WO2022210397A1 - 供試魚処置設備およびその供試魚処置設備を使用する供試魚の処置方法 - Google Patents
供試魚処置設備およびその供試魚処置設備を使用する供試魚の処置方法 Download PDFInfo
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- WO2022210397A1 WO2022210397A1 PCT/JP2022/014556 JP2022014556W WO2022210397A1 WO 2022210397 A1 WO2022210397 A1 WO 2022210397A1 JP 2022014556 W JP2022014556 W JP 2022014556W WO 2022210397 A1 WO2022210397 A1 WO 2022210397A1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/90—Sorting, grading, counting or marking live aquatic animals, e.g. sex determination
- A01K61/95—Sorting, grading, counting or marking live aquatic animals, e.g. sex determination specially adapted for fish
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K1/00—Housing animals; Equipment therefor
- A01K1/06—Devices for fastening animals, e.g. halters, toggles, neck-bars or chain fastenings
- A01K1/0613—Devices for immobilisation or restraint of animals, e.g. chutes
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K29/00—Other apparatus for animal husbandry
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Definitions
- the present invention relates to a test fish treatment facility (fish holding device) for holding test fish and using them for various biological observations, biological experiments, etc., and furthermore, the test fish treatment equipment is used to treat the test fish. It relates to a method of treating test fish (method of administering to fish) in which a predetermined treatment is given to a test fish.
- the method of administering the test substance to the test fish includes a method of mixing the test substance with food and throwing it into the tank and letting the test fish eat it, and a method of dissolving the test substance in breeding water and absorbing it through the gills (e.g. , fish acute toxicity test conforming to OECD test guideline 203), etc. are known.
- the method of administration in which feed containing the test substance is put into a water tank has the problem that it is difficult to handle the liquid test substance, and furthermore, leftover food occurs, making it impossible to accurately determine the intake of test fish. .
- these administration methods are different from animal experiments, such as mice, where the oral administration method of administering the test substance directly into the digestive tract is adopted, so it is not possible to appropriately compare with the animal experiments. There's a problem. For these reasons, it is desired to adopt an oral administration method in which a test substance is directly administered to the digestive organs of test fish.
- zebrafish have attracted a great deal of attention as experimental animals.
- damage to the test fish is minimized, that is, damage to the body surface etc.
- An important issue is to hold the test fish as gently as possible without inflicting bruising or causing symptoms of burns. Therefore, conventionally, a fish retainer has been proposed in which a synthetic resin plate is bent into a U shape and a flexible material such as sponge or nonwoven fabric is attached to the inner surface side for retaining the test fish (for example, Patent Document 1). reference).
- the present invention has focused on such conventional problems, and its object is to suppress damage to the test fish as much as possible and to reliably hold the test fish in a predetermined posture.
- An object of the present invention is to provide a fish treatment facility and, further, to provide a method for treating test fish using the test fish treatment facility.
- a first characteristic configuration of the test fish treatment equipment comprises a holder for holding the test fish,
- the holder is provided with a posture-maintaining groove for maintaining the test fish in a predetermined posture, and at least the inner surface of the posture-maintaining groove is made of a flexible material.
- the holder for holding the test fish is provided with the posture maintaining groove for maintaining the test fish in a predetermined posture. It is performed in a state located within. Since at least the inner surface of the attitude-maintaining groove is made of a flexible material, the test fish can be gently held by the flexible inner surface of the attitude-maintaining groove. Even if the fish tries to act violently, it is difficult to act violently because it is located in the posture maintenance groove, and as a result, it is possible to suppress damage to the test fish. In addition, since the test fish can be held in a resting state while moisturizing the test fish in the holding fixture, it is possible to repetitively orally administer to the test fish without anesthesia while greatly suppressing damage to the test fish. or parenteral administration.
- a second characteristic configuration of the test fish treatment equipment according to the present invention is that the holder is made of a material having flexibility, water permeability, and water retention.
- the material that constitutes the holder is not only flexible but also water-permeable.
- the test fish will remain in the attitude maintenance groove, contributing to the reduction of damage to the test fish.
- it also has water retention, so that when the test fish is maintained in the posture maintenance groove, the posture maintenance groove is in an appropriate water retention state. It is also possible to reduce damage.
- the entire holder is made of a material having such performance, for example, a sponge, it is easier to maintain the posture than when the sponge is adhered to the inner surface of the posture maintaining groove to form the holder.
- the tools are easy and inexpensive to manufacture.
- the third characteristic configuration of the test fish treatment equipment according to the present invention is that the posture maintenance groove is tapered toward the tip side and inclined downward toward the tip side.
- the test fish flowing into the posture-maintaining groove together with the water gradually approaches the predetermined posture toward the tip side. is induced to As a result, the test fish moved toward the tip side along the flow of water due to its habit, and was gradually sandwiched by the inner surface of the posture-maintaining groove and maintained in a predetermined posture. effectively suppressed.
- the fourth characteristic configuration of the equipment for treating test fish according to the present invention is that the groove for maintaining posture is formed with the same width from the front end side to the rear end side, and has an upper groove portion that opens upward and continues downward. and a lower groove portion wider than the upper groove portion.
- the test fish flowed out together with the water into the upper groove part that opens upward in the attitude maintaining groove is smoothly received in the upper groove part while maintaining its attitude, and the fish is formed below the upper groove part. It is possible to smoothly guide the test fish to the lower groove portion wider than the upper groove portion and hold the test fish in the lower groove portion with less burden.
- a fifth characteristic configuration of the apparatus for treating test fish according to the present invention is that the depth of the attitude-maintaining groove is set to be greater than the height of the test fish held in the attitude-maintaining groove. be.
- a sixth characteristic configuration of the equipment for treating test fish according to the present invention is that the holder includes a rear end side holding portion and a front end side holding portion that bisect the posture maintaining groove in the longitudinal direction, and both holding portions are The point is that they are configured to be separable.
- the holder has a rear end side holding portion and a front end side holding portion that bisect the posture maintaining groove in the longitudinal direction, and both holding portions are separable.
- the width of the groove can be set appropriately. For example, by setting the width at the tip end of the rear end side holding part to a width where only the mouth or head of the test fish protrudes from the rear end side holding part to the tip side, it is possible to maintain the posture of the rear end side holding part. By holding the test fish in the groove, the mouth and head of the test fish can be easily and reliably observed.
- the posture maintenance groove of the tip side holding part is provided.
