Fertile ovum recovery support system

Abstract

To provide a system capable of improving work efficiency when collecting fertilized eggs of fish.SOLUTION: The fertile ovum collection supporting system 1 is a fertile ovum collection supporting system that supports collection of fertile ovum of fish, and includes a water tank 2 that accommodates a male z1 and a female z2 of fish, an imaging unit 3 that is installed below the water tank 2 and captures a bottom surface image of the water tank 2, an illumination unit 4 that irradiates the water tank 2 with light, and a processing device 5 that counts the fertile ovum r based on the bottom surface image of the water tank 2 captured by the imaging unit 3 in a state where the illumination unit 4 is turned on.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an embryo collection support system for counting fertilized eggs of fish such as zebrafish. [Background technology]

[0002] In recent years, zebrafish have been widely used as a model experimental animal for vertebrates in fields such as pharmaceutical research and toxicity research. Zebrafish are small tropical fish, about 5 cm in length, characterized by dark blue stripes on the surface of their bodies as adults. Zebrafish are easy to raise and breed; more than 100 fertilized eggs can be spawned by pairing a male and female.

[0003] However, handling fertilized fish eggs, such as zebrafish eggs, involves complex procedures due to their small size and fragility. Various techniques have been proposed to simplify the procedures for processing fertilized fish eggs.

[0004] For example, Patent Document 1 describes a small fish fertilized egg processing device that includes a multi-well plate having a large number of wells separated by partitions erected on a transparent bottom plate, each well having an open top, a water supply and drainage means for controlling the water supply and drainage to each well, and a predetermined fertilized egg arrangement means for inserting small fish fertilized eggs one by one into each well. This small fish fertilized egg processing device is said to be able to perform the necessary processing on fish fertilized eggs at high speed while minimizing damage to the eggs. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2022-066093 Summary of the Invention [Problem to be solved by the invention]

[0006] When using fertilized fish eggs in various research studies, the eggs are stimulated before they begin nuclear division and the subsequent changes are observed. Therefore, the eggs must be collected promptly after fertilization (for example, within 45 minutes after fertilization).

[0007] However, identifying and collecting fertilized eggs immediately after fertilization requires skilled techniques. Traditionally, parental male and female zebrafish are raised under a 14-hour light period and a 10-hour dark period per day until the day before egg collection. During the light period on the day before egg collection, technicians in charge of rearing observe the behavior of the male and female zebrafish and rely on experience and intuition to predict the number of eggs laid the next day and manage egg collection. Therefore, depending on the technician's experience, there is a risk of inconsistency in predicting and managing the number of eggs laid. In addition, it is necessary to monitor the aquarium to immediately collect fertilized eggs above a certain number, which is not very efficient.

[0008] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a system that can improve work efficiency when collecting fertilized fish eggs. [Means for solving the problem]

[0009] The system of the present invention is a fertilized egg collection support system that supports the collection of fertilized fish eggs, and is characterized by comprising an aquarium that houses male and female fish, an imaging means that is installed below the aquarium and captures images of the bottom of the aquarium, an illumination means that irradiates light toward the aquarium, and a processing device that counts fertilized eggs based on the images of the bottom of the aquarium captured by the imaging means with the illumination means turned on.

[0010] The processing device generates a composite image from a plurality of the bottom images taken multiple times within a predetermined time (for example, two or more times within 10 seconds), and counts the fertilized eggs based on the composite image. Specifically, the processing device generates a composite image by averaging the brightness of the pixels constituting each of the plurality of bottom images, and performs threshold determination based on a predetermined brightness threshold for the pixels constituting the composite image to identify and count the fertilized eggs.

[0011] The processing device is characterized in that it generates the composite image based on a plurality of bottom images taken immediately after the lighting means is turned on after being turned off (for example, within 10 seconds from the time the lighting means is turned on).

[0012] The lighting means is characterized in that it illuminates the aquarium from diagonally above the aquarium.

[0013] The tank is characterized in that a net member is provided at the bottom thereof to separate the area in which the fish swim underwater from the area in which the fertilized eggs are housed.

