Methods for anesthetizing, observing, and reviving small fish.

Abstract

Conventional methods of anesthetizing fish are effective for edible fish such as sea bream and rainbow trout when transferring them to different tanks or during transport, but they are ineffective for small fish like medaka and are not suitable for experiments or observations. [Solution] This method of anesthetizing medaka fish involves adding 10g of dry ice to 500ml of fresh water in a tank containing the fish. This allows the medaka to be anesthetized for approximately 10 minutes, enabling observation of their bodies slowly while they are still alive, without causing them pain or stress.

Description

[Technical Field]

[0001] The present invention relates to an anesthesia method and a recovery method required when observing the bodies of small fish, particularly medaka, while they are alive. [Background Art]

[0002] For transporting live fish and shellfish (live fish transport), there exists an invention described in Patent Document 1 below as a technology that enables long-distance transportation of live fish by sedating fish and shellfish in an anesthesia tank filled with anesthetic water having predetermined carbon dioxide concentration and oxygen concentration, and then storing the sedated fish and shellfish in a dense state in a maintenance tank filled with maintenance water that provides an environment different from that of the anesthesia tank.

[0003] Further, Patent Document 2, which is an improvement on the foregoing, discloses a technology that enables an anesthetic effect on fish and shellfish to be exhibited at an early stage by dissolving carbon dioxide in a mixed water of seawater and freshwater and adjusting the salinity concentration. [Prior Art Documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent No. 6236575 [Patent Document 2] Japanese Patent No. 6655734 [Summary of the Invention] [Problems to be Solved by the Invention]

[0005] However, the inventions of Patent Documents 1 and 2 are anesthetic methods that exhibit an effect when transferring edible fish such as red sea bream and rainbow trout between water tanks or during transportation, and do not provide an effect on small fish such as medaka, thus are not suitable for experiments, observations, and the like. [Means for Solving the Problems]

[0006] To solve the aforementioned problems, the invention of claim 1 is a method for anesthetizing small fish, characterized by adding 8g to 12g of dry ice to 500ml of water containing almost no carbon dioxide, such as tap water and spring water (hereinafter referred to as "fresh water"), in a tank containing small fish, and the invention of claim 2 is a method for observing small fish, characterized by anesthetizing the small fish using the above anesthesia method, and then visually inspecting, palpating, and observing the small fish. [Effects of the Invention]

