Methods for anesthetizing, observing, and awakening small fish

Abstract

Conventional fish anesthesia methods are effective for edible fish such as red sea bream and rainbow trout when transferring them between tanks or when transporting them, but they are not effective for small fish such as killifish, and are not suitable for experiments or observations. [Solution] This method of anesthetizing medaka, a small fish, involves adding 10 g of dry ice to 500 ml of fresh water in an aquarium containing medaka, making it possible to anesthetize the small fish for approximately 10 minutes, and allowing the body of the medaka to be observed slowly while it is still alive, without causing pain or stress to the fish.

Description

[Technical Field]

[0001] The present invention relates to anesthesia and awakening methods required for observing the bodies of small fish, particularly medaka, while they are still alive. [Background technology]

[0002] The invention described in Patent Document 1 below is a technology that enables long-distance transportation of live fish (transportation of live fish) by tranquilizing the fish in an anesthesia tank made of anesthetic water with specified carbon dioxide and oxygen concentrations, and then overcrowding the fish in a maintenance tank made of maintenance water in an environment different from that of the anesthesia tank.

[0003] Furthermore, Patent Document 2, which is an improved version of this technology, is a technology that enables the anesthetic effect on seafood to be exerted early by dissolving carbon dioxide in a mixture of seawater and freshwater and adjusting the salinity. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6236575 [Patent Document 2] Patent No. 6655734 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the inventions of Patent Documents 1 and 2 are anesthesia methods that are effective for edible fish such as red sea bream and rainbow trout when transferring them between tanks or when transporting them, but they are not effective for small fish such as medaka, and are not suitable for experiments or observations. [Means for solving the problem]

[0006] In order to solve the above problem, the invention of claim 1 is as follows: How to anesthetize medakaThe invention of claim 2 is characterized in that dry ice is added to an aquarium containing killifish in a ratio of 8g to 12g per 500ml of tap water or spring water that contains almost no carbon dioxide (hereinafter referred to as "fresh water"). How to observe medaka The method is characterized in that the medaka is anesthetized by the anesthesia method, and the medaka is visually inspected, palpated, and observed. [Effects of the Invention]

