JP7591275B2 - Method and device for determining sex of chicken eggs before hatching using near-infrared light - Google Patents

Description

The present invention relates to a method for determining the sex of chicken eggs before they hatch, and a device for determining the sex.

Laying hens are sexed by feather identification after hatching (21 days after incubation). As a result of the sexing, males are usually culled. In recent years, it is estimated that 100 million male chicks are culled each year in Japan and 6 billion worldwide. The culling of male chicks after hatching is a major problem from the perspective of animal welfare.

A known method for determining the sex of eggs before they hatch is to use a laser beam to create a hole of 0.3 mm or less in the eggshell on the 9th day after incubation, collect allantoic fluid through the hole, and then detect estrone sulfate in the allantoic fluid by colorimetry to determine the sex of the eggs.

In the case of chickens, it is believed that embryos acquire a sense of pain after seven days of incubation. Therefore, if it is possible to determine the sex of eggs by the seventh day after incubation, eggs that are likely to become male can be removed early before they hatch, essentially avoiding the culling of male chicks. In addition, by selecting and incubating eggs that are likely to become female, it is possible to reduce the cost of incubation and to improve the efficiency of egg-laying hens production.

For example, Patent Document 1 describes a method for determining the condition of eggs, the method including the steps of irradiating the eggs with multiple light pulses having a wavelength in the range of 400 to 1500 nm, a width in the range of about 0.5 to about 500 picoseconds, and an intensity in the range of about 0.1 to about 100 mJ, detecting reflected light of at least a portion of the multiple light pulses, and analyzing the detected reflected light to classify the eggs into at least one gender.

Patent Document 2 describes a non-invasive method for detecting the current state of a chicken egg, the method including the steps of obtaining a test measurement image by measuring the amount of light of at least one predetermined wavelength corresponding to the reflected or transmitted light of the egg using a hyperspectral camera, comparing the test measurement image with a control measurement image, obtaining at least one spectrum of the egg in a predetermined wavelength range using the hyperspectral camera, and comparing the spectra using a neural network algorithm, and evaluating the current state of the egg using the comparison result. The document describes that the egg spectrum in a wavelength range of about 300 to about 2500 nm can be used to determine the sex of the egg on the 12th day after incubation.

Patent document 3 describes a non-destructive testing device for hatching eggs, which includes a light irradiating unit that irradiates light onto the hatching eggs, a light detecting unit that detects the intensity of light that has passed through the hatching eggs, and a gender determining unit that performs preliminary gender discrimination based on the intensity of light that has passed through the hatching eggs at a first point in time that is a predetermined period of time after the start of incubation.

U.S. Pat. No. 10,705,066 U.S. Pat. No. 9,435,732 Patent No. 6723597

As mentioned above, a method for sexing chicken eggs before hatching is known. However, there are several problems with the conventional sexing methods. For example, in the case of a method in which a hole is formed in the eggshell to collect allantoic fluid and sexing is performed, the hatching rate may decrease due to the formation of the hole and/or the collection of allantoic fluid. Patent documents 1 to 3 describe a method for sexing by non-destructive spectroscopic means. However, the method described in Patent document 1 requires irradiation of light pulses over a wide wavelength range from the visible light region to the near-infrared light region and detection of reflected light, so that the method may take a long time to perform. The method described in Patent document 2 requires the use of special means such as measurement by a hyperspectral camera and processing by a neural network algorithm, so that the method may be expensive to perform. In addition, there was no known method for sexing eggs by non-destructive spectroscopic means with high accuracy before the seventh day after incubation, when the embryo is thought to acquire pain sensation.

Therefore, the present invention aims to provide a means for determining sex with high accuracy by non-destructive spectroscopic means without using special means, before the seventh day after incubation, when embryos are thought to acquire pain sensation.

The inventors have investigated various means for solving the above problems. They have discovered that it is possible to determine the sex of an egg with high accuracy by irradiating light onto an egg 3 to 6 days after incubation, acquiring a near-infrared spectrum in the long wavelength region of the light that is transmitted through the egg or is reflected within the egg and then emitted outside the egg, and analyzing the spectral data. The inventors have completed the present invention based on the above findings.

That is, the present invention includes the following aspects and embodiments.
(1) A method for determining the sex of a chicken egg before hatching, comprising the following steps:
a light irradiation step of irradiating chicken eggs 3 to 6 days after incubation with light having a wavelength ranging from the visible light region to the near infrared light region;
a light detection step of detecting light emitted outside the egg as a result of the light irradiated in the light irradiation step passing through the egg or being reflected within the egg;
a spectrum acquiring step of acquiring the visible and near-infrared spectra of the light detected in the light detecting step; and a sex determining step of determining the sex of the egg based on the spectral data in the wavelength region of 1700 to 2500 nm in the visible and near-infrared spectra acquired in the spectrum acquiring step;
The method comprising:
(2) The method according to the above embodiment (1), wherein in the sex-determining step, the sex of the chicken egg is determined based on spectral data in a wavelength region ranging from 400 to 900 nm.
(3) The light irradiated in the light irradiation step is a light irradiated from above the egg in a direction parallel to the long axis line connecting the blunt end and the sharp end, the long axis line passing through the animal pole or embryo, on a plane parallel to the vertical plane including the long axis line, to a chicken egg arranged so that the long axis line is perpendicular to a horizontal plane,
The method according to embodiment (1) or (2), wherein the light detected in the light detection step is light that passes through a chicken egg arranged so that a long axis line connecting the blunt end and the sharp end is perpendicular to a horizontal plane, and is emitted to the side of the chicken egg, and is light that is emitted in a direction perpendicular to the long axis line on a horizontal plane perpendicular to the long axis line so as to pass through the yolk of the chicken egg.
(4) The light irradiated in the light irradiation step is a light irradiated in a direction perpendicular to the long axis line connecting the blunt end and the sharp end of the egg, the long axis line being parallel to a horizontal plane, from below the egg and passing through the animal pole or embryo, on a plane parallel to a vertical plane including the long axis line,
The method according to embodiment (1) or (2), wherein the light detected in the light detection step is light that is reflected inside a chicken egg placed so that a long axis line connecting the blunt end and the sharp end is parallel to a horizontal plane and is emitted downward from the egg, and is emitted in a direction at 45° to the long axis line on a plane parallel to a vertical plane containing the long axis line so as to pass through the yolk of the chicken egg.
(5) A device for determining the sex of a chicken egg before it is hatched, comprising the following means:
a light irradiation means for irradiating the eggs with light having a wavelength ranging from the visible light region to the near infrared light region;
a light detection means for detecting light emitted outside the egg as a result of the light irradiated by the light irradiating means passing through the egg or being reflected within the egg;
a spectrum acquiring means for acquiring the visible and near-infrared spectra of the light detected by the light detecting means; and a sex determining means for determining the sex of the egg based on the spectral data in the wavelength region of 1700 to 2500 nm in the visible and near-infrared spectra acquired by the spectrum acquiring means;
The apparatus comprising:
(6) The device according to embodiment (5), wherein the sex-determining means further determines the sex of the chicken egg based on spectral data in a wavelength region ranging from 400 to 900 nm.
(7) The light irradiating means is arranged so as to irradiate a chicken egg, which is arranged so that a long axis line connecting the blunt end and the sharp end is perpendicular to a horizontal plane, with light in a direction parallel to the long axis line on a plane parallel to a vertical plane including the long axis line, so as to pass through the animal pole or embryo from above the chicken egg,
The device according to embodiment (5) or (6), wherein the light detection means is arranged to detect light that is emitted to the side of a chicken egg by passing through the egg, the egg being arranged so that the long axis line connecting the blunt end and the sharp end is perpendicular to a horizontal plane, and that is emitted in a direction perpendicular to the long axis line on a horizontal plane perpendicular to the long axis line so as to pass through the yolk of the chicken egg.
(8) The light irradiating means is arranged so as to irradiate a chicken egg, which is arranged so that a long axis line connecting the blunt end and the sharp end is parallel to a horizontal plane, with light in a direction perpendicular to the long axis line on a plane parallel to a vertical plane including the long axis line so as to pass through the animal pole or embryo from below the chicken egg,
The device according to embodiment (5) or (6), wherein the light detection means is arranged to detect light emitted downward from an egg by reflection inside the egg, the egg being arranged so that the long axis connecting the blunt end and the sharp end is parallel to a horizontal plane, and the light is emitted in a direction at 45° to the long axis on a plane parallel to a vertical plane containing the long axis so as to pass through the yolk of the egg.