- a seventh characteristic configuration of the equipment for treating test fish according to the present invention is that the holder comprises a front holding portion, an intermediate holding portion, and a rear holding portion arranged along the longitudinal direction of the posture maintaining groove, and the intermediate holding portion are positioned between the front holding portion and the rear holding portion, the three holding portions are configured to be separable, and the tip of the posture maintaining groove is located between the front holding portion and the intermediate holding portion. It is provided only in the intermediate holding portion of the front holding portion and the intermediate holding portion so as to be positioned at the boundary between the front holding portion and the intermediate holding portion, and is tapered toward the distal end side in plan view.
- the tip of the posture-maintaining groove is positioned at the boundary between the front holding portion and the intermediate holding portion, the test fish in which the posture-maintaining groove is moved to the tip side along the water flow. can be received at the front holding portion.
- the front holding part and the intermediate holding part By separating the front holding part and the intermediate holding part while receiving the test fish in this way, if the head of the test fish is on the tip side, the mouth or head of the test fish can be placed in the position maintenance groove. It can be observed from the tip. Therefore, it is possible to easily and reliably observe the mouth and head of the test fish.
- the length of the intermediate retention part equal to the body length of the test fish and separating the intermediate retention part and the rear retention part, even if the head of the test fish is on the rear end side, The head can be observed from the rear end of the posture groove. Further, the presence of the rear holding portion makes it easier to position the guiding means with respect to the intermediate holding portion by, for example, placing the guiding means on the rear holding portion.
- the eighth characteristic configuration of the test fish treatment equipment according to the present invention comprises guiding means for guiding the test fish together with water to the holder, Said guiding means comprises an inlet at one end into which the test fish is introduced together with water, and an outlet at the other end through which the test fish are discharged together with the water into the attitude maintenance groove. in the point.
- the guiding means for guiding the test fish together with the water to the holder is provided, and the guiding means is provided with an inlet into which the test fish is introduced together with the water at one end thereof. , the other end is equipped with an outlet for the test fish to flow out to the attitude maintenance groove together with water, so the test fish can be placed in the water by simply putting the test fish together with the water from the inlet of the guidance means. Together with it, it flows toward the holder side and enters into the attitude maintenance groove, so that even in the process up to the attitude maintenance groove, damage to the test fish can be greatly suppressed.
- the guide means is composed of a pipe member having the inlet at one end and the outlet at the other end, A pipe-shaped outflow portion disposed above and adjacent to the attitude maintaining groove is connected to the outflow port, the outflow portion has a slit facing along the attitude maintaining groove, and from the slit The point is that the test fish is flowed into the posture maintenance groove together with the water in a predetermined posture.
- the pipe-shaped outflow part arranged adjacent to the upper position maintaining groove and connected to the outflow port has a slit facing along the position maintaining groove, and the water flows through the slit. Since the test fish flows out into the attitude maintenance groove together with the test fish in a predetermined attitude, the transfer of the water and the test fish from the outflow port to the attitude maintenance groove is smooth and reliable, and the damage to the test fish is minimized during that time. is suppressed.
- a tenth characteristic configuration of the test fish treatment equipment according to the present invention is a first water tank containing the test fish before being introduced into the inlet, and containing the test fish released from the holding by the holding fixture. and a second water tank.
- the test fish stored in the first water tank can be taken out together with the water and immediately put into the inlet of the guiding means. Moreover, the test fish released from the holding by the holder can be immediately accommodated in the second water tank. Therefore, it is possible to reduce the burden associated with transporting the test fish.
- An eleventh characterizing configuration of the test fish treatment equipment according to the present invention is provided with guiding means for guiding the test fish to be subjected to a predetermined treatment together with water to a predetermined treatment section,
- the guiding means has an inlet into which the test fish are introduced together with water at one end thereof, and an outlet through which the test fish are discharged together with the water into the treatment section at the other end. at the point.
- the guiding means for guiding the test fish together with the water to the holder is provided, and the guiding means is provided with an inlet into which the test fish is introduced together with the water at one end thereof. At the other end, the test fish is provided with an outlet for flowing out the test fish together with the water into the treatment section such as the above-mentioned posture maintenance groove. , the test fish flows to the holder side together with the water and enters the treatment section, and damage to the test fish can be greatly suppressed even in the process up to the treatment section.
- the twelfth characteristic configuration of the test fish treatment equipment according to the present invention is that the guiding means is composed of a pipe member having the inlet at one end and the outlet at the other end.
- the guide means is composed of a pipe member having an inlet at one end and an outlet at the other end, for example, it is composed of a gutter-shaped member with an open upper surface. Compared to , there is no risk of the test fish jumping out during guidance, and not only is the test fish guided reliably, but the degree of freedom in setting the route up to the treatment section is increased.
- the thirteenth characteristic configuration of the test fish treatment equipment according to the present invention is provided with water flow forming means for supplying water to the inlet to form a water flow in the pipe member.
- water is supplied to the inlet of the guide means by the water flow forming means, and a water flow is formed in the pipe member connected to the inlet.
- the test fish put into the pipe member can be guided to the outflow port by the water flow formed in the pipe member, and flow out from the outflow port to the treatment section.
- a fourteenth characteristic configuration of the test fish treatment equipment according to the present invention is that a funnel-shaped funnel part for guiding the test fish together with water to the input port is connected to the input port.
- the funnel-shaped funnel part that guides the test fish together with the water is connected to the inlet, by using the funnel-shaped funnel part, it is possible to supply water to the inlet. The inflow of test fish becomes easier.
- a fifteenth characteristic configuration of the test fish treatment facility according to the present invention is that the treatment section is installed and includes a water receiving section for receiving water discharged from the outflow port.
- the water receiving part since the water receiving part is provided, even if the test fish discharged from the outlet falls off from the treatment part, the dropped test fish can be stored in the water receiving part without burden. Can be accommodated in water. Furthermore, if a water level maintaining means for maintaining the water level of the water receiving part at a predetermined set water level while draining the water stored in the water receiving part is provided, the treatment part immersed in the water stored in the water receiving part is provided. It is possible to keep the wetness of the water in a constant state and keep the test fish held in the treatment section in an appropriate moisturizing state.
- a first characteristic configuration of the method for treating test fish according to the present invention is to use a test fish treatment facility having any one of the first to tenth characteristic configurations to dispose of a test fish held by the holder. It is a point to take a predetermined treatment for.
- the test fish treatment facility having any one of the first to tenth characteristic configurations when holding the test fish, the damage to the test fish is suppressed as much as possible and the predetermined treatment is performed. It can be held securely in position. In this state, the test fish held by the holder is subjected to a predetermined treatment, so that the treatment can be performed easily and reliably. In addition, since the test fish can be held in a resting state while moisturizing the test fish in the holding fixture, it is possible to repetitively orally administer to the test fish without anesthesia while greatly suppressing damage to the test fish. or parenteral administration.