[0007] According to the present invention, it is possible to quickly anesthetize small fish, particularly medaka, with significantly less effort, and it is possible to slowly observe the bodies of small fish while they are still alive, without causing pain or stress to the medaka. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows a preliminary experiment 1 according to an embodiment of the present invention, in which a doughnut-shaped water channel was created using circular water tanks (large and small) and dumbbells (2 kg). [Figure 2] 1 is a graph showing the change in carbon dioxide concentration in experimental carbonated water, which is the result of preliminary experiment 1 according to an embodiment of the present invention. [Figure 3] 1 is a graph showing the change in carbon dioxide concentration in experimental carbonated water, which is the result of Experiments 1, 3, 5 and 7 according to an embodiment of the present invention. [Figure 4] FIG. 10 is a diagram showing a preliminary experiment 2 according to an embodiment of the present invention, in which a small aquarium was wrapped in black paper to prevent external influences when observing danger avoidance behavior. [Figure 5] 10 is a graph showing the change in carbon dioxide concentration due to the experimental dry ice block, which is the result of Experiments 2, 4, 6 and 8 according to an embodiment of the present invention. [Figure 6] 1 is a graph showing the change in sensory rheotaxis of medaka fish when carbonated water is used, which is the result of an experiment according to an embodiment of the present invention. [Figure 7]1 is a graph showing the results of an experiment according to an embodiment of the present invention, showing changes in sensory rheotaxis and reaction of medaka fish when carbonated water is used. [Figure 8] 1 is a graph showing the change in sensory rheotaxis of medaka fish when dry ice was intermittently used, which is the result of an experiment according to an embodiment of the present invention. [Figure 9] 1 is a graph showing the results of an experiment according to an embodiment of the present invention, illustrating the changes in sensory rheotaxis and response of medaka fish when dry ice was intermittently used. [Figure 10] 1 is a graph showing the change in visual rheotaxis of medaka fish when carbonated water is used, which is the result of an experiment according to an embodiment of the present invention. [Figure 11] 1 is a graph showing the results of an experiment according to an embodiment of the present invention, illustrating changes in visual rheotaxis and reaction of medaka fish exposed to carbonated water. [Figure 12] 1 is a graph showing the results of an experiment according to an embodiment of the present invention, illustrating changes in visual rheotaxis and reaction of medaka fish exposed to carbonated water. [Figure 13] 1 is a graph showing the results of an experiment according to an embodiment of the present invention, illustrating changes in the visual rheotaxis and reaction of medaka fish when dry ice was intermittently used. [Figure 14] 1 is a graph showing the results of an experiment according to an embodiment of the present invention, showing changes in visual and sensory rheotaxis and responses of medaka fish when dry ice was intermittently used. [Figure 15] 1 is a graph showing the change in visual rheotaxis of medaka fish when dry ice was intermittently used, which is the result of an experiment according to an embodiment of the present invention. [Figure 16] 1 is a graph showing the change in the reaction of medaka fish when carbonated water is used, which is the result of an experiment according to an example of the present invention. [Figure 17] 1 is a graph showing the change in the reaction of medaka fish when dry ice was used intermittently, which is the result of an experiment according to an embodiment of the present invention. [Figure 18] 1 is a graph showing the change in the reaction of medaka fish when dry ice was used intermittently, which is the result of an experiment according to an embodiment of the present invention. [Figure 19]1 is a graph showing the change in the reaction of medaka fish when one block of 5 g of dry ice is continuously used, which is the result of an experiment according to an embodiment of the present invention. [Figure 20] 1 is a graph showing the change in the reaction of medaka fish when one block of 5 g of dry ice is continuously used, which is the result of an experiment according to an embodiment of the present invention. [Figure 21] 1 is a graph showing the change in the reaction of medaka fish when 10 g of dry ice per block is continuously used, which is the result of an experiment according to an embodiment of the present invention. [Figure 22] 1 is a graph showing the change in the reaction of medaka fish when 10 g of dry ice per block is continuously used, which is the result of an experiment according to an embodiment of the present invention. [Figure 23] 1 is a graph showing the change in the reaction of medaka fish when 10 g of dry ice per block is continuously used, which is the result of an experiment according to an embodiment of the present invention. [Figure 24] 1 is a graph showing the change in the reaction of medaka fish when 10 g of one block of dry ice is continuously used, which is the result of an experiment according to an embodiment of the present invention. [Figure 25] 1 is a graph showing the change in the reaction of medaka fish when two 10 g blocks of dry ice are continuously used, which is the result of an experiment according to an embodiment of the present invention. [Figure 26] 1 is a graph showing the change in the reaction of medaka fish when four 10 g blocks of dry ice were continuously used, which is the result of an experiment according to an embodiment of the present invention. [Figure 27] 1 is a graph showing the change in the reaction of medaka fish when one block of 15 g of dry ice is continuously used, which is the result of an experiment according to an embodiment of the present invention. [Figure 28] 1 is a graph showing the change in the reaction of medaka fish when one block of 15 g of dry ice is continuously used, which is the result of an experiment according to an embodiment of the present invention. [Figure 29] FIG. 11 is a summary of graphs 6 to 9 showing the changes and reactions in sensory rheotaxis of medaka fish when carbonated water and dry ice are used, which are the results of an experiment according to an embodiment of the present invention. [Figure 30]10 to 13 are graphs showing the changes in visual rheotaxis and reactions of medaka fish when carbonated water and dry ice are used, which are the results of experiments according to an embodiment of the present invention. [Figure 31] This figure summarizes graphs Figures 14 to 18, which show the results of experiments related to an embodiment of the present invention, and show the changes in visual and sensory rheotaxis and responses of medaka fish when carbonated water or intermittent dry ice was used. [Figure 32] FIG. 1 is a graph summarizing the results of experiments according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. The following embodiments are merely illustrative for explaining the present invention, and are not intended to limit the present invention to these embodiments. Furthermore, the present invention can be modified in various ways without departing from the spirit of the present invention. Furthermore, the same components in each drawing will be designated by the same reference numerals whenever possible, and redundant explanations will be omitted. [Example]

[0010] <1> Fish to be anesthetized In the present invention, the fish to be anesthetized include all small fish, but medaka, which was the subject of the experiment, is preferred.

[0011] <2> anesthetic water In the present invention, the anesthetic water is characterized by having a carbon dioxide concentration, relative to fresh water, that produces an anesthetic effect on small fish and shellfish, particularly medaka, to be anesthetized.

[0012] <3> Anesthetic water production procedure The procedure for producing anesthetic water is simply to add solid carbon dioxide, commonly known as "dry ice," to fresh water, and the amounts are explained below.