[0014] The processing device is characterized in that it notifies the outside when the number of counted fertilized eggs is equal to or greater than a predetermined number (for example, 100). [Effects of the Invention]

[0015] The system of the present invention is a fertilized egg collection support system that supports the collection of fertilized fish eggs, and includes an aquarium, an imaging means for capturing images of the bottom of the aquarium, an illumination means for illuminating the aquarium, and a processing device for counting fertilized eggs based on the images of the bottom of the aquarium captured by the imaging means with the illumination means turned on. This makes it possible to identify fertilized fish eggs to be used in various research projects immediately after fertilization, thereby improving work efficiency when collecting fertilized fish eggs. Furthermore, by counting the fertilized eggs with the processing device, it is possible to automatically determine when a certain number of fertilized eggs have been produced, thereby reducing the workload during the collection work and leading to support in the collection of fertilized eggs.

[0016] The processing device acquires multiple images taken multiple times within a specified period of time, generates a composite image by averaging the brightness of the pixels that make up each image, and performs threshold judgment on the pixels that make up the composite image based on a specified brightness threshold to identify and count the fertilized eggs.By combining images taken continuously, the influence of fish shadows can be minimized, the fertilized eggs can be identified with high accuracy, and the number can be counted more accurately.

[0017] The processing device generates a composite image based on multiple bottom images taken immediately after the lighting means is turned on after being turned off, so that by intentionally moving the fish, the influence of fish shadows can be further eliminated in generating the composite image, improving the identification of fertilized eggs.

[0018] The lighting means illuminates the aquarium from diagonally above, so that the light can be directed at the fertilized eggs while suppressing reflections from a net member that may be installed below the aquarium. Also, the light from the lighting means can be prevented from being directly captured by the imaging means.

[0019] The processing device sends an external alert when the number of counted fertilized eggs is equal to or greater than a predetermined number, allowing technicians and others to quickly become aware of the presence of a large number of fertilized eggs and encouraging them to immediately begin collection work. [Brief explanation of the drawings]

[0020] [Figure 1] 1 is a schematic diagram of an example of an embryo collection support system of the present invention. [Figure 2] FIG. 2 is a plan view of the system of FIG. 1. [Figure 3] 10 is a photograph showing an example of a raw bottom image taken by an imaging unit. [Figure 4] FIG. 10 is a diagram illustrating an image of generating a composite image. [Figure 5] FIG. 10 is a flow diagram of an example of image processing. [Figure 6] 1 is a flowchart showing an example of the steps of a method for supporting collection of fertilized fish eggs. DETAILED DESCRIPTION OF THE INVENTION

[0021] The embryo collection support system of the present invention will be described below with reference to the drawings. The embryo collection support system of the present invention is used to collect fertilized fish eggs. Note that the type of fish is not particularly limited in the present invention, and examples include small fish such as zebrafish, medaka, guppies, and goldfish.

[0022] Figure 1 shows a block diagram of an example of an embryo collection support system of the present invention. As shown in Figure 1, the embryo collection support system 1 of the present invention has an aquarium 2, an area camera 3 as an imaging means, a lighting means 4, and a processing device 5. In Figure 1, it is preferable to operate the embryo collection support system 1 with the aquarium 2, area camera 3, and lighting means 4 entirely covered with a blackout curtain or the like so that external light does not enter the area camera 3. The processing device 5 is configured to be able to communicate with the area camera 3 and lighting means 4.

[0023] The aquarium 2 is an underwater tank that houses male fish z1 and female fish z2, and contains target fish and water suitable for the fish (freshwater, seawater, etc.). In FIG. 1, the aquarium 2 has a rectangular parallelepiped shape with an open top, and is rectangular when viewed from the bottom. In FIG. 1, the longitudinal direction of the rectangle when viewed from the bottom corresponds to the X-axis direction, and the lateral direction (depth direction) corresponds to the Y-axis direction. The up-down direction of the aquarium 2 corresponds to the Z-axis direction.

[0024] The water tank 2 is preferably made of a transparent material, such as acrylic resin, polycarbonate resin, or glass.

