JP7061740B2 - Fertilized egg retention structure and fertilized egg inspection method using it - Google Patents

Description

The present invention relates to a fertilized egg holding structure and a fertilized egg inspection method using the same.

The use of fertilized eggs is drawing attention in the medical and pharmaceutical fields. For example, after a predetermined chemical solution is administered to a fertilized egg of zebrafish, the action and effect of this chemical solution are determined by spectroscopically and imagingly inspecting the hatched zebrafish.

Patent Document 1 discloses a technique for spectroscopically and imagingly inspecting a large number of zebrafish individually housed in a large number of zebrafish wells. For better handling efficiency, a large number of zebrafish wells are formed in a matrix on the upper surface of the zebrafish plate.

Patent Document 2 discloses a technique for optically and imagingly inspecting a large number of zebrafish fertilized eggs individually housed in a large number of fertilized egg wells. It is necessary to prevent the displacement of the fertilized egg in drug administration, spectroscopy and imaging. For this reason, the fertilized egg must be held in a fertilized egg well formed to be slightly larger in diameter than the fertilized egg. For efficient handling, a large number of fertilized egg wells are formed in a matrix on the upper surface of the fertilized egg plate.

After completing various operations on each fertilized egg arranged in the fertilized egg well of the fertilized egg plate, the fertilized egg is transferred from the fertilized egg well of the fertilized egg plate to the zebrafish fry well of the zebrafish fry plate. Be transferred. The fertilized egg has been grown in this zebrafish well for a predetermined period, and the influence of the drug is examined spectroscopically and imagewise.

However, fertilized zebrafish eggs with a very small diameter, for example about 1 mm, have a very thin and vulnerable egg wall. Therefore, it was not easy to transfer the fertilized egg from the fertilized egg plate to the zebrafish plate. The complexity of handling such fine fertilized eggs has increased processing costs.

In addition, the mortality rate of fertilized eggs increased due to damage to the egg wall during transfer work. This increase in fertilized egg mortality led to waste of expensive chemicals and a decrease in productivity. In the end, the difficult work process of transferring the fertilized egg from the fertilized egg plate to the zebrafish plate has hindered the spread of the zebrafish fertilized egg utilization technology, although it has various advantages.

Japanese Patent No. 6180134 Japanese Unexamined Patent Publication No. 2018-072094

An object of the present invention is to simplify fertilized egg processing techniques such as zebrafish and improve productivity.

The fertilized egg holding structure of the present invention can be obtained by detachably attaching a fertilized egg holding structure to a hatching holding plate having a well for individually accommodating a hatching such as a zebrafish. Precisely hold the fertilized egg for a predetermined period of time in a predetermined small area in the hatching well.

For example, at the end of this period during which the fertilized egg is subjected to optical separation and imaging and administration of the drug solution to the fertilized egg, the fertilized egg retention structure is removed from the hatchery retention plate. This allows the hatch to grow to the required size in the widened hatch well. Generally, after the hatch has grown in the hatch well, the required spectroscopy and imaging information is taken from the hatch.

After all, the fertilized egg can be subjected to optical separation, imaging inspection, and injection with high efficiency and accuracy while the displacement of the fertilized egg is restricted by the fertilized egg holding structure. In addition, the fertilized egg retention structure can be removed from the hatchery retention plate after completion of these treatments to ensure sufficient volume in the hatchet well for the hatchet to grow.

The small area, called the fertilized egg retention area, is compartmentalized on the transparent bottom surface of the well used in hatchery testing. This makes it possible to carry out a fertilized egg test using a hatchery test device. In the spectroscopic and imaging inspection of fertilized eggs, the inspection area is limited, so that the inspection time can be shortened. As is well known, the inspection of a fertilized egg is most preferably performed for a short time immediately before the fertilized egg hatches. Therefore, when a large number of fertilized eggs are sequentially separated and image-tested, the test time that can be assigned to one fertilized egg becomes very short.

Further, a fertilized egg holding structure capable of restricting the horizontal movement of the fertilized egg newly establishes a fertilized egg holding region at a predetermined position in the hatching well for a required time. As a result, the work of transferring the fertilized egg from the fertilized egg holding plate to the hatched body holding plate, which has been conventionally required, can be omitted, and the productivity can be improved. Furthermore, by drastically reducing the mortality rate of fertilized eggs, the productivity of the processing work can be significantly improved.

Preferably, the fertilized egg holding structure has a hanging wall that hangs from a frame resting on the upper end surface of the hatching holding plate into each hatching well, and the lower end of the hanging wall is the fertilized egg. Regulate lateral slippage. Thereby, by raising the frame portion, a large number of hanging walls connected to the frame portion can be removed from each hatching well all at once.