[0013] The experimental results described below suggest that dry ice is a preferable anesthesia method because it requires less effort. This is because dry ice has a high reproducibility of carbon dioxide concentration in water. In the experiment described below, carbonated water and dry ice had the same mortality rate and were equally safe, but when adding a large amount of carbonated water at once to increase the carbon dioxide concentration, the carbon dioxide concentration in the water increases more rapidly than with dry ice, making it less safe.

[0014] The simplest, quickest, and safest method of anesthetizing medaka, a small fish, is to add one block of dry ice to 500 ml of fresh water containing the medaka. One block of dry ice weighs approximately 8 to 12 g, and most preferably 10 g.

[0015] After the dry ice has melted sufficiently, the medaka is observed thoroughly, and if it has stopped breathing for more than one minute, it can be revived by returning it to fresh water. With the method of the present invention, the medaka is anesthetized in about 10 minutes after the dry ice is added, and then it can be returned to fresh water and observed under a microscope for about 20 minutes.

[0016] Below, we will show experiments that demonstrate the basis for the above conclusion. The medaka species used in the experiment were Mikino Medaka (Experiments 3 to 9) Albino Mikino Medaka (Experiment 9) Blue Medaka (Experiment 9) Tricolor transparent scale medaka (Experiment 9) The underwater carbon dioxide concentration measuring device used was the CGP-31 (manufactured by DKK Toa Corporation), and the carbonated water producing device used was the SodaStream E-TERRA (SodaStream Corporation).

[0017] <4> Experiment 1 (preliminary experiment) (1) As shown in Figure 1, a donut-shaped waterway was created using a circular aquarium (large) 1, a small aquarium (small) 2, and a dumbbell 3 (2 kg). (2) Pour 1 L of water W into the created water channel, create a water flow using an underwater motor, and begin measuring the underwater carbon dioxide concentration while stirring. (3) Inject 53 ml of carbonated water (carbonated water concentration 5%) into the water channel and start measuring the time. (4) After that, every 7 minutes, 58 ml of carbonated water (10% concentration), 65 ml of carbonated water (15% concentration), 74 ml of carbonated water (20% concentration), 83 ml of carbonated water (25% concentration), 96 ml of carbonated water (30% concentration), 110 ml of carbonated water (35% concentration), 128 ml of carbonated water (40% concentration), 151 ml of carbonated water (45% concentration), and 182 ml of carbonated water (50% concentration) were injected repeatedly, and the carbon dioxide concentration in the water was measured.

[0018] In Experiment 1, as shown in Figure 2, the carbon dioxide concentration in the water increased each time carbonated water was added. From Figure 2, it appears that the carbon dioxide concentration in the water increases when carbonated water is added, but immediately afterwards, the concentration begins to slowly decrease. Figure 3 shows the results of Experiment 1 and Experiments 3, 5, and 7, which will be described later. Even when using carbonated water made with the same carbonated water maker, there was considerable variation in the way the carbon dioxide concentration in the water increased.

[0019] <5> Experiment 2 (preliminary experiment) (1) As shown in Figure 4, a small aquarium 2 was wrapped in black paper 4 to prevent other influences when observing danger avoidance behavior. (2) 500 ml of water was placed in the small tank 2, and the measurement of the carbon dioxide concentration in the water was started while stirring with a stirrer and a stirrer. (3) Dry ice was added and the time measurement started, and the measurement of the carbon dioxide concentration in the water continued.

[0020] In another preliminary experiment, Experiment 2, the amount of dry ice was divided into 5g, 10g, and 15g. For the 10g and 15g blocks, the test was conducted for both a single block weighing 10g or 15g and two or more blocks totaling 10g or 15g. Figure 5 shows the results of Experiment 2 and Experiments 4, 6, and 8, which are described below. As the weight increased from 5g to 10g to 15g, the rate at which the carbon dioxide concentration in the water rose became faster and the maximum concentration increased, but the pattern of increase in carbon dioxide concentration was similar for each weight. For 10g and 15g blocks, the carbon dioxide concentration rose faster and the maximum concentration increased when two or more blocks were used. Furthermore, as the dry ice became smaller, it began to float to the surface, and the carbon dioxide concentration began to decrease at this point.