- an oral tube is inserted into the oral cavity of the test fish, which is maintained in a predetermined posture by the posture-maintaining groove of the holder.
- Oral administration is performed by directly orally administering the substance to the test fish.
- the test substance enclosed in a syringe or the like is directly applied to the test fish through an oral tube inserted into the oral cavity of the test fish while the test fish is securely held in a predetermined posture by the holder.
- Oral administration enables easy and reliable oral administration.
- Direct oral administration includes methods such as the conventional method of mixing the test substance with food and throwing it into the tank and having the test fish eat it, or the method of dissolving the test substance in breeding water and allowing it to be absorbed through the gills. Differently, oral administration is performed by administering the test substance directly to the oral cavity of the test fish.
- Direct oral administration of the test substance to the test fish may be carried out using a syringe with an oral tube such as an oral sonde while the test fish is under general anesthesia.
- an oral tube such as an oral sonde
- the conventional method of anesthetizing the test fish means anesthetizing the test fish every day. Such an evaluation test is difficult because a great stress is applied.
- direct oral administration can be performed without anesthetizing the test fish while minimizing damage. This makes it easy to evaluate the effect of the test substance on the test fish, and furthermore, it becomes possible to conduct an experiment in which a small amount of the test substance is given to the test fish every day to evaluate the effect.
- the predetermined treatment is to inject the test substance into the test fish maintained in a predetermined posture by the posture maintaining groove of the holder.
- the point is parenteral administration to test fish.
- parenteral administration is performed by administering the test substance to the test fish by injecting it into the test fish while the test fish is securely held in a predetermined posture by the holder.
- Parenteral administration includes, for example, intraperitoneal administration using injection and intravenous administration.
- the test fish in carrying out such parenteral administration, if the test fish is rotated around the front-rear axis by the holder so that the abdomen of the test fish is exposed to the upper opening of the posture-maintaining groove, the test fish can be administered. Injection can be easily performed into the abdomen of the test fish.
- FIG. 1 The perspective view which shows the state of parenteral administration to a test fish.
- Schematic diagram of the entire test fish treatment facility A plan view of a fish holder in another embodiment Front view of a holder (intermediate holding portion) in another embodiment Front view of a holder (intermediate holding portion) in another embodiment Front view of a holder (intermediate holding portion) in another embodiment Front view of a holder (intermediate holding portion) in another embodiment
- the apparatus for treating test fish of the present invention comprises a holder 1 for holding a relatively small test fish F (see FIG. 2) for biological experiments, such as zebrafish, and Guiding means 2 composed of a pipe member 2c for guiding the test fish F together with water to the holder 1 is provided.
- the pipe member 2c is transparent and A flexible synthetic resin pipe is used.
- the holder 1 is generally rectangular in plan view and made of a material having flexibility, water permeability, and water retentivity, such as sponge, to hold the test fish F by force. It has a posture maintaining groove 3 with a bottom and an upward opening which maintains a predetermined posture in a natural state without any looseness.
- the grooves 3 for maintaining the posture of the holder 1 are arranged so that the longitudinal length L, which is the length in the longitudinal direction, is longer than the body length of the test fish F. , is provided over the entire length of the holder 1 from the rear end to the tip.
- the test fish F is a zebrafish with a body length of about 40 mm
- the width of the groove for maintaining posture 3 is such that the rear end has a maximum width W1 wider than the test fish F, and the tip has a minimum width W2 narrower than the test fish F. As shown in FIG.
- the bottom 3a of the posture maintaining groove 3 is configured to be inclined downward toward the tip side in a side view.
- the holder 1 has a rear end side holding portion 1a and a front end side holding portion 1b that bisect the posture maintaining groove 3 in the longitudinal direction. Then, as shown in FIGS. 2 and 5, the lateral width of the posture maintaining groove 3 at the tip end of the rear end side holding portion 1a is the same as that of the test fish F at the posture maintaining groove 3 of the rear end side holding portion 1a. is maintained in a predetermined posture, the mouth or head of the test fish F is set to have an intermediate width W3 that protrudes from the rear end side holding portion 1a toward the tip side.
- the guiding means 2 for guiding the test fish F is composed of a pipe member 2c, as shown in FIG. At the other end of the pipe member 2c, there is provided an outlet 2b through which the test fish F flows out to the posture maintaining groove 3 together with water.
- a funnel-shaped funnel portion 4 for guiding the test fish F together with water into the pipe member 2c is connected to the inlet 2a, and a pipe-shaped outlet 5 is connected to the outlet 2b.
- a portion 5 is arranged above and adjacent to the posture maintaining groove 3 .
- the outflow part 5 is provided with a tapered slit 5a for flowing out the test fish F together with the water into the posture maintaining groove 3 in a predetermined posture. As shown in FIG.
- the guide means 2 and the holder 1 are arranged facing each other from above along the posture maintaining groove 3 of the holder 1, and have the funnel portion 4 and the outflow portion 5.
- the guide means 2 is installed such that the funnel portion 4 is positioned upward and the outflow portion 5 is positioned downward.
- the funnel portion 4 and the outflow portion 5 are configured separately from the pipe member 2c, but one or both of them may be configured integrally with the pipe member 2c.
- the pipe member 2c as the guide means 2 is arranged in a downwardly inclined state from the inlet 2a to the outlet 2b.
- the inclined state of the pipe member 2c is appropriately set to a state in which the test fish F introduced into the inlet 2a can be appropriately guided to the outlet 2b while reducing the burden on the test fish F.
- the vertical height between the inlet 2a and the outlet 2b in the pipe member 2c is within the range of 100 to 300 mm. If the height of the pipe member 2c is less than 100 mm, the test fish F introduced into the inlet 2a may stop in the middle of the pipe member 2c and not reach the outlet 2b.
- the horizontal distance between the inlet 2a and the outlet 2b in the pipe member 2c is within the range of 150 to 300 mm.
- the distance of the pipe member 2c is less than 150 mm, the moving distance of the test fish F introduced into the inlet 2a toward the outlet 2b may be too short and the direction of the test fish F may not be fixed.
- the distance of the pipe member 2c exceeds 300 mm, the movement distance of the test fish F introduced into the inlet 2a toward the outlet 2b becomes excessive, resulting in an excessive burden on the test fish F.