Preferably, the hanging wall has a conical or pyramidal shape with open tops and bottoms. Thereby, the fertilized egg introduced into the hatching well can be automatically guided by the hanging wall to a predetermined fertilized egg holding region formed on the transparent bottom surface of the hatching well.

According to the fertilized egg inspection method using the fertilized egg holding structure of the present invention, the fertilized egg is set in a predetermined area on the transparent bottom surface of the hatched well using the fertilized egg holding structure, and then each fertilized egg is separated. Inspect optically and image-wise. The fertilized egg holding structure is then removed from the hatch holding plate and the hatch is grown in the well. Then, the hatched body in the well is examined spectroscopically and imagewise. This can improve the productivity of spectroscopy and imaging of fertilized eggs and hatched bodies.

According to suitable fertilized egg segmentation and imaging tests, the substantially circular region corresponding to the fertilized egg is divided into a substantially circular scutellum region and other yolk regions, and the scutellum region and the yolk region are determined to be different from each other. Good or bad is judged by the method. This makes it possible to accurately and quickly determine the quality of the fertilized egg.

FIG. 1 is a partial plan view showing a zebrafish fry holding plate. FIG. 2 is a partial cross-sectional view of the zebrafish fry holding plate shown in FIG. FIG. 3 is a partial plan view showing a fertilized egg holding structure placed on a zebrafish holding plate. FIG. 4 is a partial cross-sectional view showing a fertilized egg holding structure placed on a zebrafish holding plate. FIG. 5 is a flowchart showing the fertilized egg inspection process. FIG. 6 is a diagram showing the spectral spectral intensities of normal eggs and dead eggs. FIG. 7 is a photographic image of a normal egg. FIG. 8 is a photographic image of a dead egg.

Examples of adopting zebrafish as hatchers are described with reference to the drawings. FIG. 1 is a partial plan view showing a part of the zebrafish fry holding plate 1. FIG. 2 is a vertical cross-sectional view taken along the line AA of the zebrafish fry holding plate 1 shown in FIG.

The rectangular thick plate-shaped zebrafish fry holding plate 1 is composed of a resin frame 2 and a thin transparent resin plate 3 adhered to the bottom surface of the resin frame 2. In addition, in order to reinforce the transparent resin plate 3, a resin plate for reinforcing the transparent resin plate 3 may be added at a site where the zebrafish and its fertilized egg do not interfere with the optical separation and image observation. It is possible.

The resin frame 2 has 96 breeding wells 21 and 96 drainage wells 22 formed in a matrix. The breeding well 21 and the drainage well 22 are each formed in the thickness direction of the resin frame 2, the upper ends thereof are opened, and the lower ends thereof are shielded by the transparent resin plate 3.

Twelve breeding wells 21 each having a space for breeding zebrafish are arranged in the horizontal direction and eight in the vertical direction. In FIG. 1, only one rearing well 21 is fully illustrated.

The breeding well 21 is partitioned by a rectangular transparent bottom 23 on which the zebrafish 4 can lie down, and partition walls 24A, 24B, 24C, and 24D erected from the periphery of the transparent bottom 23. In FIGS. 1 and 2, the zebrafish 4 lies on a transparent bottom 23 having a vertically long rectangular shape.

The lower portions of the two partition walls 24A and 24B in contact with the long side of the transparent bottom portion 23 are obliquely provided. This allows the zebrafish 4 to be guided to the transparent bottom 23 when the water in the rearing well 21 is drained. The remaining two bulkheads 24C and 24D tangent to the short side of the transparent bottom 23 are installed vertically.

Each drainage well 22 is individually adjacent to each breeding well 21. The partition walls 24A and 24B separate the drainage well 22 having a rectangular upper end opening from the breeding well 21. The lower end of the partition wall 24B has a communication hole 25 that communicates the breeding well 21 on the left side and the drainage well 22 on the right side. When the water in the drainage well 22 is sucked upward, the water in the breeding well 21 flows to the drainage well 22 through the communication hole 25. As a result, a descending water flow is formed in the breeding well 21. This descending stream urges the zebrafish downwards. In FIG. 2, water is injected into the breeding well 21 and the drainage well 22 up to the water surface 5.

FIG. 3 is a partial plan view showing a fertilized egg holding structure in which a fertilized egg holding structure 7 is placed on a zebrafish holding plate 1. The zebrafish holding plate 1 hidden below the fertilized egg holding structure 7 is shown by a broken line. FIG. 4 is a vertical cross-sectional view taken along the line BB of FIG.