[0025] The aquarium 2 is an aquarium for mating males z1 and females z2 contained therein. Fertilized eggs r released by mating have a density slightly higher than that of water and gradually sink to the bottom of the aquarium 2. The fertilized eggs r are transparent spheres with a diameter of approximately 1 mm. In FIG. 1, a net member 21 is provided at the bottom of the aquarium 2. The net member 21 is made of, for example, a wire mesh and separates the area underwater where fish swim (the area above the net member 21) from the area where the fertilized eggs r are contained (the area below the net member 21). For example, zebrafish have a biological tendency to prey on their own fertilized eggs r immediately after spawning. Therefore, by partitioning the bottom of the aquarium 2 with the net member 21, it serves as an egg collection isolation case that separates the paired adults z1 and z2 from the fertilized eggs r.

[0026] The mesh size of the net member 21 is set to a size that allows the fertilized eggs r to pass through but prevents the adults z1 and z2 from passing through. The material of the net member 21 is not particularly limited, and a metal material (e.g., stainless steel) or a resin material can be used. In order to suppress reflections from the metal material when photographing with the area camera 3, for example, the surface of the net member 21 may be black. The net member 21 is a member made by weaving fibers of the above-mentioned metal or resin material, so that the fertilized eggs r are smoothly guided by the curved fiber surface at the edge of the opening, and adhesion of the fertilized eggs r to the net member 21 can be suppressed. Furthermore, the net member 21 has a minute three-dimensional surface shape that diffuses light, thereby suppressing directional reflection of light toward the area camera and reducing adverse effects on the imaging means.

[0027] There are no particular limitations on the size or shape of the aquarium 2. In FIG. 1, the aquarium 2 has a protrusion 22 that protrudes outward from the upper part of one of the side surfaces facing each other in the X-axis direction. This protrusion 22 serves as a hook for lifting the aquarium 2 or securing it to a frame or the like, for example. The periphery of this protrusion 22 is preferably covered with black tape or the like, which makes it easier to prevent light from being caught in the bottom image.

[0028] Furthermore, in order to control the timing of mating, a partition plate may be provided in aquarium 2 to separate an area housing males z1 from an area housing females z2. The partition plate is provided, for example, to separate the area in aquarium 2 into left and right areas in the X-axis direction. In this case, the partition plate may be automatically moved upward at a desired timing to connect the two areas.

[0029] As shown in FIG. 1, the area camera 3 is installed below the aquarium 2 with its lens 31 facing upward. An image (image data) of the bottom of the aquarium 2 captured by the area camera 3 is sent to the processing device 5. The bottom image is an image showing the bottom of the aquarium 2 as viewed from above, and an image including the entire bottom surface 2a of the aquarium 2 is captured so that all fertilized eggs r fit within the image. The area camera 3 and processing device 5 are connected by wire or wirelessly.

[0030] The area camera 3 can capture an image of an object in, for example, the visible light range. In this case, the area camera 3 captures an image while irradiated with light from the lighting means 4, thereby obtaining a visible image. In the area camera 3, it is preferable to provide a polarizing plate on the illumination side of the lens 31. This makes it easier to suppress diffuse reflections from the aquarium 2, water, etc. The area camera 3 may also be capable of capturing images in the infrared range or near-infrared range.

[0031] The area camera 3 is installed so as to be positioned at the center of the longitudinal length of the aquarium 2. Note that although Figures 1 and 2 show a configuration in which one area camera 3 is installed for one aquarium 2, for example, one area camera 3 may be installed for multiple aquariums.

[0032] As shown in FIG. 1, the lighting means 4 emits light toward the aquarium 2 when turned on. The lighting means 4 emits light under the control of, for example, a processing device 5. The light source of the lighting means 4 includes a light-emitting element, such as a white LED, and a drive circuit. The wavelength of the light emitted from the light source is, for example, 300 nm to 750 nm. The configuration of the light source is not particularly limited, and light-emitting elements such as red LEDs, green LEDs, and blue LEDs may be used, or a light bulb may be used instead of an LED. The lighting means 4 and processing device 5 are connected by wire or wirelessly.