- the funnel portion 4 is connected to a water supply pipe 15 for supplying water SW into the funnel portion 4 .
- the water supply pipe 15 functions as water flow forming means for supplying water to the inlet 2a of the guide means 2 to form a water flow WF within the pipe member 2c. Since the water flow WF is formed in the pipe member 2c connected to the inlet 2a, the test fish F introduced into the inlet 2a can be fed while minimizing the burden on the test fish F moving in the pipe member 2c.
- the water flow WF formed in the pipe member 2c guides the water to the outflow port 2b, and allows the water to flow out from the outflow port 2b into the posture maintaining groove 3 of the holder 1.
- FIG. Further, in the plan view shown in FIG.
- the direction of water supply by the water supply pipe 15 inside the funnel portion 4 is the circumferential direction with the inlet 2a as the central axis.
- the water flow WF swirls in the funnel 4 and flows into the inlet 2a, so that the entire inner wall surface of the funnel 4 is kept wet, and the burden on the test fish F to be introduced is reduced. can do.
- a test fish treatment facility is installed as shown in FIG. Then, since the water flows downward inside the pipe member 2c, the test fish F swims with its head downward according to its habit, and passes through the slit 5a of the outflow part 5 in a predetermined posture, that is, the head. toward the tip side holding portion 1b, it enters the posture maintaining groove 3 together with water. In the posture maintaining groove 3, the water further flows toward the tip side of the holder 1, so that the test fish F is also flowed together and finally stopped while being clamped by the posture maintaining groove 3.
- the test fish F is maintained in a predetermined posture in the posture maintenance groove 3 of the rear end side holding portion 1a, and the oral sonde 8 (oral tube) is attached to the test fish F. ) allows direct oral administration of the test substance.
- test substance is enclosed in a syringe 7, the tip 8a of the oral probe 8 attached to the syringe 7 is inserted into the oral cavity of the test fish F, and the test substance is injected through the oral probe 8 into the test fish F. orally administered directly to At this time, since the test fish F is held so that only its mouth or head protrudes from the rear end side holding portion 1a toward the tip side, the gill lids of the test fish F are held by the posture maintaining grooves 3. blocked. Therefore, the directly orally administered test substance is prevented from leaking out of the body of the test fish F through the gills, and substantially the entire amount of the test substance is ingested by the test fish F.
- the test substance is not limited to a liquid substance, and may be in the form of jelly, paste or solid. Alternatively, the substance coated with the test substance may be dispersed in water or the like and orally administered directly to the test fish F.
- At least the tip portion 8a of the oral probe 8, which is inserted from the mouth of the test fish F, is made of a flexible material, so that the burden on the test fish F during insertion is reduced. Furthermore, since the proximal end of the distal end portion 8a is provided with a mark 8b that enables visual confirmation of the range to be inserted from the mouth of the test fish F, even an inexperienced experimenter can safely and stably enter the oral cavity directly. administration becomes possible.
- the flexible oral probe 8 and syringe 7 can be manufactured by integral molding.
- the oral probe 8 and syringe 7 are integrally manufactured, it is possible to prevent the retention of the test substance such as the administration agent at the joint between the oral probe 8 and syringe 7, and the joint can be prevented from being damaged during direct oral administration. You can prevent it from coming off.
- Example 1 For each of the direct oral administration methods of Examples and Comparative Examples, an effect evaluation experiment was conducted to determine the relationship between the concentration of the test substance and the mortality rate by administering the test substance to the test fish under the following experimental conditions.
- Test fish Number of individuals: zebrafish, 4 to 5 individuals / 1 tank x 6
- Test substance 3 concentrations (10 mg/kg, 100 mg/kg, 1000 mg/kg) Potassium dichromate solution
- Dose per individual 20 ⁇ L
- FIG. 6 shows the relationship between the concentration of the test substance and the mortality rate obtained in this experiment.
- Example 2 Regarding the direct oral administration method of the Examples, an effect evaluation experiment was conducted to determine the relationship between the concentration of the test substance and the mortality rate by administering the test substance to the test fish under the following experimental conditions.
- Test fish Number of individuals: Zebrafish, 5 individuals / 1 tank x 4
- Test substance Potassium dichromate solution at four concentrations (10 mg/kg, 100 mg/kg, 500 mg/kg, 1000 mg/kg)
- Dose per animal 20 ⁇ L
- FIG. 7 shows the relationship between the concentration of the test substance and the mortality rate obtained in this experiment.
- the holder 1 has a front holding portion 1c, an intermediate holding portion 1d, and a rear holding portion 1e arranged along the longitudinal direction of the posture maintaining groove 3, and the intermediate holding portion 1d is arranged with the front holding portion 1c. It is provided in a state of being positioned between the rear holding portion 1e. Further, the holder 1 is configured such that three holding portions, a front holding portion 1c, an intermediate holding portion 1d, and a rear holding portion 1e, are separable.
- the posture maintaining groove 3 is provided only in the intermediate holding portion 1d among the front holding portion 1c, the intermediate holding portion 1d, and the rear holding portion 1e.
- the posture maintaining groove 3 is provided over the entire length from the rear end to the front end of the intermediate holding portion 1d. That is, the posture maintaining groove 3 has its tip positioned at the boundary between the front holding portion 1c and the intermediate holding portion 1d, and its rear end positioned at the boundary between the intermediate holding portion 1d and the rear holding portion 1e. .
- the posture-maintaining groove 3 is formed so that the longitudinal length L is equal to or slightly longer than the body length of the test fish F. For example, when the test fish F is a zebrafish with a body length of about 40 mm, it is desirable to set the length L of the posture maintaining groove 3 to about 50 mm.
- the posture maintaining groove 3 includes an upper groove portion 3b that opens upward from the upper end surface of the intermediate holding portion 1d, and a portion below the upper groove portion 3b that continues to the upper groove portion 3b. and a lower groove portion 3c formed in the . Both the upper groove portion 3b and the lower groove portion 3c are provided over the entire length of the intermediate holding portion 1d from the rear end to the front end of the intermediate holding portion 1d.
- the width of the upper groove portion 3b is the same at any position in the longitudinal direction, and is narrower than the width of the test fish F.
- the width of the lower groove portion 3c is wider than the width of the upper groove portion 3b.
- the shape of the lower groove portion 3c when viewed in the longitudinal direction is formed in a circular shape, and the lower groove portion 3c is formed in a conical shape that tapers toward the tip side.
- the vertical width (depth) of the attitude-maintaining groove 3 is larger than the vertical width (the height of the test fish F) of the test fish F held in the attitude-maintaining groove 3 at any position in the longitudinal direction. set to large.