The present invention makes it possible to provide a means for determining sex with high accuracy using non-destructive spectroscopic means without using special means, before the seventh day after incubation, when embryos are thought to acquire pain sensation.

FIG. 1 is a flow chart showing the steps of a method for determining the sex of a chicken egg before hatching according to one embodiment of the present invention. FIG. 2 is a configuration diagram showing one embodiment of a device for determining the gender of a chicken egg before hatching according to one aspect of the present invention. FIG. 3 is a schematic diagram showing one embodiment of a device for determining the gender of a chicken egg before hatching according to one aspect of the present invention. FIG. 4 is a schematic diagram showing one embodiment of a device for determining the gender of a chicken egg before hatching according to one aspect of the present invention. FIG. 5 is a schematic diagram showing one embodiment of a device for determining the gender of a chicken egg before hatching according to one aspect of the present invention. FIG. 6 is a schematic diagram showing one embodiment of a device for determining the gender of a chicken egg before hatching according to one aspect of the present invention. FIG. 7 is a schematic diagram showing one embodiment of a device for determining the gender of a chicken egg before hatching according to one aspect of the present invention. FIG. 8 is a schematic diagram showing one embodiment of a device for determining the gender of a chicken egg before hatching according to one aspect of the present invention. FIG. 9 is an example of visible and near-infrared spectra measured by a transmission method. A is the visible and near-infrared spectrum, and B is its second derivative spectrum. In A, the horizontal axis is wavelength (nm) and the vertical axis is absorbance. In B, the horizontal axis is wavelength (nm) and the vertical axis is the intensity of the second derivative value. Figure 10 shows an example of visible and near infrared spectra measured by the reflectance method. A shows the visible and near infrared spectrum, and B shows the second derivative spectrum. In A, the horizontal axis is wavelength (nm) and the vertical axis is absorbance. In B, the horizontal axis is wavelength (nm) and the vertical axis is the intensity of the second derivative value.

1. How to determine the sex of chicken eggs before they hatch
In a conventional method for sexing chicken eggs before hatching, for example, when a hole is formed in the eggshell to collect allantoic fluid, the hatching rate may decrease due to the formation of the hole and/or collection of allantoic fluid. Patent documents 1 to 3 describe a method for sexing by non-destructive spectroscopic means. However, the method described in Patent document 1 requires irradiation of light pulses and detection of reflected light over a wide wavelength range from the visible light region to the near-infrared light region, so that the method may take a long time to carry out. The method described in Patent document 2 requires the use of special means such as measurement by a hyperspectral camera and processing by a neural network algorithm, so that the method may be expensive to carry out. In addition, a method for sexing eggs by non-destructive spectroscopic means with high accuracy before the seventh day after incubation, when the embryo is thought to acquire pain sensation, has not been known.

The inventors have discovered that it is possible to determine the sex of a chicken egg with high accuracy by irradiating the egg with light 3 to 6 days after incubation, obtaining a near-infrared spectrum in the long wavelength region of the light that is transmitted through the egg or is reflected within the egg and emitted outside the egg, and analyzing the spectral data. Therefore, one aspect of the present invention relates to a method for determining the sex of a chicken egg before it hatches.

Figure 1 shows a flow chart illustrating each step in one embodiment of the method of this aspect. As shown in Figure 1, the method of this aspect includes a light irradiation step (S1), a light detection step (S2), a spectrum acquisition step (S3), and a sex determination step (S4). Each step will be described in detail below.

[1-1. Light irradiation process]
The method of this embodiment includes a light irradiation step (step S1) of irradiating a chicken egg with light having a wavelength ranging from the visible light region to the near-infrared light region.

In each aspect of the present invention, "visible light" means light having a wavelength in the wavelength range of 400 to 900 nm, including the wavelength range of 400 to 750 nm corresponding to visible light, and "near-infrared light" means light having a wavelength in the wavelength range of 900 to 2500 nm. For example, "light having a wavelength in the visible light range to the near-infrared light range" means light having a wavelength in the wavelength range of 400 to 2500 nm. The light irradiated in this step (hereinafter also referred to as "irradiation light") preferably has a wavelength in the wavelength range of 400 to 2500 nm, more preferably has a wavelength in the range of 400 to 900 nm and the range of 1700 to 2500 nm. The irradiation light may be light having all wavelengths continuously in the wavelength range, or may be light having a part of the wavelengths (e.g., a specific wavelength) in the wavelength range. By irradiating light having a wavelength in the wavelength range, it is possible to perform sexing with high accuracy in the sexing step described below. In addition, by selectively irradiating light having a specific wavelength, for example, a wavelength in the wavelength range of 400 to 900 nm and a wavelength in the wavelength range of 1700 to 2500 nm, the wavelength sweep range can be narrowed and the time required to perform this step can be shortened.

In each aspect of the present invention, chicken eggs refer to fertilized eggs for use in the production of egg-laying hens.

The chicken eggs used in this process are usually chicken eggs on the 3rd to 6th day after incubation, for example the 4th to 6th day after incubation, and particularly the 5th or 6th day after incubation. In the egg yolk of the chicken eggs at this stage, the animal pole or an embryo differentiated therefrom is formed. The animal pole or embryo contains blood and/or various components such as protein and fat. However, it is believed that the animal pole or embryo at this stage has not yet acquired a sense of pain. Therefore, by analyzing the egg yolk of a chicken egg at this stage, particularly the blood and/or various components contained in the animal pole or embryo, it is possible to determine the sex with high accuracy before the embryo acquires a sense of pain.

In this step, the irradiation light can be applied to the egg from various directions. For example, in one embodiment, the irradiation light is applied to the egg arranged so that the long axis line connecting the blunt end and the sharp end is perpendicular to the horizontal plane, from above the egg in any direction (for example, in a direction in the range of 0 to 50° with respect to the long axis line, particularly in a direction parallel to the long axis line on a plane parallel to the vertical plane including the long axis line) so as to pass through the animal pole or embryo (hereinafter, also referred to as the "first embodiment"). In another embodiment, the irradiation light is applied to the egg arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane, from the side of the egg in any direction (for example, in a direction in the range of 0 to 90° with respect to the long axis line, particularly in a direction perpendicular to the long axis line on a plane parallel to the horizontal plane including the long axis line) so as to pass through the animal pole or embryo (hereinafter, also referred to as the "second or fourth embodiment"). In another embodiment, the irradiated light is light that is irradiated from above the egg in any direction (e.g., in a direction in the range of 40 to 90° with respect to the long axis, particularly in a direction perpendicular to the long axis on a plane parallel to a vertical plane including the long axis) so as to pass through the animal pole or embryo, on a chicken egg arranged such that the long axis connecting the blunt end and the sharp end is parallel to a horizontal plane (hereinafter, also referred to as "third or sixth embodiment"). In another embodiment, the irradiated light is light that is irradiated from below the egg in any direction (e.g., in a direction in the range of 40 to 90° with respect to the long axis, particularly in a direction perpendicular to the long axis on a plane parallel to a vertical plane including the long axis) so as to pass through the animal pole or embryo, on a chicken egg arranged such that the long axis connecting the blunt end and the sharp end is parallel to a horizontal plane (hereinafter, also referred to as "fifth embodiment"). In a preferred first embodiment, the irradiating light is light that is irradiated to a chicken egg arranged so that the long axis line connecting the blunt end and the sharp end is perpendicular to the horizontal plane, in a direction parallel to the long axis line on a plane parallel to the vertical plane including the long axis line, so as to pass through the animal pole or embryo from above the egg. In a preferred second or fourth embodiment, the irradiating light is light that is irradiated to a chicken egg arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane, in a direction perpendicular to the long axis line on a plane parallel to the horizontal plane including the long axis line, so as to pass through the animal pole or embryo from the side of the egg. In a preferred third or sixth embodiment, the irradiating light is light that is irradiated to a chicken egg arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane, in a direction perpendicular to the long axis line on a plane parallel to the vertical plane including the long axis line, so as to pass through the animal pole or embryo from above the egg. In a preferred fifth embodiment, the irradiated light is a light that is irradiated from below the egg in a direction perpendicular to the long axis on a plane parallel to a vertical plane including the long axis, so as to pass through the animal pole or embryo, on a chicken egg placed so that the long axis connecting the blunt end and the sharp end is parallel to a horizontal plane. In each of the embodiments exemplified above, it is preferable to confirm the position of the animal pole or embryo of the egg in advance with an egg candling device, and to place the egg so that the animal pole or embryo is in the direction of incidence of the irradiated light. By carrying out this step so that the irradiated light passes through the animal pole or embryo, information on the blood and/or various components contained in the animal pole or embryo can be obtained in each step described below, and sexing can be performed with high accuracy.