[0021] <6> Experiment 3 (Observation of sensory rheotaxis when using carbonated water) Sensory rheotaxis is the habit of swimming against the current of water. (1) As shown in Figure 1, a donut-shaped water channel was created using circular water tanks (large and small) and dumbbells (2 kg). (2) 1 L of water and killifish (only Mikino killifish were used in Experiments 3-8; see Table 1 below) were placed in the created waterway, and measurements of the carbon dioxide concentration in the water were started while creating a water current with an underwater motor. (Nets were strung up in front of and behind the underwater motor, but in early experiments, anesthetized killifish were swept through gaps in the net and caught in the motor, resulting in their deaths. Therefore, in all subsequent experiments, the motor itself was wrapped in netting.) (3) 53 ml of carbonated water (carbonated water concentration 5%) was poured into the water channel, and time measurement was started. (4) Six minutes after the injection of carbonated water, sensory rheotaxis was observed for one minute. (5) After that, every 7 minutes, 58 ml of carbonated water (10% concentration), 65 ml of carbonated water (15% concentration), 74 ml of carbonated water (20% concentration), 83 ml (25% concentration), 96 ml of carbonated water (30% concentration), 110 ml (35% concentration), 128 ml (40% concentration), 151 ml (45% concentration), and 182 ml (50% concentration) were injected, and step (4) was repeated.

[0022] <7> Experiment 4 (Observation of sensory rheotaxis using dry ice) (1) As shown in Figure 1, a donut-shaped water channel was created using circular water tanks (large and small) and dumbbells (2 kg). (2) We placed 1L of water and medaka fish in the created waterway, and began measuring the underwater carbon dioxide concentration while creating a water current with an underwater motor. (3) Dry ice was thrown into the waterway and the time measurement started. (The dry ice was surrounded by a net to prevent the killifish from touching it.) (4) After 2 minutes, the dry ice was removed from the channel, and 4 minutes later, sensory rheotaxis was observed for 1 minute. (5) Then, steps (3) and (4) were repeated.

[0023] In Experiments 3 and 4 above, we confirmed the stage at which sensory rheotaxis is lost due to an increase in the carbon dioxide concentration in the water. Figures 6 and 7 show the results when carbonated water was used, and Figures 8, 9, and 14 show the results when dry ice was used. Medaka that had stopped swimming in the absence of water flow were awakened by the stimulation of the water flow and appeared to swim against the current.

[0024] It took longer for sensory rheotaxis to disappear when carbonated water was used, but this may be because the medaka gradually became accustomed to the increased carbon dioxide concentration in the water as it gradually increased.On the other hand, when dry ice was used, the carbon dioxide concentration increased more quickly, which may be because the medaka became anesthetized more quickly.

[0025] <8> Experiment 5 (Observation of visual rheotaxis when using carbonated water) Visual rheotaxis is the swimming habit of following the surrounding scenery, and when stripes are rotated around the tank, medaka follow the stripes. (1) As shown in Figure 1, a donut-shaped water channel was created using circular water tanks (large and small) and dumbbells (2 kg). (2) We placed 1L of water and medaka fish in the created waterway, and began measuring the underwater carbon dioxide concentration while creating a water current with an underwater motor. (3) 53 ml of carbonated water (carbonated water concentration 5%) was poured into the water channel, and time measurement was started. (4) To observe visual rheotaxis, the striped cardboard was rotated around the tank, which interfered with the underwater carbon dioxide meter, making continuous measurements impossible. Therefore, before observing visual rheotaxis, the underwater motor was stopped, 40 cc of water was transferred from the water channel to the measurement cell, and the carbon dioxide concentration measurement was resumed. (5) As shown in Figure 2, 6 minutes after the injection of carbonated water, the striped pattern was rotated for 1 minute to observe visual rheotaxis. (6) After that, every 7 minutes, 58 ml of carbonated water (10% concentration), 65 ml of carbonated water (15% concentration), 74 ml of carbonated water (20% concentration), 83 ml of carbonated water (25% concentration), 96 ml of carbonated water (30% concentration), 110 ml of carbonated water (35% concentration), 128 ml of carbonated water (40% concentration), 151 ml of carbonated water (45% concentration), and 182 ml of carbonated water (50% concentration) were injected, and steps (4) and (5) were repeated.

[0026] <9> Experiment 6 (Observation of visual rheotaxis when dry ice is used) (1) As shown in Figure 1, a donut-shaped water channel was created using circular water tanks (large and small) and dumbbells (2 kg). (2) We placed 1L of water and medaka fish in the created waterway, and began measuring the underwater carbon dioxide concentration while creating a water current with an underwater motor. (3) Dry ice was dropped into the waterway and the time measurement began. (4) After 2 minutes, the dry ice was removed from the waterway. (5) After that, the underwater motor was stopped, 40 cc of water was transferred from the waterway to the measuring cell for concentration measurement, and measurement of the carbon dioxide concentration was resumed. (6) Three minutes after the dry ice was removed, the stripes were rotated for one minute to begin visual rheotaxis observation. (7) After that, steps (3)-(6) were repeated every 7 minutes.