[0033] In FIG. 1, a pair of lighting means 4, 4 are provided spaced apart in the X-axis direction of the aquarium 2. Each lighting means 4 is provided to illuminate the aquarium 2 from diagonally above the aquarium 2, and more specifically, the light axis O of the light source is tilted downward at an angle θ with respect to the horizontal plane. The angle θ is, for example, 20° to 60°, and preferably 30° to 40°. By providing the lighting means 4 in this manner, it becomes easier to reduce reflections by the net member 21 while directing light toward the fertilized eggs r. Furthermore, it is possible to prevent the light from the lighting means 4 from being directly captured by the area camera 3.

[0034] As shown in Figure 1, the processing device 5 is an information processing device mainly composed of a microcomputer including a well-known CPU, ROM, RAM, etc. The processing device 5 counts the fertilized eggs r based on the image of the bottom of the aquarium 2 captured by the area camera 3 with the lighting means 4 turned on. The processing device 5 operates as various functional units by executing software stored in the memory unit.

[0035] 1, the processing device 5 has an illumination control unit 51, a counting unit 52, and an alarm unit 53. For convenience, the processing device 5 is illustrated separately from the area camera 3 in FIG. 1, but the area camera 3 also has a built-in microcomputer, and all or part of the functions of the processing device 5 may be provided by the microcomputer in the area camera 3.

[0036] The lighting control unit 51 controls the turning on / off of the lighting means 4. For example, when counting the fertilized eggs r, the timing of turning on and off the lighting means 4 is set in advance, as shown in Fig. 6 described later, and the lighting control unit 51 turns on and off the lighting means 4 based on this.

[0037] FIG. 3 shows an example of a raw bottom image (before image processing) captured by the area camera 3. As shown in FIG. 3, in addition to the fertilized eggs, netting members and fish shadows can be observed in the bottom image. The fertilized eggs are transparent and appear whitish when illuminated by the light from the lighting means. The fish shadows are also captured in the bottom image and appear whitish due to the light reflected by the body surface of the fish. As the fish shadows appear whitish in addition to the fertilized eggs, it is preferable to be able to identify the fertilized eggs separately from the fish shadows.

[0038] For example, in order to eliminate the influence of fish shadows and improve the identification of fertilized eggs, the bottom image captured by the area camera can be processed by the processing device 5 to make it easier to identify the fertilized eggs. Specifically, it is preferable that the processing device 5 generates a composite image by, for example, superimposing multiple bottom images captured multiple times within a predetermined time, and counts the fertilized eggs based on the composite image.

[0039] In an aquarium, fish are generally constantly moving, whereas fertilized eggs that sink to the bottom do not move. Taking this difference into account, the influence of fish shadows can be eliminated by combining multiple images of the bottom. In other words, when observing a common location across multiple bottom images, fertilized eggs will be observed in almost all images because they do not move, whereas fish shadows will only be observed in some of the images. Therefore, smoothing the combined image makes it easier to identify only the fertilized eggs.

[0040] The specific process for generating a composite image is not particularly limited, and a known method can be adopted. For example, for a plurality of bottom surface images, parameters (such as brightness and color parameters) of each pixel constituting each image can be calculated, and an arithmetic average of the parameters for the plurality of bottom surface images can be calculated for each pixel, and a composite image can be generated based on the calculated arithmetic average.

[0041] The multiple bottom images are, for example, images taken at least twice within 10 seconds, more specifically, at one-second intervals at least twice, preferably at one-second intervals at least five times. For example, in a configuration in which eight bottom images are taken at one-second intervals (see FIG. 4), the eight bottom images are combined to generate a single composite image. As shown in FIG. 4, in the eight bottom images, the fertilized eggs are observed in the same position in all images, whereas the position of the fish shadows changes from image to image. For example, in this case, in the composite image, the fish shadows in each image are so faint that they are barely observable, thereby improving the identifiability of the fertilized eggs.