In each aspect of the present invention, "parallel," "perpendicular," and "orthogonal" mean that lines and/or surfaces are in a completely or approximately parallel, perpendicular, or orthogonal positional relationship.

This process is usually carried out with the egg fixed so that the egg and the irradiated light are positioned as described above. For this reason, it is preferable to use an egg fixing member in this process to position the egg as described above. When an egg fixing member is used, the egg is placed, for example, on the upper surface of the egg fixing member.

In each embodiment of the present invention, "passing through the animal pole or embryo" and "passing through the yolk" mean that the light passes through at least a portion of the animal pole or embryo in the egg, or the yolk. A position passing through the animal pole or embryo is, for example, 15 to 30 mm from the sharp end of the egg.
The term "position passing through the egg yolk" refers to, for example, a range of 20 to 60 mm, typically a range of 27 to 52 mm, from the sharp end of the egg.

This process is preferably carried out by irradiating light inside a container that contains the eggs and the egg fixing member. The container is preferably a member that can substantially block external light, such as a dark box. By carrying out this process inside the container, the effects of external light can be substantially suppressed, and sexing can be performed with higher accuracy.

[1-2. Optical detection process]
The method of this embodiment includes a light detection step (step S2) of detecting light emitted outside the egg as a result of the light irradiated in the light irradiation step passing through the egg or being reflected within the egg.

In each aspect of the present invention, the light emitted outside the egg by transmitting the light irradiated in the light irradiation step through the egg may be referred to as "transmitted light", and the light emitted outside the egg by reflecting the light irradiated in the light irradiation step within the egg may be referred to as "reflected light". Although the transmitted light and reflected light can be selected based on a combination of the light irradiation position in the light irradiation step and the light detection position in this step, it is difficult to strictly separate the transmitted light and the reflected light from each other. Therefore, in each aspect of the present invention, the transmitted light may contain a certain proportion of reflected light, and the reflected light may contain a certain proportion of transmitted light. In addition, in each aspect of the present invention, an embodiment in which the transmitted light emitted outside the egg is detected to determine the sex of the egg may be referred to as the "transmission method", and an embodiment in which the reflected light emitted outside the egg is detected to determine the sex of the egg may be referred to as the "reflection method".

The transmitted or reflected light detected in this step preferably has a wavelength in the wavelength range of 400 to 2500 nm, and more preferably has a wavelength in the range of 400 to 900 nm and the range of 1700 to 2500 nm. The transmitted or reflected light may be light having all wavelengths in the wavelength range continuously, or may be light having some wavelengths (e.g., specific wavelengths) in the wavelength range. By detecting the transmitted or reflected light having a wavelength in the wavelength range, it is possible to perform sex determination with high accuracy in the sex determination step described below. In addition, by selectively detecting the transmitted or reflected light having a specific wavelength, for example, a wavelength in the range of 400 to 900 nm and the range of 1700 to 2500 nm, it is possible to narrow the wavelength sweep range and shorten the time required to perform this step.

In this step, light emitted in various directions relative to the egg can be detected. For example, in one embodiment, the detected light is light (transmitted light) that passes through an egg arranged so that the long axis line connecting the blunt end and the sharp end is perpendicular to a horizontal plane and is emitted to the side of the egg, and that passes through the yolk of the egg in any direction (for example, in a direction in the range of 40 to 90° relative to the long axis line, particularly in a direction perpendicular to the long axis line on a horizontal plane perpendicular to the long axis line) (hereinafter, also referred to as "first embodiment"). In another embodiment, the detected light is light (transmitted light) emitted downward from a chicken egg arranged so that the long axis connecting the blunt end and the sharp end is parallel to a horizontal plane, and is emitted in any direction (for example, in a direction in the range of 40 to 90° with respect to the long axis, particularly in a direction perpendicular to the long axis on a plane parallel to a vertical plane including the long axis) so as to pass through the yolk of the chicken egg (hereinafter, also referred to as the "second or third embodiment"). In another embodiment, the detected light is light (transmitted light) emitted laterally from a chicken egg arranged so that the long axis connecting the blunt end and the sharp end is parallel to a horizontal plane, and is emitted in any direction (for example, in a direction in the range of 0 to 90° with respect to the long axis, particularly in a direction perpendicular to the long axis on a plane parallel to a horizontal plane including the long axis) so as to pass through the yolk of the chicken egg (hereinafter, also referred to as the "fourth embodiment"). In another embodiment, the detected light is light (reflected light) emitted downward from a chicken egg by reflection inside the chicken egg arranged so that the long axis line connecting the blunt end and the sharp end is parallel to a horizontal plane, and is emitted in any direction (for example, in a direction in the range of 40 to 90° to the long axis line, particularly in a direction of 45° to the long axis line on a plane parallel to a vertical plane including the long axis line) so as to pass through the yolk of the chicken egg (hereinafter, also referred to as the "fifth embodiment"). In another embodiment, the detected light is light (reflected light) emitted upward from a chicken egg by reflection inside the chicken egg arranged so that the long axis line connecting the blunt end and the sharp end is parallel to a horizontal plane, and is emitted in any direction (for example, in a direction in the range of 40 to 90° to the long axis line, particularly in a direction perpendicular to the long axis line on a plane parallel to a vertical plane including the long axis line) so as to pass through the yolk of the chicken egg (hereinafter, also referred to as the "sixth embodiment"). In a preferred first embodiment, the detected light is light (transmitted light) that passes through a chicken egg arranged so that the long axis line connecting the blunt end and the sharp end is perpendicular to the horizontal plane and is emitted to the side of the egg, and is emitted in a direction perpendicular to the long axis line on the horizontal plane perpendicular to the long axis line so as to pass through the yolk of the egg. In a preferred second or third embodiment, the detected light is light (transmitted light) that passes through a chicken egg arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane and is emitted to the bottom of the egg, and is emitted in a direction perpendicular to the long axis line on a plane parallel to a vertical plane including the long axis line so as to pass through the yolk of the egg. In a fourth preferred embodiment, the detected light is light (transmitted light) that passes through an egg arranged so that the long axis connecting the blunt end and the sharp end is parallel to the horizontal plane and is emitted to the side of the egg, and is emitted in a direction perpendicular to the long axis on a plane parallel to the horizontal plane and including the long axis so as to pass through the yolk of the egg. In a fifth preferred embodiment, the detected light is light (reflected light) that is reflected inside the egg arranged so that the long axis connecting the blunt end and the sharp end is parallel to the horizontal plane and is emitted in a direction at 45° to the long axis on a plane parallel to the vertical plane and including the long axis so as to pass through the yolk of the egg. In a sixth preferred embodiment, the detected light is light (reflected light) that is reflected inside an egg placed so that the long axis line connecting the blunt end and the sharp end is parallel to a horizontal plane and is emitted upward from the egg, and is emitted in a direction perpendicular to the long axis line on a plane parallel to a vertical plane including the long axis line so as to pass through the yolk of the egg. In each of the embodiments exemplified above, it is preferable to confirm the position of the yolk of the egg in advance with an egg candling device, and to place the egg so that the transmitted light or reflected light passes through the yolk. By carrying out this step so that the transmitted light or reflected light passes through the yolk, information on the blood and/or various components contained in the animal pole or embryo can be obtained in each step described below, and sexing can be performed with high accuracy.