The fertilized egg holding structure 7 is separately placed in the square frame portion 71 placed on the upper end surface of the zebrafish holding plate 1 shown by the broken line, and from the square frame portion 71 into each breeding well 21 of the zebrafish holding plate 1. It consists of a large number of hanging walls 72. The plate-shaped square frame portion 71 has a rectangular shape substantially equal to that of the rectangular zebrafish holding plate 1. The square frame portion 71 has a square opening 73 formed at the same position as each well 1. The fertilized egg is thrown into this opening 73.

The hanging wall 72 is slanted downward from the peripheral edge of the opening 73 of the square frame portion 71. In other words, the hanging wall 72 consists of a quadrangular pyramid-shaped cylinder with openings at both ends. The lower part of the hanging wall 72 reaches around the square fertilized egg holding region 8 located in the center of the transparent bottom 23. Of the four slopes of the hanging wall 72, a pair of slopes located above the rectangular transparent bottom 23 have vertical protrusions 74A and 74B facing each other across the fertilized egg holding region 8.

The lower ends of the vertical protrusions 74A and 74B substantially reach the rectangular transparent bottom 23. The vertical protrusions 74A and 74B prohibit the fertilized egg 9 on the fertilized egg holding region 8 from being displaced along the transparent bottom 23. In other words, the pyramidal drooping wall 72 guides the fertilized egg 9 onto the fertilized egg holding region 8 and restricts the fertilized egg from laterally displaced from the fertilized egg holding region 8.

In this embodiment, the lower portions of the two partition walls 24A and 24B in contact with the long side of the transparent bottom portion 23 are formed diagonally, but the fertilized egg holding region 8 is formed by vertically lowering the lower ends of the partition walls 24A and 24B. It is also possible to further suppress the lateral displacement of the fertilized egg 9 inside. Further, in this embodiment, the breeding well 21 can be formed into a substantially conical shape. In this case, the hanging wall 72 can also have a substantially conical shape.

Next, a process for processing zebrafish and fertilized eggs using the fertilized egg holding structure described above will be described. First, a fertilized egg holding structure composed of a zebrafish holding plate 1 to which the fertilized egg holding structure 7 is attached is prepared. Next, the fertilized egg of the zebrafish is thrown into the opening 73 of the square frame portion 71 of the fertilized egg holding structure 7, and the water of the drainage well 22 is sucked upward to form a descending water flow in the breeding well 21. As a result, the fertilized egg 9 is set in the fertilized egg holding region 8.

Next, the chemical solution is administered to the fertilized egg 9, and the fertilized egg 9 is subjected to spectroscopic analysis and image inspection. Next, the fertilized egg holding structure 7 is removed upward from the zebrafish holding plate 1. The zebrafish hatched from the fertilized egg 9 are then grown in the expanded rearing well 21 for a predetermined time. Next, the zebrafish 4 that has become asphyxial due to the mixing of the anesthetic into water is laid down on the transparent bottom 23. This lying down is performed by draining the drainage well 22. The zebrafish 4 is then spectroscopically and image-tested.

The spectroscopic and imaging process of the fertilized egg 9 will be described in detail with reference to the flowchart shown in FIG. First, in step S100, the spectral signal and the image signal of the fertilized egg 9 are acquired. The spectroscopic and image sensors in the illustration convert the spectral spectrum emitted from the fertilized egg 9 by the ultraviolet rays applied to the fertilized egg holding region 8 into an electric signal. This electrical signal is called a spectral signal. Next, the two-dimensional image sensor in the figure outputs an image signal from the fertilized egg 9 to the image processor in the figure.

In the next step S102, it is determined whether or not the intensity of the spectral signal is within a predetermined range. If the intensity of the spectral signal is within a predetermined range, in step S104, a substantially circular fertilized egg region is extracted from the image signal, and the fertilized egg region is further divided into a substantially circular scutellum region and this scutellum region. Separate into the surrounding ring-shaped yolk region. Next, in step S106, it is determined whether or not the scutellum region is normal, and in step S108, it is determined whether or not the yolk region is normal. Next, if all of these determination results are normal, the fertilized egg is determined to be normal (step S110), and if not, the fertilized egg is determined to be defective (step S112).

According to this embodiment, in order to determine the quality of the fertilized egg based on the three results of the spectral intensity determination, the image determination of the scutellum region, and the image determination of the yolk region, the defective fertilized egg is automatically determined more precisely than before. be able to.

First, the details of the spectral intensity determination will be described in more detail with reference to FIGS. 6-8. FIG. 6 is a diagram showing spectral intensities obtained from normal eggs and dead eggs. It can be seen that the spectral intensity obtained from normal eggs is almost half that obtained from dead eggs. FIG. 7 shows a photographic image of a normal egg, and FIG. 8 shows a photographic image of a dead egg and subsequently a dead egg. It can be seen that a black cloud-like region that does not exist in the normal egg image exists in the dead egg image. It is presumed that the reason why the dead egg has high spectral intensity is due to the existence of this black cloud-like region.