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

[0008] [Figure 1] This figure shows a preliminary experiment 1 according to an embodiment of the present invention, in which a donut-shaped water channel was created using circular water tanks (large and small) and dumbbells (2 kg). [Figure 2] This graph shows the change in carbon dioxide concentration using experimental carbonated water, which is the result of preliminary experiment 1 according to an embodiment of the present invention. [Figure 3] This graph shows the changes in carbon dioxide concentration due to the experimental carbonated water, representing the results of experiments 1, 3, 5, and 7 according to the embodiments of the present invention. [Figure 4] This figure shows a preliminary experiment 2 according to an embodiment of the present invention, illustrating how a small aquarium is wrapped in black paper to prevent external influences when observing danger avoidance behavior. [Figure 5] This graph shows the results of experiments 2, 4, 6, and 8 according to embodiments of the present invention, illustrating the change in carbon dioxide concentration due to the experimental dry ice block. [Figure 6] This graph shows the results of an experiment according to an embodiment of the present invention, illustrating the change in the perceptual fluidity of medaka fish when using carbonated water. [Figure 7]This graph shows the results of an experiment according to an embodiment of the present invention, illustrating the changes in the sensory fluotaxis and response of medaka fish using carbonated water. [Figure 8] This graph shows the results of an experiment according to an embodiment of the present invention, illustrating the change in the perceived flowability of medaka fish when dry ice is used intermittently. [Figure 9] This graph shows the results of an experiment according to an embodiment of the present invention, illustrating the changes in the sensory fluotaxis and response of medaka fish when dry ice is intermittently used. [Figure 10] This graph shows the results of an experiment according to an embodiment of the present invention, illustrating the change in the visual flow of medaka fish when using carbonated water. [Figure 11] This graph shows the results of an experiment according to an embodiment of the present invention, illustrating the changes in the visual fauna and response of medaka fish using carbonated water. [Figure 12] This graph shows the results of an experiment according to an embodiment of the present invention, illustrating the changes in the visual fauna and response of medaka fish using carbonated water. [Figure 13] This graph shows the results of an experiment according to an embodiment of the present invention, illustrating the changes in the visual fluidity and response of medaka fish when dry ice is intermittently used. [Figure 14] This graph shows the results of an experiment according to an embodiment of the present invention, illustrating the changes in visual and sensory fluotaxis and response of medaka fish when dry ice is intermittently used. [Figure 15] This graph shows the results of an experiment according to an embodiment of the present invention, illustrating the change in the visual flowability of medaka fish when dry ice is used intermittently. [Figure 16] This graph shows the results of an experiment according to an embodiment of the present invention, illustrating the change in the reaction of medaka fish when carbonated water is used. [Figure 17] This graph shows the results of an experiment according to an embodiment of the present invention, illustrating the changes in the response of medaka fish when dry ice is used intermittently. [Figure 18] This graph shows the results of an experiment according to an embodiment of the present invention, illustrating the changes in the response of medaka fish when dry ice is used intermittently. [Figure 19]It is a graph showing changes in the response of medaka when one block of 5 g dry ice is used consecutively, which is the result of an experiment according to an embodiment of the present invention. [Figure 20] It is a graph showing changes in the response of medaka when one block of 5 g dry ice is used consecutively, which is the result of an experiment according to an embodiment of the present invention. [Figure 21] It is a graph showing changes in the response of medaka when one block of 10 g dry ice is used consecutively, which is the result of an experiment according to an embodiment of the present invention. [Figure 22] It is a graph showing changes in the response of medaka when one block of 10 g dry ice is used consecutively, which is the result of an experiment according to an embodiment of the present invention. [Figure 23] It is a graph showing changes in the response of medaka when one block of 10 g dry ice is used consecutively, which is the result of an experiment according to an embodiment of the present invention. [Figure 24] It is a graph showing changes in the response of medaka when one block of 10 g dry ice is continuously used, which is the result of an experiment according to an embodiment of the present invention. [Figure 25] It is a graph showing changes in the response of medaka when two blocks of 10 g dry ice are continuously used, which is the result of an experiment according to an embodiment of the present invention. [Figure 26] It is a graph showing changes in the response of medaka when four blocks of 10 g dry ice are continuously used, which is the result of an experiment according to an embodiment of the present invention. [Figure 27] It is a graph showing changes in the response of medaka when one block of 15 g dry ice is continuously used, which is the result of an experiment according to an embodiment of the present invention. [Figure 28] It is a graph showing changes in the response of medaka when one block of 15 g dry ice is continuously used, which is the result of an experiment according to an embodiment of the present invention. [Figure 29] It is a diagram summarizing Figures 6 to 9, which show changes in and responses of the sensory rheotaxis of medaka when carbonated water and dry ice are used, which is the result of an experiment according to an embodiment of the present invention. [Figure 30]Figures 10 to 13 summarize the results of experiments according to embodiments of the present invention, showing the changes in the visual flowability and responses of medaka fish using carbonated water and dry ice. [Figure 31] Figures 14 to 18 summarize the results of experiments according to embodiments of the present invention, showing the changes in visual and sensory fluotaxis and response of medaka fish when using carbonated water or intermittently dry ice. [Figure 32] This figure combines graphs showing the experimental results according to embodiments of the present invention. [Modes for carrying out the invention]

[0009] The following examples of the present invention will be described with reference to the attached drawings. The following examples are illustrative for explaining the present invention and are not intended to limit the present invention to these examples alone. Furthermore, the present invention can be modified in various ways without departing from its essence. In addition, the same reference numerals are used for the same components in each drawing whenever possible, and redundant explanations are omitted. [Examples]

[0010] <1> Fish to be anesthetized In this invention, all small fish are included as subjects for anesthesia, but the medaka used in the experiment is preferred.

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

[0012] <3> Procedure for manufacturing anesthetic water The procedure for preparing anesthetic water is simply to add solid carbon dioxide, also known as "dry ice," to fresh water, and the quantities will be explained below.

[0013] Based on the experimental results described later, it is preferable to use dry ice as an anesthetic method because it requires less effort. This is because dry ice offers high reproducibility of carbon dioxide concentration in water. In the experiments described later, the mortality rate was the same for carbonated water and dry ice, indicating equivalent safety. However, when a large amount of carbonated water is added at once to increase the carbon dioxide concentration, the concentration in the water rises more rapidly than with dry ice, making it less safe.

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

[0015] After the dry ice has completely dissolved, the medaka can be carefully observed, and any medaka that have stopped breathing for more than one minute can be revived by returning them to fresh water. In the method of the present invention, the medaka are anesthetized approximately 10 minutes after the dry ice is introduced, and then they can be returned to fresh water for about 20 minutes of microscopic observation.