[0027] Visual rheotaxis, confirmed in Experiments 5 and 6 above, is the habit of swimming as if following the surrounding scenery. When a circular aquarium with no water flow is surrounded by patterned cardboard or the like and the pattern is moved, the fish swim as if following the pattern. This is thought to create the illusion of water flow, and medaka have the habit of trying to stay in the same position so as not to be swept away by water currents.

[0028] Although not included in this experiment, in a previous experiment, when a pattern was moved in the same direction as the water flow in an aquarium with water flow, it was confirmed that medaka swam following the pattern rather than against the water flow. It has been found that visual rheotaxis is stronger than sensory rheotaxis, and that medaka are more strongly influenced by visual information than by the sensations on the body surface that sense the flow.

[0029] In this study, we also conducted experiments on visual rheotaxis, and as shown in Figures 10 to 15, it became clear that visual rheotaxis disappeared more quickly than sensory rheotaxis once anesthesia began to take effect.

[0030] Although medaka do not close their eyes, it seems that anesthesia prevents them from gathering visual information. As shown in Figures 10 to 15, it took longer for visual rheotaxis to disappear when carbonated water was used. This is thought to be because, as with sensory rheotaxis, medaka become accustomed to a gradual increase in carbon dioxide concentration, slowing the onset of anesthesia, while a sudden increase in carbon dioxide concentration causes anesthesia to occur more quickly.

[0031] <10> Experiment 7 (Observation of the reaction when carbonated water is used) (1) The small aquarium was wrapped in black paper to prevent external visual influences when observing risky behavior (see (b) and (c) below). (2) 500 ml of water and a killifish were placed in a small aquarium, and the carbon dioxide concentration in the water was measured while stirring with a stirrer. (The stirrer was surrounded by a net to prevent the killifish from touching it.) (3) 26.5 ml of carbonated water was poured in and the time measurement was started. (4) After 5 minutes, the following steps (a)-(d) were carried out for 2 minutes and observed. (a) Feeding the fish (b) Hitting the tank (c) Holding his hand over the tank (d) Touching the killifish with a stick (5) After that, every 7 minutes, 29 ml of carbonated water (10% concentration), 32.5 ml of carbonated water (15% concentration), 37 ml of carbonated water (20% concentration), 41.5 ml of carbonated water (25% concentration), 48 ml of carbonated water (30% concentration), 55 ml of carbonated water (35% concentration), 64 ml of carbonated water (40% concentration), 75.5 ml of carbonated water (45% concentration), and 91 ml of carbonated water (50% concentration) were injected, and step (4) was repeated.

[0032] <11> Experiment 8 (Observation of reactions when dry ice is used) (1) The small aquarium was wrapped in black paper to prevent external visual influences when observing danger avoidance behavior (see (b) and (c) below). (2) 500 ml of water and a killifish were placed in a small aquarium, and measurements of the carbon dioxide concentration in the water were started while stirring with a stirrer and a stirrer. (3) Add dry ice and start timing. (4) (Intermittent use of dry ice) After 2 minutes, the dry ice was removed from the waterway, and after 3 minutes, the following (a)-(d) were carried out for 2 minutes and observed. (Continuous use of dry ice) The dry ice was not collected, and after 5 minutes, the following (a)-(d) were carried out over a 2-minute period and observed. (a) Feeding the fish (b) Hitting the tank (c) Holding his hand over the tank (d) Touching the killifish with a stick (5) Then, steps (3) and (4) were repeated.

[0033] <12> Experiment 9 (Observation of medaka fish using dry ice as anesthesia) (1) 500 ml of fresh water and one medaka (Medaka medaka, Albino Medaka, Tricolor transparent scale medaka, Blue medaka) were placed in a small aquarium, and measurements of the carbon dioxide concentration in the water were started. (2) 10g of dry ice was placed in the tank and the medaka were observed. (3) When the medaka stopped moving even when lightly stimulated (even when lightly touched with a stick), the medaka was transferred to a zippered plastic bag containing 5 ml of water and observed under a microscope. (4) The movements of the medaka's heart and gills were confirmed, and the blood flow in the arteries and veins of the medaka's fins was observed. After that, the entire body of the medaka was observed for about 20 minutes.