[0042] Furthermore, it is preferable that the multiple bottom images are taken immediately after the lighting means is turned on (for example, within 20 seconds, and preferably within 10 seconds). When the lighting means is turned on after being turned off, the fish are startled by the light and become active. Intentionally moving the fish in this way makes it easier to eliminate fish shadows from the composite image, which ultimately leads to improved identification of the fertilized eggs.

[0043] In an embodiment in which fertilized eggs are counted based on a composite image, it is preferable to perform image processing on the composite image. Figure 5 shows a flow chart of an example of this image processing. The image processing shown in Figure 5 is performed by a processing device.

[0044] In Figure 5, the composite image is first subjected to gray conversion. In this step, the image is converted into a grayscale image. Note that the gray conversion process is not particularly limited, and any known method can be used.

[0045] Next, dilation and erosion are performed on the gray-converted composite image. For example, dilation replaces the maximum luminance value in the vicinity of the pixel of interest with the luminance value of the pixel of interest, while erosion replaces the minimum luminance value in the vicinity of the pixel of interest with the luminance value of the pixel of interest. In other words, dilation increases the area with high luminance values, while erosion increases the area with low luminance values.

[0046] In this step, the dilation or erosion process may be performed independently, or a closing process, which involves dilating and eroding the same number of times, or an opening process, which involves eroding and expanding the same number of times, may be performed. The closing process is primarily suitable for removing black noise, and the opening process is primarily suitable for removing white noise. This dilation and erosion process can remove fine patterns, for example, to eliminate the effects of light flicker in the composite image.

[0047] Next, blobs are detected from the image that has been subjected to the expansion and contraction processing. In blob detection, the image is first binarized. Specifically, a binarization process is performed to convert the image into two levels of color tone, thereby generating a two-level color tone image (e.g., a black and white image). For example, binarization compares the parameters of each pixel with different shades of gray with a predetermined threshold, and converts pixels within one range to white and pixels within the other range to black, thereby generating a black and white image. In this way, by performing binarization and converting to a black and white image, the outlines of blobs are clarified, making it easy to identify fertilized eggs with high accuracy.

[0048] Then, multiple blobs contained in the binarized image are detected. Here, "blob" refers to interconnected white areas contained in the binarized image. In other words, the binarized image contains multiple white masses, and each white mass corresponds to a blob.

[0049] The detected blobs may be labeled, and a feature value may be calculated for each blob. Examples of feature values ​​include the area and circularity of the blob. Because fertilized eggs are spherical and are detected with a certain area when viewed from the bottom, the identification of fertilized eggs can be further improved by taking the area and circularity of the blob into account as feature values. For example, blobs with an area or circularity less than a predetermined value may be removed from the detected blobs.

[0050] When detecting blobs, blobs with an area smaller than a predetermined value or a circularity smaller than a predetermined value may be prevented from being detected. Also, a predetermined region may be excluded from the blob detection region. For example, when the container for the aquarium is formed by injection molding or the like, a gate mark is formed in the center of the bottom surface of the aquarium, as shown in FIG. 3. This gate mark appears whitish and may be mistaken for a fertilized egg, so it is preferable to exclude it from the detection region.

[0051] Then, as shown in FIG. 5, the number of blobs is counted by the counting unit 52 (see FIG. 1), and the number of blobs is calculated as the number of fertilized eggs.

[0052] It should be noted that the image processing method in Fig. 5 is not limited to the method described above. In Fig. 5, for example, gray conversion is performed on the composite image, but binarization processing may also be performed on the composite image. In this case, expansion and contraction are performed on the binarized image. For example, in the expansion processing, if there is even one white pixel near the pixel of interest, the pixel of interest is replaced with white, and in the contraction processing, if there is even one black pixel near the pixel of interest, the pixel of interest is replaced with black.

[0053] 1, the notification unit 53 has a function of notifying an external party when the number of counted fertilized eggs is equal to or greater than a predetermined number, etc. The notification means is not particularly limited, and means such as displaying on a monitor, notifying by sound or voice, notifying by a lamp display, or sending an email notification to an external terminal or the like can be appropriately used.

[0054] 6 is a flow chart showing an example of the procedure of the embryo collection support method using the embryo collection support system of the present invention. This method is carried out, for example, by automatic operation.