- the vertical width (depth) of the lower groove portion 3c is wider than the vertical width (height of the test fish F) of the test fish F at any position in the longitudinal direction.
- the entire height direction of the test fish F is accommodated in the lower groove portion 3c of the posture maintaining groove 3, so that the test fish F is held in a more rested state in the lower groove portion 3c.
- the damage to the test fish F can be greatly suppressed.
- the depth of the lower groove portion 3c is within the range of 5 to 25 mm. If the depth of the lower groove portion 3c is less than 5 mm, it may be difficult for the test fish F to enter the lower groove portion 3c. On the other hand, when the depth of the lower groove portion 3c is more than 25 mm, the test fish F entering the lower groove portion 3c is held in the vertical posture when it entered, making it difficult to take the horizontal posture. be.
- the width of the lower groove portion 3c is wider than the width of the test fish F at any position in the longitudinal direction.
- the rear holding portion 1e is sized such that its upper end surface is located below the upper end surface of the intermediate holding portion 1d.
- the rear holding portion 1e is formed with a concave portion 11 recessed downward from the upper end surface thereof.
- the bottom of the concave portion 11 is inclined downward toward the tip side, and widened toward the tip side.
- the concave portion 11 is formed in a shape along the outer shape of the pipe member 2c, which is inclined downward toward the tip side.
- the recess 11 is formed such that the lower end of the recess 11 is located above the upper end of the lower groove portion 3c.
- the intermediate holding portion 1d has an upper portion 12 and a lower portion 13 located below the upper portion 12.
- the upper part 12 is narrower in width than the lower part 13 . Therefore, the upper portion 12 is easier to deform in the width direction than the lower portion 13, and as shown in FIGS. It is easy to spread to That is, as shown in FIG. 11, the outflow portion 5 is inserted into the upper groove portion 3b of the posture maintaining groove 3 while widening the upper portion 12 in the width direction, and the slit 5a of the outflow portion 5 is positioned directly above the lower groove portion 3c. will be located.
- the upper portion side of the upper groove portion 3b can be formed wide. Further, since the width of the upper portion 12 is formed narrow as described above, the lower portion 13 is easily deformed so as to be bent upward. Therefore, the intermediate holding portion 1d is easily deformed into a round shape by bending the lower portion 13 upward and compressing the upper portion 12 in the width direction. As a result, the test fish F can be held by compressing the lower groove portion 3c from both sides in the width direction and the lower groove portion 3c, and the upper groove portion 3b can be compressed in the width direction and closed.
- the thickness T of the upper portion 12 is within the range of 5 to 40 mm. If the thickness T of the upper portion 12 is less than 5 mm, the compressive force transmitted to the test fish F held in the lower groove portion 3c when the upper portion 12 is pinched from both the left and right sides is too large. can be too burdensome.
- the compressive force transmitted to the test fish F held in the lower groove portion 3c when the upper portion 12 is pinched from both the left and right sides is too small. can not be held well, and the gill cover of the test fish F may not be closed well.
- the front holding portion 1c is shaped so that its upper end surface is at the same height as the upper surface of the intermediate holding portion 1d. Therefore, the front holding portion 1c closes the upper groove portion 3b and the lower groove portion 3c from the front end side in a state in which the front holding portion 1c is arranged in contact with the front end side of the intermediate holding portion 1d.
- the pipe member 2c is placed on the concave portion 11 of the rear holding portion 1e while the tip of the outflow portion 5 is in contact with the front holding portion 1c.
- the outflow portion 5 is installed in the posture maintaining groove 3 with its lower portion sandwiched by the upper portion 12 of the intermediate holding portion 1d, and the slit 5a extends along the lower groove portion 3c of the posture maintaining groove 3. are arranged so as to face each other from above.
- the treatment method for the test fish F of the present embodiment is configured as a method for applying a predetermined treatment to the test fish F held by the holder 1 using the test fish treatment equipment described above. It is In such a method for treating the test fish F, the above-described equipment for treating the test fish F is used. can be held. In this state, the test fish F held by the holder 1 is subjected to a predetermined treatment, so that the treatment can be performed easily and reliably. In addition, since the test fish F can be held in a resting state while moisturizing the test fish F in the holder 1, damage to the test fish F can be greatly suppressed, and the test fish F can be repeatedly exposed to the test fish F without anesthesia.
- Predetermined treatments such as oral administration and parenteral administration can be performed.
- the treatment method for the test fish F as shown in FIG.
- the oral administration method and the parenteral administration method are described below.
- test fish treatment equipment is installed as shown in FIGS. 10 and 12, and the test fish F is put into the funnel-shaped funnel portion 4 (see FIG. 1) together with water. Then, since the water flows downward inside the pipe member 2c, the test fish F swims with its head downward according to its habit, and enters a predetermined posture through the slit 5a of the outflow part 5, that is, It enters into the posture maintaining groove 3 of the holder 1 with its head directed toward the distal end side, and stops with its head in contact with the front holding portion 1c.
- the test fish F can be directly orally administered with a syringe 7 equipped with an oral probe 8 .
- the test fish F enters the posture maintaining groove 3 in a posture opposite to the predetermined posture, that is, with the tail fin facing the tip side. stop in contact.
- test fish F can be directly orally administered with a syringe 7 equipped with an oral probe 8 .
- test administration method A parenteral administration method of administering a test substance to the test fish F by injection using a test fish treatment facility employing the holder 1 of the present embodiment will be described.
- the test fish treatment equipment is installed as shown in FIGS. 10 and 12, and the test fish F is held in the posture maintaining groove 3 of the intermediate holding portion 1d of the holder 1 in the same manner as in the oral administration method described above. do.
- FIG. 14 when the injection site where the injection needle 9a of the injection 9 is inserted is the abdomen Fa of the test fish F, the burden on the test fish F is not directly touched.
- intraperitoneal administration can be performed by inserting the injection needle 9a of the injection 9 into the abdomen Fa of the test fish F, which is maintained in a predetermined posture in the posture-maintaining groove 3, to administer the test substance.
- intravenous administration can also be performed by inserting the injection needle 9a of the injection 9 into the vein of the test fish F.
- FIG. 15 shows the overall configuration of a test fish processing facility 100 in which the holder 1 and the guiding means 2 described so far are integrated together with other facility configurations.
- the test fish processing facility 100 contains the first water tank 21 that houses the test fish F before being introduced into the inlet 2a of the guide means 2, and the test fish F released from the holding by the holder 1.