This step is preferably carried out by detecting light inside the container described above. By carrying out this step inside the container, the influence of external light can be substantially suppressed, and sexing can be performed with higher accuracy.

[1-3. Spectrum acquisition process]
The method of this aspect includes a spectrum acquisition step (step S3) of acquiring the visible and near-infrared spectra of the light detected in the light detection step.

This step is carried out by generating visible and near-infrared spectra based on the light detected in the light detection step. Examples of means for generating visible and near-infrared spectra include visible and near-infrared spectroscopic devices that are commonly used in the art. The visible and near-infrared spectroscopic devices are usually connected to a data analysis device that stores a program for analyzing the spectral data in addition to a control program for the visible and near-infrared spectroscopic device. Therefore, by using the visible and near-infrared spectroscopic device, it is possible to generate visible and near-infrared spectra and analyze the spectral data in a short period of time.

The visible and near-infrared spectra acquired in this process may be the spectra as they are, or may be second-derivative spectra obtained by subjecting the spectra to second-derivative processing. Second-derivative spectra are preferred because they can reduce the effects of baseline changes, etc. By acquiring second-derivative spectra in this process, sexing can be performed with high accuracy.

[1-4. Sex determination process]
The method of this embodiment includes a sex determining step (step S4) of determining the sex of the chicken egg based on the spectral data of the visible and near-infrared spectra acquired in the spectrum acquiring step.

In this step, as a means for determining the sex of the chicken eggs based on the spectral data, for example, a multivariate analysis known in the art can be applied. Examples of the multivariate analysis include principal component analysis and partial least squares discriminant analysis (PLS discriminant analysis).
Examples of the sex determination method include PLS-DA (partial linear linear analysis). When this step is carried out using principal component analysis, the following procedure may be followed. First, a sex determination model is created. A light irradiation step, a light detection step, and a spectrum acquisition step are carried out using a predetermined number of chicken eggs whose sexes are known, to obtain standard visible and near-infrared spectra for each sex. The acquired visible and near-infrared spectra are preferably second derivative spectra. The sex determination of the chicken eggs used to obtain the standard visible and near-infrared spectra may be carried out by applying a sex determination means known in the art. Examples of known sex determination means include a genetic analysis method in which samples of chicken egg embryos and blood are collected, and DNA extracted from the collected samples is used to determine the sex by multiplex PCR using sex-specific primers, and a method in which the collected samples are analyzed by an instrument to determine the sex based on the component concentrations (e.g., hormone concentrations) in the samples. The sex determination means exemplified above may be carried out by cracking the chicken eggs used after obtaining the standard visible and near-infrared spectra, and collecting samples. Next, a principal component analysis is performed on the standard spectral data groups for both sexes. In the standard principal component space for both sexes, it is preferable to detect outliers using Mahalanobis distance to obtain a principal component score plot from which the outliers have been removed. The obtained principal component score plot can be used as a sex determination model. Next, a light irradiation step, a light detection step, and a spectrum acquisition step are performed on the egg to be measured to obtain a visible and near-infrared spectrum. The obtained visible and near-infrared spectrum is preferably a second derivative spectrum. A principal component analysis is performed on the obtained spectral data of the visible and near-infrared spectrum, and the obtained principal component score is applied to the principal component space of the sex determination model to determine the sex. In this case, examples of means for applying the principal component score of the egg to be measured to the principal component space of the sex determination model include the residual variance method in the principal component space, the maximum distance method by wavelength, and the Mahalanobis distance in the principal component space.

In this step, the creation of a sex determination model by multivariate analysis and the sex determination of the measurement subject may be performed, for example, using a data analysis device such as a computer installed with commercially available multivariate analysis software commonly used in the technical field, or using a data analysis device such as a computer connected to the visible and near-infrared spectrometer used in the spectrum acquisition step. The data analysis device connected to the visible and near-infrared spectrometer usually stores a program for performing multivariate analysis of spectral data. Therefore, by performing this step using a data analysis device connected to the visible and near-infrared spectrometer, sex determination can be performed at low cost.

In this process, the sexing model may be created using multivariate analysis each time this process is performed. However, it is preferable to store the data of the sexing model created in advance in a data analysis device, and to call up and use the model from the data analysis device when this process is performed on the eggs to be measured. In this embodiment, the time required for sexing can be shortened.

In this process, it has been found that sex determination can be performed with high accuracy by performing sex determination based on spectral data in the wavelength range of 1700 to 2500 nm. The wavelength range of the spectral data used in this process is 1700 to 2500 nm, preferably 1700 to 2200 nm or 1800 to 2500 nm, and more preferably 1800 to 2200 nm. The spectral data used in this process may be spectral data of light having all wavelengths belonging to the wavelength range continuously, or may be spectral data of light having some wavelengths (e.g., specific wavelengths) belonging to the wavelength range. The wavelength range belongs to the near-infrared light range on the long wavelength side. From the spectral data in the wavelength range on the long wavelength side, information on various components such as proteins and fats contained in the eggshell and inside the chicken egg can be obtained. It is thought that the composition of various components contained inside the chicken egg, especially the animal pole of the egg yolk or the embryo, differs slightly depending on the sex. Therefore, by carrying out this process based on spectral data in the near-infrared light region on the long wavelength side of the above range, it is possible to determine the sex of the egg with high accuracy based on slight differences in the composition of the various components contained in the eggshell and inside the egg.

In this process, it is preferable to determine the sex of the egg based on spectral data in the wavelength range of 400 to 900 nm. The further spectral data used in this embodiment may be spectral data of light having all wavelengths in the wavelength range continuously, or may be spectral data of light having some wavelengths (e.g., specific wavelengths) in the wavelength range. The wavelength range belongs to the visible light range. From the spectral data in the visible light range, color information of the blood and the like contained in the eggshell surface and inside the egg can be obtained. It is considered that the color of the blood and the like contained in the inside of the egg, particularly the animal pole of the egg yolk or the embryo, differs slightly depending on the sex. Therefore, by carrying out this process based on the spectral data in the visible light range in addition to the long-wavelength near-infrared light range, it is possible to determine the sex with higher accuracy based on the slight difference in the composition of various components contained in the eggshell and inside the egg, as well as the slight difference in the color of the blood and the like contained in the eggshell surface and inside the egg.

When sexing eggs using the method of this aspect, it has been found that the accuracy of the sexing can vary depending on the positional relationship between the light irradiation step and the light detection step, the size of the egg, and/or the equipment used to carry out the steps (e.g., a visible and near-infrared spectrometer). In the method of this aspect, in the first, second, third, and fourth embodiments, mainly transmitted light is detected, and in the fifth and sixth embodiments, mainly reflected light is detected. Therefore, by selecting an appropriate embodiment based on the positional relationship between the light irradiation step and the light detection step, the size of the egg, and/or the equipment used to carry out the steps, sexing can be carried out with greater accuracy.

2. A program for carrying out a method for determining the sex of chicken eggs before they are hatched.
Another aspect of the present invention relates to a program for carrying out a method for determining the sex of chicken eggs before hatching.

The program of this embodiment can be used to execute the method of determining the sex of a chicken egg before hatching according to one embodiment of the present invention on a data analysis device (e.g., a computer). The program of this embodiment includes a light irradiation step (S1), a light detection step (S2), a spectrum acquisition step (S3), and a sex determination step (S4). Each step corresponds to each step of the method of determining the sex of a chicken egg before hatching according to one embodiment of the present invention described above.

<3. Device for determining the gender of chicken eggs before they hatch>
Another aspect of the invention relates to an apparatus for determining the sex of chicken eggs before they hatch.