Next, the details of the image determination of the scutellum region will be described in more detail with reference to FIGS. 7 and 8. The substantially circular fertilized egg is clearly divided into an approximately circular scutellum region and a ring-shaped yolk region. Furthermore, it is understood that the scutellum region has a dark ring-shaped contour. It is understood that in the dead egg, the outer contour of the scutellum region is obscured by the black cloud-like region. According to this embodiment, it is determined whether or not the outer peripheral contour that divides the scutellum region is circular over the entire circumference, and if the outer peripheral contour is circular, the outer peripheral contour is determined to be normal.

Next, it can be seen that many small regions are densely packed in the scutellum region of normal eggs as a result of cell division. This dense subregion group means that the image signal obtained from within the scutellum region contains many high frequency signals. Therefore, according to this embodiment, if the integrated value of the high frequency signal among the image signals in the scutellum region is equal to or higher than a predetermined value, it is determined that the scutellum region is normal.

Next, the details of the image determination of the yolk region will be described in more detail with reference to FIGS. 7 and 8. It can be seen that the yolk region of the normal egg shown in FIG. 7 does not include a region having a peculiar shape and has a uniform brightness. On the other hand, the yolk region of the dead egg shown in FIG. 8 includes a dark cloud-like region.
That is, 1) the size and density of scutellum cells are non-uniform.
2) The outer circumference of the scutellum cell group is extremely irregular, and sometimes dents are observed.
3) A blank cannon is found on the outer circumference of the scutellum cell group.
4) The boundary between scutellum cells and yolk cells is irregular.
5) Diffuse black zones spread widely from the boundary between scutellum cells and yolk cells to both sides.
6) Particulate structures and blanks are found in the yolk cell region.
Therefore, according to this embodiment, if the change in brightness of the yolk region is equal to or less than a predetermined level, it is determined that the yolk region is normal.

After all, the quality judgment of the fertilized egg of this example was made by paying attention to the fact that the defective egg develops a black cloud-like region straddling the scutellum region and the yolk region. Furthermore, it was noted that a large number of small regions are generated in the scutellum region through normal cell division. By these image judgments, it became possible to automatically judge a defective fertilized egg accurately and promptly.

Claims (7)

Wells with an upper end opening capable of accommodating a hatched body hatched from a fertilized egg are arranged in a matrix, and each of the wells has a hatched body holding plate having a transparent bottom surface on which the hatched body can be placed, and from above the well. By hanging in the well and forming a fertilized egg holding region slightly wider than the size of the fertilized egg on the transparent bottom surface, the fertilized egg is formed from the fertilized egg holding region along the transparent bottom surface. It has a fertilized egg retention structure that regulates lateral deviation and
The fertilized egg holding structure is a structure for holding a fertilized egg, which is detachably set on the hatched body holding plate. The fertilized egg holding structure has a frame portion placed on the upper end surface of the hatched body holding plate, and a hanging wall hanging from the frame portion into each well.
The structure for holding a fertilized egg according to claim 1, wherein the hanging wall substantially reaches the transparent bottom surface portion. The fertilized egg holding structure according to claim 2, wherein the hanging wall has a conical shape or a pyramid shape in which the upper end and the lower end are open. The structure for holding a fertilized egg according to claim 1, wherein the hatched body is a zebrafish. A hatchery holding plate in which wells having an upper end opening having a transparent bottom surface on which a hatched body hatched from a fertilized egg can be placed are arranged in a matrix, and a hatching body holding plate that hangs from above the well into the well and onto the transparent bottom surface. The first step of preparing a fertilized egg holding structure for partitioning a fertilized egg holding region slightly wider than the size of the fertilized egg and setting the fertilized egg in the fertilized egg holding region.
The second step of obtaining fertilized egg information from the fertilized egg in the fertilized egg holding region optically and imagewise, and
The third step of determining the quality of the fertilized egg based on the fertilized egg information, and
The fourth step of removing the fertilized egg holding structure from the hatched body holding plate before the hatching of the fertilized egg, and the fourth step.
The fifth step of optically and image-acquiring hatchery information from the hatchet grown in the well from which the fertilized egg holding structure was removed.
A fertilized egg inspection method characterized by sequentially executing. In the third step, the substantially circular region corresponding to the fertilized egg is divided into a substantially circular scutellum region and other egg yolk regions, and the quality of the scutellum region and the yolk region is determined by different determination methods. The fertilized egg inspection method according to claim 5. The fertilized egg inspection method according to claim 5, wherein the hatched body is a zebrafish.

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