between the sexes of birds to distinguish.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• US 5876942 [0004, 0004]
• WO 9749806 [0006]
• - US 6396938 [0008]
• - US 6493566 B1 [0008]
• WO 9814781 [0011]
• WO 2006130728 [0015]

Cited non-patent literature

• - KBC Appa Rao, D. Mohan and SM Totey: Polymerase chain reaction and its applications: Special emphasis on its role in embryo sexing, Biotechnology Advances 12, 1994 341-55 [0003]
• - KK De Mattos, SN Del Lama, ARC Salles, MR Francisco, A. Medaglia, AM Tomasulo, FMS Carmo, JMC Pereira and MA De Oliveira: Sex determination of parrots Amazona a. aestiva and Amazona amazonica using the polymerase chain reaction, Journal of Experimental Zoology, 281, 1998, 217-219 [0004]
• - T. Minematsu, M. Sugiyama, Y. Tohma, A. Tajima and Y. Kanai: Simplified DNA extraction methods for sexing chick embryos, Journal of Poultry Science, 41, 2004, 147-154 [0004]
• - L. Huynen, CD Millar and Lambert: A DNA test to sex ratite birds, Molecular Ecology, 11, 2002, 851-856 [0004]
• - LG Bermudez-Humaran, A. Garcia-Garcia, CH Leal-Garza, VM Riojas-Valdes, G. Jaramillo-Rangel and R. Montes-De-Oca-Luna: Molecular sexing of monomorphic endangered macaw birds, Journal of Experimental Zoology , 292, 2002 677-680 [0004]
• - Y. Itoh, M. Suzuki, A. Ogawa, I. Munechika, K. Murata and S. Mizuno: Identification of the wide range of Carinatae birds by PCR using primer sets EE0.6 and its related sequences, Journal of Heredity, 92, 2001, 315-321 [0004]
• - K. Makondo, GS Amiridis, IA Jeffcoate, PJ O'Shaughnessy, JS Boyd, C. Paterson and L. Robertson: Use of the polymerase chain reaction to sex the bovine fetus using cells recovered by ultrasound-guided fetal fluid aspiration, Animal Reproduction Science, 49, 1997, 125-133 [0004]
• - BC Robertson and NJ Gemmell: PCR-based sexing in conservation biology: wrong answers from an accurate methodology ?, Conservation Genetics, 7, 2006, 267-271 [0007]
• - G. TH. Kaal, sexual characteristics in birds, Verlag M. & H. Scharper Hannover 1982 [0009]
• DV Gill, HA Robertson and TW Betz; In vivo estrogen synthesis by the developing chicken (Gallus gallus) embryo, General and Comparative Endocrinology, 49, 1983, 176-86 [0011]
• - A. Herzog: Clinical Cytogenetics of Pets, JF Lehmann's Fachbuchhandlung, 1995, 200-205 [0012]
• JM Benevides and GJ Thomas Jr .: Raman spectral studies of nucleic acids. Part XXV. Characterization of DNA structures by Raman spectroscopy: high-salt and low-salt forms of double helical poly (dG-dC) in water and D2O solutions and application to B, Z and A-DNA, Nucleic Acids Research, 11, 1983, 5747 -61 [0014]
• JM Benevides, G. Chan, X.-J. Lu, WK Olson, MA Weiss and GJ Thomas: Protein-Directed DNA Structure, I. Raman Spectroscopy of a High Mobility Group Box with Application to Human Sex Reversal, Biochemistry, 39, 2000, 537-547. [0015]
• DI Ellis, D. Broadhurst, SJ Clarke and R. Goodacre: Rapid identification of closely related muscle foods by vibrational spectroscopy and machine learning, Analyst (Cambridge, United Kingdom), 130, 2005, 1648-1654 [0016]
• M. Veronelli, G. Zerbi and R. Stradi: In Situ Raman Spectra of Carotenoids in Bird's Feathers, Journal of Raman Spectroscopy, 26, 1995, 683-92 [0016]

Claims (16)

Method for determining the sex of birds, characterized in that DNA-relevant cell material of the sexually determined bird is examined with light and the molecular vibrations are measured, that detects the spectrum of molecular vibrations resulting from the light and with predetermined and gender-specific DNA structures of comparing the reference spectra representing the species of interest and that based on the DNA content of the cell material based on the DNA content of the genetic material of the bird is taken from this spectral comparison. Method according to claim 1, characterized in that that the molecular vibrations by application of Raman spectroscopy be measured. Method according to claim 1, characterized in that that the molecular vibrations by application of the UV resonance Raman spectroscopy be measured. Method according to claim 1, characterized in that that the molecular vibrations by application of IR spectroscopy be measured. Method according to claim 1, characterized in that that the molecular vibrations by applying the absorption method terahertz spectroscopy. Method according to claim 1, characterized in that that the DNA-relevant cell material from the shaft of a young Feather of the bird is taken. A method according to claims 2 to 5, characterized in that the DNA-relevant cell material on a support, for example a quartz substrate, a CaF ₂ substrate or silicon carrier for Raman spectroscopic studies or KRS5, CaF ₂ substrate, ZnSe carrier or silicon carrier for IR spectroscopic investigations, is prepared on the surface of which the light is focused to measure the molecular vibrations. Process according to claims 2 to 5, characterized characterized in that as a light source for measuring the molecular vibrations Laser radiation for Raman spectroscopic investigations and MIR-Globar or Nernst-Stift for IR-spectroscopic Investigations is used. Method according to claim 1, characterized in that that the light is used to measure the molecular vibrations of the DNA-relevant cell material from unsheathed birds through the eggshell to the embryo or the germinal disc is focused and that the spectrum of the through the molecular vibrations resulting radiation in the egg with a guided through the shell Probe is measured. Method according to claim 9, characterized in that that for the light to measure the molecular vibrations of the DNA-relevant cell material and for the passage the probe for measuring the spectrum at least a microscopic small hole is drilled through the eggshell. Method according to claim 3, characterized that for measuring the molecular vibrations of the DNA-relevant Cell material laser radiation having a wavelength preferably of 244 nm or 257 nm is used. Method according to claim 1, characterized in that that the DNA-relevant cell material to avoid its thermal overload during the measurement of the molecular vibrations, for example by rotation and displacement or vibration, is moved. Method according to claim 1, characterized in that that the measured spectrum of the DNA-relevant cell material with Reference spectra compared with the one to be taken Gender assignment were determined statistically. Method according to claim 1, characterized in that that the comparison of the measured spectrum with the reference spectra as well as the gender assignment computationally done. A method according to claim 1, characterized in that the signals, (the signal pattern) of the spectra of the DNA-relevant cell material for comparison with the reference spectra by means of unmonitored Klas analysis methods, which include, in particular, the hierarchical cluster analysis and the principal component analysis. Method according to claim 1, characterized in that that the signals, (the signal pattern) of the spectra of the DNA-relevant Cell material for comparison with the reference spectra using monitored Classification methods, in particular the methods K Nearest Neighbor Classification (KNN), Nearest Mean Classifier (NMC), linear discriminant analysis (LDA), artificial neural networks (ANN - Artificial Neural Networks) as well as Support Vector Machines (SVM), to be analyzed.