Figure 2 shows a block diagram of one embodiment of the device for determining the gender of a chicken egg before hatching according to this embodiment. As shown in Figure 2, the device 100 of the present invention includes a light irradiation means 4 that irradiates a chicken egg 1 with light having a wavelength ranging from the visible light region to the near-infrared light region, a light detection means 6 that detects light emitted outside the chicken egg 1 when the light irradiated by the light irradiation means 4 passes through the chicken egg 1 or is reflected within the chicken egg 1, a spectrum acquisition means that acquires the visible and near-infrared spectra of the light detected by the light detection means 6, and a sex determination means that determines the gender of the chicken egg 1 based on the spectral data in the visible and near-infrared spectra acquired by the spectrum acquisition means. By using this device having the above configuration, it is possible to determine the gender of a chicken egg before hatching.

The device 100 of the present invention preferably includes an egg fixing member 3. In the device 100 of the present invention, the egg 1 is placed on the upper surface of the egg fixing member 3 so that, for example, its lower surface is in contact with the egg fixing member 3. In one embodiment, the egg 1 is placed on the upper surface of the egg fixing member 3 so that the long axis line connecting the blunt end and the sharp end is perpendicular to the horizontal plane. In another embodiment, the egg 1 is placed on the upper surface of the egg fixing member 3 so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane. By including the egg fixing member 3, the egg 1 can be held in the position exemplified above while the visible and near-infrared spectra are measured by the device of this embodiment. This can improve the accuracy of sex determination by the device of this embodiment.

The device 100 of this embodiment preferably includes a storage member 10 that stores the egg 1, the egg fixing member 3, the light irradiating means 4, and the light detecting means 6. The storage member 10 is preferably a member that can substantially block external light, such as a dark box. By storing the above members and means inside the storage member 10, the influence of external light can be substantially suppressed, and the accuracy of sex determination can be further improved.

The light irradiation means 4 is preferably a light irradiation element selected from the group consisting of a halogen lamp, a light emitting diode (LED), and an EverGlo ceramic. The light irradiation means 4 is connected to the light source 5, for example, by an optical fiber cable or the like. The light source 5 is used to oscillate light in the desired wavelength region described above. The irradiated light may be dispersed by the light source 5 or may be dispersed by the light irradiation means 4. In the device of this embodiment, the number of light irradiation means may be one or more, and is preferably one.

The light detection means 6 is preferably a light detection element selected from the group consisting of silicon, PbS (lead sulfide), InGaAs (indium gallium arsenide) and arsenide. The light detection means 6 is connected to the detector 7, for example, by an optical fiber cable or the like. The detector 7 is used to detect light in the desired wavelength region described above. In the device of this embodiment, the number of light detection means may be one or more, and is preferably one or two.

In the device of this embodiment, the light irradiating means and the light detecting means may be arranged as separate components or as a single component.

The spectrum acquisition means is an instrument including a detector 7 and is used to generate visible and near infrared spectra based on the light detected by the light detection means 6. The spectrum acquisition means is preferably a spectroscopic instrument 8 including a light source 5 in addition to the detector 7, such as a visible and near infrared spectroscopic device typically used in the art.

The sex determination means may be a data analysis device 9 that is typically used in the technical field for analyzing spectral data of visible and near-infrared spectra. The data analysis device typically has a central control unit, a spectral data creation unit, an analysis processing unit, a measurement data storage unit, a sex determination model storage unit, an input unit, and an output unit. The data analysis device typically stores a program for performing multivariate analysis of the spectral data. The data analysis device is preferably in the form of a computer having, for example, a central processing unit, a storage device such as a hard drive, an input device, and an output device. In this case, in addition to the control program for the near-infrared spectroscopic device, a program for executing the method for determining the sex of a chicken egg before hatching according to one embodiment of the present invention is installed in the computer, and by executing the method, it is possible to easily achieve measurement of the visible and near-infrared spectra of the chicken egg to be measured and sex determination at low cost.

When using the device of this embodiment to determine the gender of a chicken egg before it hatches, the light irradiating means and light detecting means can be positioned in various positions relative to the egg. Schematic diagrams showing embodiments of the device of this embodiment are shown in Figures 3 to 6.

One embodiment of the device of this aspect is shown in FIG. 3. As shown in FIG. 3, in the device 101 of this embodiment, the light irradiation means 14 is arranged to irradiate light from above the egg 11 in any direction passing through the animal pole or embryo 12 to the egg 11 arranged so that the long axis line connecting the blunt end and the sharp end is perpendicular to the horizontal plane (for example, in a direction in the range of 0 to 50° to the long axis line, particularly in a direction parallel to the long axis line on a plane parallel to a vertical plane including the long axis line), and the light detection means 16 is arranged to detect light emitted to the side of the egg 11 by passing through the egg 11 arranged so that the long axis line connecting the blunt end and the sharp end is perpendicular to the horizontal plane, and passing through the yolk of the egg 11 (for example, in a direction in the range of 40 to 90° to the long axis line, particularly in a direction perpendicular to the long axis line on a horizontal plane perpendicular to the long axis line) (hereinafter, also referred to as the "first embodiment"). In the device 101 of the first preferred embodiment, the light irradiating means 14 is arranged to irradiate light on a plane parallel to the vertical plane including the long axis of the egg 11, which is arranged so that the long axis connecting the blunt end and the sharp end is perpendicular to the horizontal plane, from above the egg 11 and passes through the animal pole or embryo 12, in a direction parallel to the long axis. The light detecting means 16 is arranged to detect light emitted to the side of the egg 11 by passing through the egg 11, which is arranged so that the long axis connecting the blunt end and the sharp end is perpendicular to the horizontal plane, and light emitted in a direction perpendicular to the long axis on the horizontal plane perpendicular to the long axis, which passes through the yolk of the egg 11. The device 101 preferably includes an egg fixing member 13. The egg 11 is placed on the upper surface of the egg fixing member 13 so that, for example, its lower surface is in contact with the egg fixing member 13.

Another embodiment of the device of this aspect is shown in Fig. 4. As shown in Fig. 4, in the device 102 of this embodiment, the light irradiation means 24 is arranged to irradiate light in any direction (for example, in a direction in the range of 0 to 90° with respect to the long axis line, particularly in a direction perpendicular to the long axis line on a plane parallel to the horizontal plane including the long axis line) from the side of the chicken egg 21 through the animal pole or embryo 22, to the chicken egg 21 arranged such that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane, and the light detection means
26 is positioned to detect light that is emitted below chicken egg 21 by passing through chicken egg 21 arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane, and that is emitted in any direction so as to pass through the yolk of chicken egg 21 (for example, in a direction in the range of 40 to 90° with respect to the long axis line, particularly in a direction perpendicular to the long axis line on a plane parallel to a vertical plane including the long axis line) (hereinafter, also referred to as the "second embodiment"). In the device 102 of the second preferred embodiment, the light irradiating means 24 is arranged to irradiate light to the egg 21 arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane in a direction perpendicular to the long axis line on a plane parallel to the horizontal plane including the long axis line, so as to pass from the side of the egg 21 through the animal pole or embryo 22, and the light detecting means 26 is arranged to detect light emitted downward from the egg 21 by passing through the egg 21 arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane, and emitted in a direction perpendicular to the long axis line on a plane parallel to the vertical plane including the long axis line, so as to pass through the yolk of the egg 21. The device 102 preferably includes an egg fixing member 23. The egg 21 is placed on the upper surface of the egg fixing member 23 so that the lower surface of the egg 21 is in contact with the egg fixing member 23, for example. It is preferable that the egg fixing member 23 is provided with a through hole for detecting the light emitted downward from the egg 21 by the light detecting means 26 .