[0016] The following is an experiment that demonstrates the basis for the above conclusion. The type of medaka used in the experiment was • Miyuki medaka (Experiments 3 to 9) Albino Miyuki medaka (Experiment 9) • Blue medaka (Experiment 9) • Tricolor transparent-scaled medaka (Experiment 9) The underwater carbon dioxide concentration meter used was the CGP-31 (manufactured by Toa DKK Co., Ltd.), and the carbonated water generator used was the SodaStream E-TERRA (SodaStream Co., Ltd.).

[0017] <4> Experiment 1 (Preliminary Experiment) (1) As shown in Figure 1, a donut-shaped waterway is created using one large circular water tank, two small circular water tanks, and three dumbbells (2 kg). (2) Add 1 liter of water W to the prepared waterway, create a water flow with an underwater motor, and start measuring the carbon dioxide concentration in the water while stirring. (3) Pour 53 ml of carbonated water into the waterway (carbonated water concentration 5%) and start measuring the time. (4) Subsequently, 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 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 was thought that although the carbon dioxide concentration in the water increased when carbonated water was added, it began to slowly decrease immediately afterward. Figure 3 shows the results of Experiment 1 and Experiments 3, 5, and 7, which will be described later. Even when using carbonated water produced with the same carbonated water maker, there was considerable variation in how the carbon dioxide concentration in the water increased.

[0019] <5> Experiment 2 (Preliminary Experiment) (1) As shown in Figure 4, the small aquarium 2 was wrapped in black paper 4 to prevent external 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 stirring bar and a stirrer. (3) Dry ice was added and the time measurement was started, and the measurement of the carbon dioxide concentration in the water was continued.

[0020] In Experiment 2, another preliminary experiment, the amount of dry ice was divided into 5g, 10g, and 15g. For the 10g and 15g amounts, the experiment was divided into cases where one block weighed 10g or 15g, and cases where two or more blocks totaled 10g or 15g. Figure 5 shows the results of Experiment 2 and Experiments 4, 6, and 8, which will be described later. As the weight increased from 5g to 10g to 15g, the rate of increase in the carbon dioxide concentration in the water increased faster and the maximum concentration also increased, but the way in which the carbon dioxide concentration increased was similar for each weight. It was found that with 10g and 15g, the carbon dioxide concentration increased faster and the maximum concentration was higher when two or more blocks were used. Also, as the dry ice became smaller, it began to float to the surface of the water, and at that point, the carbon dioxide concentration began to decrease.

[0021] <6> Experiment 3 (Observation of subjective flowability when using carbonated water) Sensory rheotaxis refers to the habit of swimming against the current of water. (1) As shown in Figure 1, a donut-shaped waterway is created using circular water tanks (large and small) and dumbbells (2 kg). (2) 1 liter of water and medaka fish (only Miyuki medaka were used in Experiment 3-8; see Table 1 below) were placed in the prepared waterway, and the measurement of the carbon dioxide concentration in the water was started while creating a water flow with an underwater motor. (Nets were stretched in front of and behind the underwater motor, but in the early experiments, there were cases where anesthetized medaka were swept away through gaps in the nets and caught in the motors and died, so in all subsequent experiments the motors themselves were wrapped in nets.) (3) 53 ml of carbonated water (5% concentration) was poured into the waterway, and time measurement was started. (4) Sensory flow patterns were observed for 1 minute starting 6 minutes after the injection of carbonated water. (5) Then, 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 the procedure (4) was repeated.

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

[0023] Experiments 3 and 4 described above investigated at what stage perceptual dysphagia is lost due to an increase in the carbon dioxide concentration in the water. Figures 6 and 7 show the results using carbonated water, while Figures 8, 9, and 14 show the results using dry ice. Even medaka that had stopped swimming in the absence of water flow appeared to be awakened by the stimulation of the water flow and swam against the current.

[0024] Using carbonated water resulted in a longer time until the loss of perceived rhozynthesis, which may be because the carbon dioxide concentration in the water increased gradually, allowing the medaka to gradually adapt to the rising carbon dioxide levels. On the other hand, using dry ice resulted in a faster increase in carbon dioxide concentration, which is thought to have led to faster anesthesia.