- a second water tank 30 is provided.
- the test fish F housed in the first water tank 21 can be taken out together with the water and immediately put into the inlet 2a of the guiding means 2.
- the test fish F released from the holding by the holder 1 can be immediately accommodated in the second water tank 30 .
- a transparent beaker for temporarily storing the test fish F taken out from the first water tank 21 before being introduced into the inlet 2a.
- a measuring water tank 22 such as a water tank is provided. This weighing water tank 22 is placed on a weighing scale 23 , and the weighing scale 23 measures the weight of the test fish F housed in the weighing water tank 22 .
- body length measurement, individual identification, male/female determination, and the like are performed by observation from the outside, captured image processing, and the like.
- individual identification and sex determination can be performed by image processing based on the captured image of the test fish F based on its face, pattern, and the like.
- Water is stored in the weighing water tank 22 so as to accommodate the test fish F without burden. This stored water is put into the inlet 2a together with the test fish F.
- a water tap for replenishing the water tank 22 with water may be provided.
- the test fish processing equipment 100 is provided with a water receiving section 25 in which the holder 1 as a treatment section is installed and water discharged from the outflow port 2b of the guiding means 2 is received.
- the water receiving portion 25 is provided with a drain port 26a leading to a drain pipe 26 for draining the stored water.
- the drain port 26a can be provided with a net for preventing the test fish F that have fallen from the holder 1 from flowing out to the drain pipe 26 side.
- a water level maintaining means for maintaining the water level of the water receiving portion 25 at a predetermined set water level can be constructed by locating the drain port 26a slightly above the bottom surface of the water receiving portion 25. FIG. By providing such a water level maintaining means, the wet state of the holder 1 installed in the water receiving portion 25 is maintained in a constant state, and the moisture content of the test fish F held in the holder 1 is appropriately maintained. kept in good condition.
- the holder 1 can be installed at a predetermined position of the water receiver 25 in such a manner that a mark is provided at a predetermined position of the water receiver 25 and the holder 1 is installed according to the mark. Further, the holder 1 can be installed at a predetermined position of the water receiver 25 in such a manner that a magnet or a magnetic material is provided at a predetermined position on the water receiver 25 and on the holder 1 so as to attract these.
- hook-and-loop fasteners may be provided on the water receiver 25 and on the holder 1 , and the holder 1 may be installed on the water receiver 25 at a predetermined position in such a manner that the fasteners are attached to each other.
- the holder 1 can be installed at a predetermined position of the water receiver 25 by forming a recess at a predetermined position of the water receiver 25 and fitting the holder 1 into the recess.
- Such a test fish processing facility 100 can be entirely covered with a cover to prevent contamination from entering. Furthermore, clean air can be supplied to the space surrounded by this cover.
- the entire holder 1 is made of sponge was shown, but it is made of other materials having flexibility, water permeability, and water retention, such as non-woven fabric. can also Furthermore, as long as it has flexibility, it can be made of materials other than sponge and non-woven fabric, such as soft rubber and synthetic cloth. In that case, not only is the entire holder 1 made of a flexible material, but also, for example, the holder 1 is made of non-flexible polystyrene foam, and the grooves 3 for maintaining the posture of the holder 1 are formed. It can also be implemented by attaching a material having flexibility or the like to the inner surface of the .
- the shape of the groove 3 for maintaining the posture of the holder 1 can be changed as appropriate according to the type and habits of the test fish F.
- the tip side is tapered, and the bottom 3a is formed on the side surface. It is not necessary to incline downward toward the distal end side, and in particular, it is preferable to change the lateral width of the posture maintaining groove 3 so as to be suitable for living body observation and living body experiment.
- the lateral width of the posture maintaining groove 3 of the holder 1 may have a shape that widens toward the distal side in plan view, or a shape that does not change between the proximal side and the distal side in plan view.
- the bottom of the posture maintaining groove 3 of the holder 1 can be shaped such that it slopes upward toward the distal end side, or it can be shaped horizontally between the proximal end side and the distal end side.
- a posture maintaining groove 3 can be formed as shown in FIGS. 16 and 17.
- the test fish F flowing out together with water into the upper groove portion 3b that opens upward in the posture-maintaining groove 3 can be smoothly carried out while maintaining its posture.
- the test fish F can be held in the lower groove portion 3c with less burden.
- the shape of the lower groove portion 3c when viewed in the longitudinal direction was circular.
- a narrow groove shape may be formed by omitting the wide lower groove portion 3c (see FIGS. 8, 9, 16 and 17).
- the holder 1 is divided into the rear end side holding portion 1a and the front end side holding portion 1b.
- An example is shown in which only the mouth or head of the test fish F protrudes from the rear end side holding portion 1a to the tip side while the posture is maintained.
- the tip side is gradually tapered so that the rear end has the maximum width W1 and the tip has the minimum width W2.
- the holder 1 does not necessarily have to be divided into two pieces, and the holder 1 as a whole can be configured as a single unit or divided into three pieces.
- the guiding means 2 was composed of a transparent and flexible synthetic resin pipe member 2c. It can also be composed of a pipe member. Further, the guiding means 2 can be constructed of a member other than the pipe member, for example, a gutter-shaped member having an open upper surface. Further, an example is shown in which the funnel-shaped funnel portion 4 is connected to the inlet 2a of the pipe member 2c, which is the guiding means 2, and the pipe-shaped outflow portion 5 is connected to the outflow port 2b.
- the outflow part 5 is not indispensable, and the funnel part 4 and the outflow part 5 can be eliminated.
- the apparatus for treating test fish according to the present invention can be used not only for direct oral administration to test fish, but also for various purposes such as various biological observations and blood sampling of test fish.
- the present invention can be suitably used as a test fish treatment facility and a test fish treatment method that can reliably hold a test fish in a predetermined posture.