Another embodiment of the device of this aspect is shown in FIG. 5. As shown in FIG. 5, in the device 103 of this embodiment, the light irradiation means 34 is arranged to irradiate light to the chicken egg 31 arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane from above the chicken egg 31 in any direction passing through the animal pole or embryo 32 (for example, in a direction in the range of 40 to 90 degrees to the long axis line, particularly in a direction perpendicular to the long axis line on a plane parallel to a vertical plane including the long axis line), and the light detection means 36 is arranged to detect light emitted downward from the chicken egg 31 by passing through the chicken egg 31 arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane, and emitted in any direction passing through the yolk of the chicken egg 31 (for example, in a direction in the range of 40 to 90 degrees to the long axis line, particularly in a direction perpendicular to the long axis line on a plane parallel to a vertical plane including the long axis line) (hereinafter, also referred to as the "third embodiment"). In the device 103 of the third preferred embodiment, the light irradiating means 34 is disposed so as to irradiate light to the egg 31 arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane, in a direction perpendicular to the long axis line, so as to pass from above the egg 31 through the animal pole or embryo 32, on a plane parallel to the vertical plane including the long axis line, and the light detecting means 36 is disposed so as to detect light emitted downward from the egg 31 by passing through the egg 31 arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane, and also emitted in a direction perpendicular to the long axis line, so as to pass through the yolk of the egg 31. The device 103 preferably includes an egg fixing member 33. The egg 31 is placed on the upper surface of the egg fixing member 33 so that, for example, its lower surface is in contact with the egg fixing member 33. It is preferable that the egg fixing member 33 has a through hole so that the light detection means 36 can detect the light emitted below the egg 31.

Another embodiment of the device of this aspect is shown in Fig. 6. As shown in Fig. 6, in the device 104 of this embodiment, the light irradiation means 44 is arranged to irradiate light to a chicken egg 41 arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane from the side of the chicken egg 41 in any direction passing through the animal pole or embryo 42 (for example, in a direction in the range of 0 to 90° with respect to the long axis line, particularly in a direction perpendicular to the long axis line on a plane parallel to the horizontal plane including the long axis line), and the light detection means 46 is arranged to detect light emitted to the side of the chicken egg 41 by passing through the chicken egg 41 arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane, and emitted in any direction passing through the yolk of the chicken egg 41 (for example, in a direction in the range of 0 to 90° with respect to the long axis line, particularly in a direction perpendicular to the long axis line on a plane parallel to the horizontal plane including the long axis line) (hereinafter, also referred to as "fourth embodiment"). In the device 104 of the fourth preferred embodiment, the light irradiating means 44 is arranged to irradiate light to the egg 41 arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane in a direction perpendicular to the long axis line on a plane parallel to the horizontal plane including the long axis line, so as to pass from the side of the egg 41 through the animal pole or embryo 42, and the light detecting means 46 is arranged to detect light emitted to the side of the egg 41 by passing through the egg 41 arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane, and emitted in a direction perpendicular to the long axis line on a plane parallel to the horizontal plane including the long axis line, so as to pass through the yolk of the egg 41. The device 104 preferably includes an egg fixing member 43. The egg 41 is placed on the upper surface of the egg fixing member 43 so that, for example, its lower surface is in contact with the egg fixing member 43.

Another embodiment of the device of this aspect is shown in FIG. 7. As shown in FIG. 7, in the device 105 of this embodiment, the light irradiation means 54 is arranged to irradiate light to the chicken egg 51 arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane from below the chicken egg 51 in any direction passing through the animal pole or embryo 52 (for example, in a direction in the range of 40 to 90 degrees to the long axis line, particularly in a direction perpendicular to the long axis line on a plane parallel to a vertical plane including the long axis line), and the light detection means 56 is arranged to detect light emitted downward from the chicken egg 51 by reflection inside the chicken egg 51 arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane, and emitted in any direction passing through the yolk of the chicken egg 51 (for example, in a direction in the range of 40 to 90 degrees to the long axis line, particularly in a direction of 45 degrees to the long axis line on a plane parallel to a vertical plane including the long axis line) (hereinafter, also referred to as the "fifth embodiment"). In the device 105 of the fifth preferred embodiment, the light irradiating means 54 is disposed to irradiate light to the egg 51 arranged such that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane in a direction perpendicular to the long axis line, so as to pass through the animal pole or embryo 52 from below the egg 51, on a plane parallel to a vertical plane including the long axis line, and the light detecting means 56 is disposed to detect light that is emitted downward from the egg 51 by reflection inside the egg 51 arranged such that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane, and that is emitted in a direction at an angle of 45° to the long axis line on a plane parallel to the vertical plane including the long axis line, so as to pass through the yolk of the egg 51. The device 105 preferably includes two light detecting means 56. The device 105 preferably includes an egg fixing member 53. The egg 51 is placed on the upper surface of the egg fixing member 53, for example, so that its lower surface is in contact with the egg fixing member 53. It is preferable that the egg fixing member 53 has a through hole for irradiating light below the egg 51 with the light irradiating means 54 and/or for detecting light emitted below the egg 51 with the light detecting means 56.

Another embodiment of the device of this aspect is shown in Fig. 8. As shown in Fig. 8, in the device 106 of this embodiment, the light irradiating means is arranged to irradiate light from above the egg 61 to the chicken egg 61 arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane in any direction passing through the animal pole or embryo 62 (for example, in a direction in the range of 40 to 90° to the long axis line, particularly in a direction perpendicular to the long axis line on a plane parallel to a vertical plane including the long axis line), and the light detecting means is arranged to detect light that is emitted above the egg 61 by reflection inside the egg 61 arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane, and that is emitted in any direction passing through the yolk of the egg 61 (for example, in a direction in the range of 40 to 90° to the long axis line, particularly in a direction perpendicular to the long axis line on a plane parallel to a vertical plane including the long axis line) (hereinafter, also referred to as "sixth embodiment"). In the device 106 of the sixth preferred embodiment, the light irradiating means is arranged to irradiate light to the egg 61 arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane in a direction perpendicular to the long axis line, so as to pass through the animal pole or embryo 62 from above the egg 61, on a plane parallel to a vertical plane including the long axis line, and the light detecting means is arranged to detect light that is emitted above the egg 61 by reflection inside the egg 61 arranged so that the long axis line connecting the blunt end and the sharp end is parallel to the horizontal plane, and that is emitted in a direction perpendicular to the long axis line, so as to pass through the yolk of the egg 61. In the device 106 of this embodiment, the light irradiating means and the light detecting means may be arranged as separate members, or the light irradiating means and the light detecting means may be arranged as a single member 64, as shown in FIG. 8. The device 106 preferably includes an egg fixing member 63. The egg 61 is placed on the upper surface of the egg fixing member 63 so that the lower surface of the egg 61 contacts the egg fixing member 63, for example.

When sexing an egg using the device of this aspect, it has been found that the accuracy of the sexing can vary depending on the positional relationship of the light irradiating means and light detecting means to the egg, the size of the egg, and/or the equipment corresponding to said means (e.g., visible and near-infrared spectroscopic device). Of the devices of this aspect, the first, second, third, and fourth embodiments mainly detect transmitted light, while the fifth and sixth embodiments mainly detect reflected light. Therefore, sexing can be performed with higher accuracy by selecting an appropriate embodiment based on the positional relationship of the light irradiating means and light detecting means, the size of the egg, and/or the equipment corresponding to said means.

As explained in detail above, by carrying out the method of determining the sex of chicken eggs before hatching according to one embodiment of the present invention, it is possible to determine the sex with high accuracy before the seventh day after incubation, when the embryo is thought to acquire a sense of pain. Therefore, by carrying out the method of one embodiment of the present invention, eggs that are likely to become male can be removed early before hatching, and the culling of male chicks can be essentially avoided. In addition, by selecting and incubating eggs that are likely to become female, it is possible to reduce the cost of incubation and to improve the efficiency of egg-laying hens production.

The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

I. Materials and Methods
[I-1: Fertilized egg]
Fertilized eggs of White Leghorn (Shirotama) breed produced at the National Research and Development Agency, National Agriculture and Food Research Organization (NARO) Livestock Research Division and Okazaki Farm of the Livestock Breeding Center were used. 321 fertilized eggs were used for the transmission method and 160 fertilized eggs were used for the reflection method.