[0025] <8> Experiment 5 (Observation of visual flow characteristics when using carbonated water) Visual dysphagia is the habit of swimming as if following the surrounding scenery, and when a striped pattern is rotated around the aquarium, the medaka will follow the stripes. (1) As shown in Figure 1, a donut-shaped waterway is created using circular water tanks (large and small) and dumbbells (2 kg). (2) 1 liter of water and medaka fish were placed in the prepared waterway, and the measurement of the carbon dioxide concentration in the water was started while creating a water flow with an underwater motor. (3) 53 ml of carbonated water (5% concentration) was poured into the waterway, and time measurement was started. (4) In order to observe the visual flow, a striped piece of cardboard is rotated around the tank, which makes it difficult to use the underwater carbon dioxide measuring device for continuous measurement. Therefore, the underwater motor was stopped before observing the visual flow, 40cc of water was transferred from the waterway to the measuring cell, and carbon dioxide concentration measurement was resumed. (5) As shown in Figure 2, the striped pattern was rotated for 1 minute 6 minutes after the carbonated water was injected, and the visual flow characteristics were observed. (6) Then, 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)-(5) were repeated.

[0026] <9> Experiment 6 (Observation of visual flowability using dry ice) (1) As shown in Figure 1, a donut-shaped waterway is created using circular water tanks (large and small) and dumbbells (2 kg). (2) 1 liter of water and medaka fish were placed in the prepared waterway, and the measurement of the carbon dioxide concentration in the water was started while creating a water flow with an underwater motor. (3) Dry ice was poured into the waterway and time measurement was started. (4) Dry ice was recovered from the waterway after 2 minutes. (5) After that, the underwater motor was stopped, 40cc of water was transferred from the waterway to the measurement cell for concentration measurement, and the measurement of carbon dioxide concentration was resumed. (6) Three minutes after the dry ice was recovered, the striped pattern was rotated for one minute to begin observing its visual flow characteristics. (7) After that, steps (3)-(6) were repeated every 7 minutes.

[0027] The visual dysphagia observed in experiments 5 and 6 above is the habit of swimming as if following the surrounding scenery. When a circular tank without water current is surrounded by patterned cardboard, and the pattern is moved, the fish swim as if following the pattern. This is thought to be because it creates the illusion of water current, and medaka have a habit of trying to stay in the same position to avoid being swept away by the current.

[0028] Although not included in this experiment, previous experiments have shown that when a pattern is moved in the same direction as the water flow in a tank with current, medaka fish swim following the pattern rather than resisting the current. It has been found that visual dysphagia is stronger than sensory dysphagia, and that fish are more strongly influenced by visual information than by the sensation of current on their body surface.

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

[0030] Although the medaka did not close their eyes, it was thought that anesthesia had prevented them from gathering visual information. As shown in Figures 10 to 15, the time until visual rhotaxis disappeared was longer when carbonated water was used. This was thought to be because, similar to the case with sensory rhotaxis, a slow increase in carbon dioxide concentration allowed the medaka to adapt, delaying the onset of anesthesia, while a rapid increase in carbon dioxide concentration resulted in faster anesthesia.

[0031] <10> Experiment 7 (Observation of the reaction when using carbonated water) (1) To prevent external visual interference when observing dangerous behavior (see (b) and (c) below), the small aquarium was wrapped in black paper. (2) 500 ml of water and medaka fish were placed in a small tank, and the measurement of the carbon dioxide concentration in the water was started while stirring with a stirring bar and a stirrer. (The stirring bar was surrounded with a net to prevent the medaka fish 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 (a)-(d) were performed for 2 minutes and observed. (a) I fed them. (b) I tapped the tank. (c) I held my hand over the tank. (d) I touched the medaka with a stick. (5) Then, 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 the procedure (4) was repeated.

[0032] <11> Experiment 8 (Observation of the reaction using dry ice) (1) To prevent external visual influences when observing the danger avoidance behaviors (see (b) and (c) below), the small aquarium was wrapped in black paper. (2) 500 ml of water and medaka fish were placed in a small tank, and the measurement of the carbon dioxide concentration in the water was started while stirring with a stirring bar and a stirrer. (3) Dry ice was added and the time measurement was started. (4) (Intermittent use of dry ice) After 2 minutes, the dry ice was retrieved from the waterway, and after 3 minutes, the following (a)-(d) were performed 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 performed for 2 minutes and observed. (a) I fed them. (b) I tapped the tank. (c) I held my hand over the tank. (d) I touched the medaka with a stick. (5) Then, steps (3)-(4) were repeated.

[0033] <12> Experiment 9 (Observation of medaka fish using anesthesia with dry ice) (1) 500 ml of fresh water and one medaka (Miyuki medaka, albino Miyuki medaka, tricolor transparent-scaled medaka, blue medaka) were placed in each small aquarium, and the measurement of the carbon dioxide concentration in the water was started. (2) 10g of dry ice was added to the tank and the medaka were observed. (3) When the medaka stopped moving even with a slight stimulus (such as being lightly touched with a stick), they were transferred to a resealable plastic bag containing 5 ml of water and observed under a microscope. (4) The movement of the medaka's heart and gills was observed, and the blood flow in the arteries and veins of the medaka's fins was checked. After that, the entire body of the medaka was observed for about 20 minutes.