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Abstract
Description
しかしながら、被験物質を含む餌を水槽に投入する投与方法では、液状の被験物質を取り扱うのが困難であり、更には、食べ残しが生じて供試魚の摂取量を正確に把握できないという問題がある。また、これらの投与方法では、被験物質を消化器官に直接投与する経口投与法が採用されるマウス等の動物実験とは条件が異なるため、当該動物実験との比較を適切に行うことができないという問題がある。
これらのことから、供試魚に対しても被験物質を消化器官に直接投与する経口投与法を採用することが望まれている。
そこで、従来、合成樹脂板をU字形に折り曲げて、供試魚を保持する内面側にスポンジや不織布などの柔軟性を有する素材を貼り付けた魚保持具が提案された(例えば、特許文献1参照)。
また、供試魚が暴れるのを抑制するために、麻酔を施して供試魚を保持することが考えられるが、その場合には、実験結果が供試魚に対する被験物質の影響であるか麻酔の影響であるかが不明となり、さらには、被験物質の反復投与を行う実験の場合には、投与の都度麻酔を施すとその影響が繰り返されることから、反復投与を行う実験ができないことになる。
更に、供試魚を保持する魚保持具は存在しても、従来、供試魚を保持するまでの過程に関しては、特別な装置類や解決策などは存在しなかった。したがって、従来では、供試魚を網などで掬ってその掬った供試魚をそのまま魚保持具により保持していたのが実情であり、魚保持具により供試魚を保持するまでの過程において、供試魚に対してかなり大きなダメージを与えていた可能性も高い。
前記保持具が、供試魚を所定の姿勢に維持する姿勢維持用溝を備えて、その姿勢維持用溝の少なくとも内面が柔軟性を有する材料で構成されている点にある。
また、保持具において供試魚を保湿しながら安静させた状態で保持することができるので、供試魚に対するダメージを大幅に抑制しながら、無麻酔で反復して供試魚に対して経口投与や非経口投与などの所定の処置を行うことができる。
そして、保持具全体が、このような性能を備えた材料、例えば、スポンジで構成されているので、姿勢維持用溝の内面にスポンジを貼着して保持具を構成する場合に比べて、保持具の作製が容易かつ廉価となる。
その結果、供試魚はその習性により水の流れに沿って先端側へと行き、徐々に姿勢維持用溝の内面により挟持されて所定の姿勢に維持されることになり、その間におけるダメージも効果的に抑制される。
また、先端側保持部位の後端側端部における横幅を供試魚の尾ひれのみが先端側保持部位から後端側へ突出する横幅に設定することで、先端側保持部位の姿勢維持用溝により供試魚を保持して供試魚の尾ひれの観察などを容易、確実に行うことができる。
前記誘導手段が、その一端に供試魚が水と一緒に投入される投入口を備え、他端に供試魚を水と一緒に前記姿勢維持用溝に流出させる流出口を備えて構成されている点にある。
前記姿勢維持用溝の上方に隣接して配置されるパイプ状の流出部が、前記流出口に接続され、その流出部が、前記姿勢維持用溝に沿って対面するスリットを備え、そのスリットから水と一緒に供試魚を前記姿勢維持用溝に所定の姿勢で流出する点にある。
前記誘導手段が、その一端に供試魚が水と一緒に投入される投入口を備え、他端に供試魚を水と一緒に前記処置部に流出させる流出口を備えて構成されている点にある。
また、保持具において供試魚を保湿しながら安静させた状態で保持することができるので、供試魚に対するダメージを大幅に抑制しながら、無麻酔で反復して供試魚に対して経口投与や非経口投与などの所定の処置を行うことができる。
尚、直接経口投与とは、従来のように餌に被験物質を混合して水槽に投入して供試魚に食べさせる方法や、被験物質を飼育水に溶解して鰓から吸収させる方法などと異なり、供試魚の口腔に対して直接的に被験物質を投与する経口投与を行うことを示す。
そこで、本発明に係る供試魚の処置方法では、供試魚に対して麻酔を施さずにダメージを極力抑制した状態で直接経口投与を行うことができる。このことで、被験物質が供試魚へ与える影響を評価しやすく、さらに、被験物質を毎日少量ずつ供試魚に与えてその影響を評価するような実験も行うことが可能となる。
尚、非経口投与とは、例えば注射を用いて行われる腹腔内投与や静脈内投与がある。また、このような非経口投与を行うにあたり、保持具にて供試魚を前後軸周りに回転させて、姿勢維持用溝の上方開口部に供試魚の腹部を露出させるようにすれば、供試魚の腹部に対して容易に注射を行うことができる。
本発明の供試魚処置設備は、図1に示すように、例えば、ゼブラフィッシュなどのような生体実験用の比較的小さな供試魚F(図2参照)を保持する保持具1と、その保持具1にまで水と一緒に供試魚Fを誘導するパイプ部材2cで構成された誘導手段2とを備えて構成され、パイプ部材2cとしては、一例として、内部が見えるように透明でかつ可撓性を有する合成樹脂製のパイプが使用される。
保持具1は、図2に示すように、全体が平面視で矩形に形成され、柔軟性、透水性、および、保水性を有する材料、例えば、スポンジで構成されて、供試魚Fを無理のない自然な状態で所定の姿勢に維持する有底で上方開口の姿勢維持用溝3を備えている。
保持具1は、この姿勢維持用溝3を長手方向に二分する後端側保持部位1aと先端側保持部位1bを備え、それら両保持部位1a、1bが分離自在に構成されている。そして、後端側保持部位1aの先端側端部における姿勢維持用溝3の横幅が、図2および図5に示すように、後端側保持部位1aの姿勢維持用溝3で供試魚Fを所定の姿勢に維持した状態で、供試魚Fの口あるいは頭のみが後端側保持部位1aから先端側へ突出する中間横幅W3になるように設定される。
流出部5は、図3に示すように、水と一緒に供試魚Fを姿勢維持用溝3に所定の姿勢で流出する先細り状のスリット5aを備え、その流出部5のスリット5aが、図4に示すように、保持具1の姿勢維持用溝3に沿って上方から対面するように配設され、漏斗部4や流出部5を備えた誘導手段2および保持具1が、図1で一部のみ示す図外の設置装置6によって、誘導手段2の漏斗部4が上方に、流出部5が下方に位置するように設置される。
なお、図示の形態では、漏斗部4と流出部5をそれぞれパイプ部材2cと別体に構成した例を示したが、いずれか一方または両方をパイプ部材2cと一体的に構成することもできる。
更に、図13に示す平面視において、漏斗部4内での給水管15による給水方向は、投入口2aを中心軸とした周方向とされている。このことで、漏斗部4内において水流WFが旋回しながら投入口2aに流れ込むことになるので、漏斗部4の内壁面全体の濡れを維持して、投入される供試魚Fの負担を軽減することができる。