[I-2: Equipment]
In the following experiments, two near-infrared spectrometers were used. For the transmission method, NIRSystem (now Foss) Model 6500 was used, and for the reflection method, Methrohm NIRS XDS MultiVial Analyzer was used. A schematic diagram of measuring visible and near-infrared spectra by the transmission method using the above-mentioned device is shown in FIG. 3. As shown in FIG. 3, in this device 101, a chicken egg 11 was placed on a sample fixing table (egg fixing member 13) 13 so that the long axis line connecting the blunt end and the sharp end was perpendicular to the horizontal plane. At this time, the position of the animal pole or embryo 12 of the chicken egg 11 was confirmed in advance with an egg candling device, and the chicken egg 11 was placed on the sample fixing table (egg fixing member 13) 13 so that the animal pole or embryo 12 was on the upper side. The light emitting element 14 was arranged so that it irradiated the chicken egg 11 with light in a direction parallel to the long axis line on a plane parallel to the vertical plane including the long axis line so as to pass through the animal pole or embryo 12 from above the chicken egg 11. The light detection element 16 was positioned so as to detect transmitted light that was emitted to the side of the egg 11 by passing through the egg 11 and that was emitted in a direction perpendicular to the long axis on a horizontal plane perpendicular to the long axis so as to pass through the yolk of the egg 11. The light irradiation element 14 and the light detection element 16 were connected by optical fiber cables to a light source and a detector, respectively, that were placed outside the dark box.

For the reflection method, a Methrohm NIRS XDS MultiVial Analyzer was used. A schematic diagram of the measurement of visible and near-infrared spectra by the reflection method using the above-mentioned device is shown in Figure 7. As shown in Figure 7, in this device 105, a chicken egg 51 was placed on a sample fixing table (egg fixing member 13) 53 so that the plane including the long axis line connecting the blunt end and the sharp end was parallel to the horizontal plane. At this time, the position of the animal pole or embryo 52 of the chicken egg 51 was confirmed in advance with an egg candling device, and the chicken egg 51 was placed on the sample fixing table (egg fixing member 13) 53 so that the animal pole or embryo 52 was on the lower side. The light emitting element 54 was arranged so that it irradiated the chicken egg 51 with light in a direction perpendicular to the long axis line on a plane parallel to the vertical plane including the long axis line, so as to pass through the animal pole or embryo 52 from below the chicken egg 51. The light detection element 56 was positioned to detect light that was reflected inside the egg 51 and emitted downward from the egg 51, and that passed through the yolk of the egg 51, and was emitted in a direction at 45° to the long axis on a plane parallel to a vertical plane including the long axis. A through hole was provided in the sample fixing stage (egg fixing member 13) 53 to allow the light irradiation element 54 to irradiate light downward from the egg 51. The light irradiation element 54 and the light detection element 56 were connected by optical fiber cables to a light source and a detector located outside the dark box, respectively.

[I-3: Measurement of visible and near infrared spectra]
On the 3rd, 4th, 5th and 6th days after incubation, the fertilized eggs were irradiated with light (halogen lamp) and the visible and near infrared spectra were measured. In both methods, one or two points per fertilized egg were measured by shifting the measurement position, and all the obtained spectra were used for identification confirmation without averaging. In both methods, the measurement range of the visible and near infrared spectra was 400 to 2500 nm.

[I-4: Sex determination by genetic analysis]
After the visible and near-infrared spectrum measurements, the eggs were cracked and the embryos and blood samples were collected. DNA was extracted from the collected samples. Using the obtained DNA samples, the sex of each egg was determined by multiplex PCR using sex-specific primers.

[I-5: Sexing based on visible and near infrared spectra]
The obtained visible and near-infrared spectra were analyzed using the software (Vision) attached to the instrument. The visible and near-infrared spectra were subjected to second-order differential processing to obtain second-order derivative spectra. Spectral data was compiled for each sex determined by genetic analysis.

In the case of the transmission method, principal component analysis was performed on the second derivative spectral data groups for both sexes. In the principal component space for both sexes, outliers were detected using Mahalanobis distance, and principal component score plots were obtained with the outliers removed. Using the obtained principal component score plots, the residual variance method in the principal component space was applied to determine the sex. The threshold value was set at 0.7.

In the case of the reflectance method, principal component analysis was performed on the second derivative spectral data groups for both sexes. In the principal component space for both sexes, outliers were detected using Mahalanobis distance, and principal component score plots were obtained with the outliers removed. Using the obtained principal component score plots, sex was determined by applying the maximum distance method by wavelength or the residual variance method in the principal component space. The threshold value was set at 5 (maximum distance method) or 0.84 (residual variance method).

<II: Results>
[II-1: Measurement of visible and near-infrared spectra]
In the transmission method, 321 fertilized eggs were used, and one to two points of the spectrum were measured for each fertilized egg. An example of the visible and near-infrared spectra measured by the transmission method is shown in Figure 9. In the figure, A is the visible and near-infrared spectrum, and B is its second derivative spectrum. In A, the horizontal axis is the wavelength (nm), and the vertical axis is the absorbance. In B, the horizontal axis is the wavelength (nm), and the vertical axis is the intensity of the second derivative value.

As shown in Figures 9A and B, absorption believed to be due to water was observed in the wavelength ranges of 950 nm, 1450 nm, and 1940 nm. The spectrum was significantly disrupted in the wavelength range of 2200 to 2500 nm. In the device used in this measurement, the detector switches at 1100 nm. For this reason, a spectral step occurred in this wavelength range in the spectra of Figures 9A and B. Therefore, the wavelength range of 1050 to 1150 nm was excluded from the qualitative analysis.

In the reflectance method, 160 fertilized eggs were measured. An example of the visible and near-infrared spectra measured by the reflectance method is shown in FIG. 10. In the figure, A is the visible and near-infrared spectrum, and B is its second derivative spectrum. In A, the horizontal axis is the wavelength (nm) and the vertical axis is the absorbance. In B, the horizontal axis is the wavelength (nm) and the vertical axis is the intensity of the second derivative value.

As shown in Figures 10A and B, absorption believed to be due to water was observed in the wavelength ranges of 1450 nm and 1940 nm. In addition, absorption believed to be due to calcium carbonate (CaCO ₃ ) was observed in the wavelength range of 2340 nm. In the visible and near-infrared spectra obtained by the reflection method, the spectral disturbance in the wavelength range of 2200 to 2500 nm observed in the visible and near-infrared spectra obtained by the transmission method was not observed. In the device used for this measurement, the detector is switched at 1100 nm. Therefore, a spectral step occurred in this wavelength range in the spectra of Figures 10A and B. Therefore, the wavelength range of 1050 to 1150 nm was excluded from the qualitative analysis.

[II-2: Sexing based on visible and near infrared spectra]
In the transmission method, 321 fertilized eggs on the 5th day after incubation were used, and the spectrum was measured at 1-2 points per fertilized egg. From the second derivative spectral data of each group of males and females, outliers suspected to be due to measurement errors were eliminated using the Mahalanobis distance in the principal component space. Using the spectral data in a specified wavelength range (female: 275 points, male: 259 points) after excluding the outliers, the residual variance method in the principal component space was applied, and sex determination was performed by setting the threshold at 0.7. The wavelength ranges used for sex determination were as follows: Test i-1: 400-1050 nm and 1150-2500 nm (all wavelength ranges from visible light to near-infrared light); Test i-2: 400-900 nm (visible light range, wavelength range described in Patent Document 3); Test i-3: 400-1050 nm (visible light range to short-wavelength near-infrared light range); Test i-4: 1150-1800 nm (shorter-wavelength near-infrared light range); Test i-5: 1700-2200 nm (longer-wavelength near-infrared light range); Test i-6: 400-1050 nm and 1150-1500 nm (wavelength range combining the conditions of Tests i-3 and i-4 (partially), wavelength range described in Patent Document 1); Test i-7: 400-1050 The wavelength ranges were 1150-1800 nm and 400-900 nm (wavelength ranges combining the conditions of tests i-3 and i-4), test i-8: 1150-2200 nm (wavelength ranges combining the conditions of tests i-4 and i-5), and test i-9: 400-900 nm and 1700-2200 nm (wavelength ranges combining the conditions of tests i-2 and i-5). The results of sexing in the transmission method are shown in Table 1.

As shown in Table 1, by using spectral data including the near-infrared region by the transmission method to determine sex, the sex determination rate exceeded 70% for both sexes (Tests i-1, and i-3 to i-9). In the near-infrared region, the sex determination rate was higher when spectral data including the long-wavelength region was used (Tests i-5 and i-9) compared to when spectral data only from the short-wavelength region was used (Test i-4).