[0034] In experiments 7 and 8 described above, we examined the order in which feeding behavior (eating food), danger avoidance behavior (moving in response to vibrations in the tank, reacting when a hand is held above the tank), swimming (normal swimming around), and escape behavior in response to external stimuli (touching the body with a stick) disappeared during the anesthesia process. As shown in Figures 16 to 18 and 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 in response to external stimuli → breathing. A summary of the series is shown in Figure 32.

[0035] Furthermore, in external stimuli, responses to light touch (mild stimulation) and strong touch (strong stimulation) were examined separately. However, in observational experiments like Experiment 9, it was considered sufficient to anesthetize the animal to the point where it became immobile with light stimulation (immobility with light stimulation).

[0036] As shown in Figures 19 to 28, each experiment focused on observing the disappearance of the response to external stimuli when the weight or number of dry ice blocks was changed. As a result, as shown in Table 1 below, it was considered that a rapid increase in the carbon dioxide concentration in the water would quickly cause medaka to stop breathing and pose a high risk of death. [Table 1]

[0037] Therefore, if breathing stopped for more than one minute, rescue was carried out by moving the fish to fresh water. If breathing stopped for one minute, the fish would resume breathing and recover after being returned to fresh water. Medaka that were anesthetized quickly due to a rapid increase in the carbon dioxide concentration in the water recovered more quickly.

[0038] Medaka fish that were kept in water with a high carbon dioxide concentration for a long time took longer to recover when returned to fresh water. Furthermore, as shown in Figures 8, 14, 18, and 19, it was found that using dry ice lowered the water temperature by 2-4 degrees Celsius in approximately 30 minutes, depending on the weight and number of pieces used.

[0039] In Experiment 9, we used Miyuki medaka, albino Miyuki medaka, tricolor transparent-scaled medaka, and blue medaka, which are considered easy to observe internally. As shown in Table 1 above, after adding 10g of dry ice to 500ml of water, all medaka were anesthetized to a degree that they would not react to mild stimuli after about 10 minutes (average 12 minutes).

[0040] The medaka were then transferred to a 5ml resealable plastic bag filled with fresh water, and observed under a microscope for approximately 20 minutes while they were still alive (breathing but not moving). We were able to observe their heartbeat, gill movement, blood flow in the blood vessels, mainly around the tail fin, and the surface of their bodies, and even locate their lateral lines.

[0041] The heartbeat could be detected from the body surface, but it was particularly clear in the pale blue medaka and the three-color transparent-scaled medaka, whose bodies are translucent.

[0042] The movement of the gills was clearly visible, but the inside of the gills could not be seen. Blood vessels in the caudal fin were confirmed in all medaka, and blood flow was observed in both arteries and veins.

[0043] Observation of the body surface revealed the presence and movement of a parasite called Gyrodactylus, which is invisible to the naked eye, and confirmed infection. This demonstrated that it can be used for screening parasitic infections.

[0044] During the search for the lateral line, no clear lateral line was found in any of the medaka, leading to the conclusion that medaka of all species lack a lateral line. Anesthetizing the fish allowed for slow observation (presumably without causing pain or stress), enabling observations that would not be possible without anesthesia. Furthermore, for observations like this, even a mild anesthetic that prevents a reaction to subtle stimuli is sufficient and therefore considered safe.

[0045] Based on the results of experiments 3 through 9 described above, it was concluded that the simplest, quickest, and safest method of anesthetizing medaka, a small fish species, 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 of anesthetizing small fish, Add 8g to 12g of dry ice to 500ml of fresh water in a tank containing small fish. A method for anesthetizing small fish, characterized by the following features.

2. The method of anesthesia according to claim 1, The aforementioned small fish is a medaka. A method for anesthetizing medaka fish, characterized by the following:

3. After inducing anesthesia in the small fish using the anesthesia method described in claim 1, The aforementioned small fish are examined under a microscope, visually, tactilely, and observed. A method for observing small fish characterized by the following features.

4. After inducing anesthesia in the small fish using the anesthesia method described in claim 1, Confirmation of parasitic infection in the aforementioned small fish A screening method for small fish characterized by the following features.

5. After inducing anesthesia in small fish using the anesthesia method described in claim 1, Within 20 minutes of the introduction, return the small fish to fresh water. A method for waking up small fish, characterized by the features described above.

Images