[0055] In Figure 6, a dark state is maintained in the aquarium or the like from the day before starting this method by covering it with a black curtain. Then, upon starting the method, the lighting means is turned on (step S11). For example, zebrafish exhibit sexual behavior in a bright environment as a biological characteristic, so turning on the light can encourage sexual behavior. In addition, an inspection timer is started when the lighting means is turned on. Then, this state is maintained until a predetermined time (for example, 10 minutes) has elapsed.

[0056] When a predetermined time has elapsed with the lighting means turned on (step S12: Yes), a preliminary test is performed prior to the main test (step S13). Specifically, fertilized eggs are detected based on the bottom image captured by the imaging means. Note that this step is positioned as a preliminary test, and the processing procedure for detecting fertilized eggs may be simpler than that of the main test. For example, it is not necessary to count the number of fertilized eggs, and the presence or absence of fertilized eggs may be detected. Furthermore, fertilized eggs may be detected based on the captured bottom image without generating a composite image. If no fertilized eggs are detected (step S13: No), this process is temporarily terminated, and step S13 is performed again after a predetermined time has elapsed using the test timer.

[0057] If fertilized eggs are detected in step S13 (step S13: Yes), the actual test is performed. In step S14, a series of lighting control steps is performed, in which the lighting means is first turned off and then turned on. Specifically, the lighting means is turned off for several seconds and then turned on. This lighting control allows the fish to move intentionally.

[0058] In the next step S15, a plurality of bottom images taken immediately after the lighting means is turned on are acquired, a composite image is generated from these bottom images, and the generated composite image is subjected to the image processing shown in Fig. 5 above, to count the number of fertilized eggs. If the number of fertilized eggs is, for example, 100 or more (step S15: Yes), an alarm is sounded to notify a technician or the like (step S16). Then, the lighting means is turned off, and the process of counting the fertilized eggs is terminated.

[0059] A technician or the like who receives the notification can then carry out the work of retrieving the fertilized eggs, thereby enabling the fertilized eggs to be retrieved promptly after fertilization.

[0060] The embryo collection support system of the present invention is not limited to the above-described configuration, and can be modified as appropriate. [Industrial Applicability]

[0061] The embryo collection support system of the present invention can improve work efficiency when collecting fertilized fish eggs, making it possible to collect large quantities of fresh fertilized eggs, for example, within 45 minutes of fertilization. Demand for fertilized fish eggs is expected to continue to grow in various research fields, and the present invention can make a significant contribution to meeting this demand. [Explanation of symbols]

[0062] 1. Embryo Collection Support System 2. Aquarium 21 Net components 22 Protrusion 3 Area camera (imaging means) 31 Lens 4 Lighting means 5 Processing equipment 51 Lighting control unit 52 Counting section 53 Information Department z1 male z2 female r fertilized egg

Claims

1. This is a fertilized egg collection support system that supports the collection of fertilized fish eggs. The system is a fertilized egg collection support system characterized by comprising: an aquarium for housing male and female fish; an imaging means installed below the aquarium for capturing images of the bottom of the aquarium; an illumination means for irradiating light toward the aquarium; and a processing device for counting fertilized eggs based on the images of the bottom of the aquarium captured by the imaging means with the illumination means turned on.

2. 2. The embryo collection support system according to claim 1, wherein the processing device generates a composite image from a plurality of bottom images taken a plurality of times within a predetermined time period, and counts the embryos based on the composite image.

3. The fertilized egg collection support system according to claim 2, characterized in that the processing device generates the composite image based on a plurality of bottom images taken immediately after the lighting means is turned on after being turned off.

4. 3. The embryo collection support system according to claim 1, wherein the lighting means illuminates the water tank from diagonally above the water tank.

5. 3. The fertilized egg collection support system according to claim 1, wherein a net member is provided at the bottom of the aquarium to separate the area in which fish swim underwater from the area in which fertilized eggs are housed.

6. 3. The embryo collection support system according to claim 1, wherein said processing device notifies an external device when the number of counted embryos is equal to or greater than a predetermined number.

Images