As disclosed in Patent Document 3, when spectral data in the visible light region only was used (Test i-2), the identification rate for both males and females was 70% or less. When spectral data in the wavelength region corresponding to the range of 400 to 1500 nm disclosed in Patent Document 1 was used (Test i-6), the identification rate for females was 78.8%, and the identification rate for males was 89.2%. In contrast, when spectral data in the wavelength region of the long-wavelength near-infrared light range of 1700 to 2200 nm was used (Test i-5), the identification rate for females was 79.2%, and the identification rate for males was 93.9%. In particular, when spectral data in the wavelength region combining the long-wavelength near-infrared light region with the visible light region was used (Test i-9), the identification rate for both males and females exceeded 90%. From the above results, it became clear that by using spectral data in the long-wavelength near-infrared light region, it is possible to determine the sex of the fish with a higher identification rate than when using the spectral data in the wavelength region disclosed in the prior art. It was also revealed that the accuracy rate can be further improved by using spectral data from a wavelength range that combines the long-wavelength near-infrared light region with the visible light region.

For the reflectance method, 160 fertilized eggs on the 6th day after incubation were used, and the spectra were measured at two points per fertilized egg. From the second derivative spectral data of each group of males and females, outliers suspected to be due to measurement errors were removed using the Mahalanobis distance in the principal component space. The spectral data in the specified wavelength range after removing the outliers (females: 156 points, males: 164 points) were used to determine the sex of the eggs by applying the maximum distance method by wavelength or the residual variance method in the principal component space, setting the threshold to 5 (maximum distance method) or 0.84 (residual variance method). The wavelength ranges used for sex determination were: Test ii-5: 1800-2500 nm (near-infrared light range on the long wavelength side), and Test ii-9: 400-900 nm and 1700-2500 nm (wavelength range combining the conditions of ii-5 with the visible light range). The results of sex determination using the reflectance method are shown in Table 2.

As shown in Table 2, when sexing by the reflectance method, a sexing rate of over 60% was achieved by applying either the maximum distance method or the residual dispersion method. In particular, when spectral data in the wavelength range of 1800-2500 nm, which is the long-wavelength near-infrared light region, was used (Test ii-5), the female identification rate was 80.6% when the residual dispersion method was applied, and the male identification rate was 64.3% when the maximum distance method was applied. These results demonstrate that by using spectral data in the long-wavelength near-infrared light region, sexing can be achieved by the reflectance method as well as the transmittance method.

When measuring the spectrum from the visible light region to the near-infrared light region of eggs 3 to 6 days after incubation, it is believed that spectral data in the visible light region will provide color information on the eggshell surface and blood and other substances contained inside the egg, while spectral data in the longer wavelength region will provide information on compounds such as proteins and fats contained in the eggshell and inside the egg. By determining sex using spectral data in a wavelength region that combines the visible light region and the longer wavelength near-infrared light region, it is believed that the accuracy of sexing can be improved by combining color information and compound information.

The present invention is not limited to the above-described embodiments, but includes various modifications. For example, the above-described embodiments have been described in detail to clearly explain the present invention, and the present invention is not necessarily limited to those having all of the configurations described. In addition, it is possible to add, delete, and/or replace part of the configuration of each embodiment with other configurations.

100, 101, 102, 103, 104, 105, 106...Devices for determining the sex of chicken eggs before hatching
1, 11, 21, 31, 41, 51, 61…Egg
12, 22, 32, 42, 52, 62...Animal pole or embryo
3, 13, 23, 33, 43, 53, 63...Egg fixing parts
4, 14, 24, 34, 44, 54...light irradiation means
64...Light irradiation and detection means
5…Light source
6, 16, 26, 36, 46, 56...Light detection means
7…Detector
8. Spectroscopic equipment
9. Data analysis equipment
10...Housing member

Claims (8)

1. A method for determining the sex of a chicken egg before hatching, comprising the steps of:
a light irradiation step of irradiating chicken eggs 3 to 6 days after incubation with light having a wavelength ranging from the visible light region to the near infrared light region;
a light detection step of detecting light emitted outside the egg as a result of the light irradiated in the light irradiation step passing through the egg or being reflected within the egg;
a spectrum acquiring step of acquiring the visible and near-infrared spectra of the light detected in the light detecting step; and a sex determining step of determining the sex of the egg based on the spectral data in the wavelength region of 1700 to 2500 nm in the visible and near-infrared spectra acquired in the spectrum acquiring step;
The method comprising: The method according to claim 1, further comprising determining the sex of the chicken eggs based on spectral data in the wavelength range of 400 to 900 nm in the sex determination step. the light irradiated in the light irradiation step is light irradiated from above the egg in a direction parallel to the long axis line connecting the blunt end and the sharp end, the long axis line passing through the animal pole or embryo, on a plane parallel to the vertical plane including the long axis line, to the egg arranged so that the long axis line is perpendicular to the horizontal plane;
3. The method according to claim 1 or 2, wherein the light detected in the light detection step is light that passes through a chicken egg that is placed so that a long axis line connecting the blunt end and the sharp end is perpendicular to a horizontal plane, and is emitted laterally from the chicken egg, and is light that is emitted in a direction perpendicular to the long axis line on a horizontal plane perpendicular to the long axis line so as to pass through the yolk of the chicken egg. the light irradiated in the light irradiation step is light irradiated from below the egg in a direction perpendicular to the long axis line connecting the blunt end and the sharp end, the long axis line passing through the animal pole or the embryo, on a plane parallel to a vertical plane including the long axis line, to the egg arranged such that the long axis line is parallel to a horizontal plane;
3. The method according to claim 1 or 2, wherein the light detected in the light detection step is light that is reflected inside a chicken egg placed so that a long axis line connecting the blunt end and the sharp end is parallel to a horizontal plane, and is emitted downward from the egg, and is emitted in a direction at an angle of 45° to the long axis line on a plane parallel to a vertical plane including the long axis line, so as to pass through the yolk of the chicken egg. 1. An apparatus for determining the sex of chicken eggs before they hatch, comprising:
a light irradiation means for irradiating the eggs with light having a wavelength ranging from the visible light region to the near infrared light region;
a light detection means for detecting light emitted outside the egg as a result of the light irradiated by the light irradiating means passing through the egg or being reflected within the egg;
a spectrum acquiring means for acquiring the visible and near-infrared spectra of the light detected by the light detecting means; and a sex determining means for determining the sex of the egg based on the spectral data in the wavelength region of 1700 to 2500 nm in the visible and near-infrared spectra acquired by the spectrum acquiring means;
The apparatus comprising: The device according to claim 5, wherein the sex determining means further determines the sex of the chicken egg based on spectral data in the wavelength range of 400 to 900 nm. the light irradiation means is disposed so as to irradiate a chicken egg with light in a direction parallel to the long axis line connecting the blunt end and the sharp end, the long axis line passing through the animal pole or the embryo from above the chicken egg, on a plane parallel to a vertical plane including the long axis line,
7. The apparatus according to claim 5 or 6, wherein the light detection means is arranged to detect light emitted laterally from a chicken egg arranged such that a long axis connecting the blunt end and the sharp end is perpendicular to a horizontal plane, and the light is emitted in a direction perpendicular to the long axis on a horizontal plane perpendicular to the long axis so as to pass through the yolk of the chicken egg. the light irradiation means is arranged so as to irradiate light to a chicken egg arranged so that a long axis line connecting the blunt end and the sharp end is parallel to a horizontal plane, in a direction perpendicular to the long axis line on a plane parallel to a vertical plane including the long axis line, so as to pass through the animal pole or embryo from below the chicken egg,
7. The apparatus according to claim 5 or 6, wherein the light detection means is arranged to detect light emitted downward from an egg by reflection inside the egg, the egg being arranged so that a long axis connecting the blunt end and the sharp end is parallel to a horizontal plane, and the light is emitted in a direction at an angle of 45° to the long axis on a plane parallel to a vertical plane including the long axis so as to pass through the yolk of the egg.