[0034] In Experiments 7 and 8 above, we confirmed the order in which feeding behavior (eating food), danger-avoidance behavior (movement in response to vibration in the tank, response when a hand is held over the tank), swimming (normal swimming), and escape behavior due to external stimuli (touching the body with a stick) disappear during the anesthesia process. As a result, as shown in Figures 16 to 18 and Figures 29 to 31, whether carbonated water or dry ice was used, the order was danger-avoidance behavior → feeding behavior → visual rheotaxis → swimming → sensory rheotaxis → escape due to external stimuli → breathing. A summary of the series is shown in Figure 32.

[0035] In addition, we checked the reactions to light touch (light stimulation) and strong touch (strong stimulation) separately for external stimulation, but in observational experiments such as Experiment 9, it was thought that anesthesia to the point where the animal became immobile in response to light stimulation (immobility in response to light stimulation) was sufficient.

[0036] As shown in Figures 19 to 28, in each experiment, observations were made mainly on the disappearance of reaction to external stimuli when the weight and number of dry ice blocks were changed. As a result, as shown in Table 1 below, it was thought that a sudden increase in the concentration of carbon dioxide in the water would cause the medaka to stop breathing quickly, putting them at high risk of death. [Table 1]

[0037] Therefore, if breathing had stopped for more than one minute, rescue was carried out by moving the fish into fresh water. If breathing had stopped for one minute, breathing resumed and the fish was revived by returning it to fresh water. Medaka that were anesthetized quickly due to a sudden increase in the carbon dioxide concentration in the water took a short time to recover.

[0038] Medaka that had been in water with a high carbon dioxide concentration for a long time took longer to recover when returned to fresh water. Also, as shown in Figures 8, 14, 18, and 19, when dry ice was used, the water temperature dropped by 2-4 degrees in about 30 minutes, depending on the weight and number of pieces.

[0039] In Experiment 9, we used the Mikino medaka, albino Mikino medaka, tricolor clear scale medaka, and blue medaka, which are thought to be easier to observe internally. As shown in Table 1 above, after adding 10g of dry ice to 500ml of water, all medaka were anesthetized to the extent that they did not react to weak stimuli within approximately 10 minutes (average 12 minutes).

[0040] The killifish was then transferred to a 5ml zippered plastic bag filled with fresh water, and for about 20 minutes it was possible to observe it alive (breathing but without moving) under a microscope. It was possible to observe the heartbeat, gill movement, blood flow in the blood vessels, mainly in the caudal fin, the surface of the body, and even find the lateral line.

[0041] The beating of the heart could be confirmed from the surface of the body, but this was particularly clear in the light-colored blue medaka and the three-scale transparent scale medaka, whose bodies are transparent.

[0042] The gill movements were clearly visible, but it was not possible to see inside the gills. The blood vessels in the caudal fins of all medaka were visible, and blood flow in both arteries and veins was confirmed.

[0043] Observation of the body surface revealed that a parasite called Gyrodactylus, which is invisible to the naked eye, was attached to the surface and moving, and infection was also confirmed, demonstrating that the method can also be used to screen for parasitic infections.

[0044] In our search for lateral lines, we were unable to find any obvious lateral lines in any of the medaka, so it was thought that medaka do not have lateral lines, regardless of species. By anesthetizing the medaka in this way, we were able to observe them slowly (probably without causing pain or stress to the fish), making it possible to observe things that would not be possible without anesthesia. Furthermore, for observations like this, anesthesia at a level that does not react to weak stimuli is sufficient and therefore considered safe.

[0045] From the results of Experiments 3 to 9 above, it was concluded that the simplest, quickest, and safest method of anesthetizing medaka, a small fish, is to add one 10g block of dry ice to 500ml of fresh water containing the medaka. [Explanation of symbols]

[0046] 1 large aquarium 2 aquariums (small) 3 dumbbells 4. Black Paper F. Medaka (small fish) W water

Claims

1. A method for anesthetizing medaka, comprising: Add 8g to 12g of dry ice to 500ml of fresh water in a tank containing medaka fish. A method for anesthetizing medaka, comprising:

2. After anesthesia is induced in the medaka by the anesthesia method according to claim 1, The medaka is subjected to microscopic examination, visual examination, palpation and observation. A method for observing medaka, characterized by:

3. After anesthesia is induced in the medaka by the anesthesia method according to claim 1, Check for parasitic infection of the medaka A method for screening medaka fish, comprising:

4. After anesthesia is induced in medaka by the anesthesia method according to claim 1, Return the medaka to fresh water within 20 minutes of the introduction. A method for awakening medaka, characterized by:

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