まず、供試魚処置設備を図1に示すように設置し、漏斗状の漏斗部4に水と一緒に供試魚Fを入れる。すると、水がパイプ部材2cの内部を下方へ向けて流れるため、供試魚Fはその習性にしたがって頭を下方に向けて泳いで行き、流出部5のスリット5aから所定の姿勢、つまり、頭を先端側保持部位1bの方へ向けた状態で水と一緒に姿勢維持用溝3へ入り込む。姿勢維持用溝3において、水は更に保持具1の先端側へと流れるため、供試魚Fも一緒に流されて、最終的に、姿勢維持用溝3で挟持された状態で停止する。
その挟持状態で保持具1の後端側保持部位1aを先端側保持部位1bから分離すれば、図5に示すように、供試魚Fの口あるいは頭のみが後端側保持部位1aから先端側へ突出するように設定されているので、後端側保持部位1aの姿勢維持用溝3に供試魚Fを所定の姿勢に維持して、その供試魚Fに経口ゾンデ8(経口チューブ)を備えたシリンジ7により被験物質の直接経口投与を行うことができる。
尚、被験物質は、液状のものに限らず、ゼリー状やペースト状や固形のものであってもよく、また、被験物質を餌に混入したもの、被験物質を微小なカプセルに封じたもの、又は試験物質をコーティングしたものを、水などに分散液に分散させた状態で供試魚Fに直接経口投与しても構わない。
尚、可撓性を有する経口ゾンデ8とシリンジ7とは一体成型で製作することができる。
経口ゾンデ8とシリンジ7とを一体成型で製作すれば、経口ゾンデ8とシリンジ7の接続部における投与剤等の被験物質の滞留を防止でき、また、直接経口投与の作業中においてその接続部が外れることを防止できる。
本発明に係る供試魚処置設備を用いた供試魚の処置方法の有効性を確認するための実験1~4について以下に説明を加える。尚、各実験で採用した直接経口投与方法は、以下のとおりである。
(直接経口投与方法)
実施例:図5に示す状態(本発明に係る供試魚処置設備を用いて保持具1で供試魚Fを保持した状態)で、供試魚Fに対して麻酔を施さずに被験物質をシリンジにて直接経口投与する。
比較例:実験用ゴム手袋を装着した手で供試魚を保持した状態で、供試魚Fに対して麻酔を施さずに被験物質をシリンジにて直接経口投与する。
実施例及び比較例の夫々の直接経口投与方法について、下記実験条件にて供試魚へ被験物質を投与して被験物質の濃度と死亡率との関係を求める影響評価実験を行った。
(実験条件)
供試魚、個体数:ゼブラフィッシュ、4乃至5個体/1水槽×6
被験物質:3通りの濃度(10mg/kg、100mg/kg、1000mg/kg)
の二クロム酸カリウム溶液
1個体あたりの投与量:20μL
(実験結果)
本実験で求めた被験物質の濃度と死亡率との関係を図6に示す。この実験結果では、実施例の直接経口投与方法を採用した場合には、比較例の直接経口投与方法を採用した場合よりも、供試魚へ投与した被験物質の濃度と死亡率との関係に相関が見られた。よって、本発明に係る供試魚処置設備を用いることで、実験用ゴム手袋を装着した手で供試魚を保持する場合よりも、より高精度に直接経口投与を行うことができるといえる。
実施例の直接経口投与方法について、下記実験条件にて供試魚へ被験物質を投与して被験物質の濃度と死亡率との関係を求める影響評価実験を行った。
(実験条件)
供試魚、個体数:ゼブラフィッシュ、5個体/1水槽×4
被験物質:4通りの濃度(10mg/kg、100mg/kg、500mg/kg、1000mg/kg)の二クロム酸カリウム溶液
1個体あたりの投与量:20μL
(実験結果)
本実験で求めた被験物質の濃度と死亡率との関係を図7に示す。この実験結果においても、上記実験1と同様に、実施例の直接経口投与方法を採用した場合に、供試魚へ投与した被験物質の濃度と死亡率との関係に相関が見られた。よって、本発明に係る供試魚処置設備を用いることで、高精度に直接経口投与を行うことができるといえる。
実施例及び比較例の夫々の直接経口投与方法において、下記の実験条件にて供試魚へ投与した被験物質の鰓からの漏洩を目視で確認する実験を行った。
(実験条件)
供試魚、個体数:ゼブラフィッシュ、4個体/1水槽×1
被験物質:メチレンブルー
(実験結果)
比較例の直接経口投与方法では、水槽内の全4個体の供試魚で鰓からのエチレンブルー
(被験物質)の漏洩が視認できた。一方、実施例の直接経口投与方法では、水槽内の全4個体の供試魚でエチレンブルー(被験物質)の漏洩は視認できなかった。よって、本発明に係る供試魚処置設備を用いることで、実験用ゴム手袋を装着した手で供試魚を保持する場合よりも、供試魚にダメージを与えることなく高精度に直接経口投与を行うことができるといえる。
実施例の直接経口投与方法において、下記の実験条件にて所定の頻度で直接経口投与を継続的に実施して供試魚へのダメージを評価する実験を行った。
(実験条件)
供試魚、個体数:ゼブラフィッシュ、10個体×2
被験物質:メチレンブルー
(実験結果)
実施例の直接経口投与方法では、10個体の供試魚に対して1回/週の頻度で直接経口投与を実施した場合、約2か月を経過しても死亡した個体は存在しなかった。更に、別の10個体の供試魚に対して3回/週の頻度で直接経口投与を実施した場合も同様に、約2か月を経過しても死亡した個体は存在しなかった。よって、本発明に係る供試魚処置設備を用いることで、供試魚にダメージを与えることなく高精度に直接経口投与を行うことができるといえる。
次に、保持具1の別の実施形態1,2について図面に基づいて説明する。尚、保持具1の別の実施形態について説明するにあたり、主に先の実施形態と異なる構成を中心に説明し、先の実施形態と同様に構成されている部分については説明を省略する。
図8に示すように、保持具1は、姿勢維持用溝3の長手方向に沿って並ぶ前保持部位1cと中間保持部位1dと後保持部位1eとを中間保持部位1dが前保持部位1cと後保持部位1eとの間に位置する状態で備えている。また、保持具1は、前保持部位1cと中間保持部位1dと後保持部位1eとの3つの保持部位が分離自在に構成されている。
姿勢維持用溝3の上下幅(深さ)は、長手方向のいずれの位置でも、当該姿勢維持用溝3に保持される供試魚Fの上下幅(供試魚Fの高さ)よりも大きいものに設定されている。更に、本実施形態では、下溝部位3cの上下幅(深さ)が、長手方向のいずれの位置でも供試魚Fの上下幅(供試魚Fの高さ)より広くなっている。このことで、供試魚Fの高さ方向全体が、姿勢維持用溝3の下溝部位3cに収まることになるので、この下溝部位3cにおいて供試魚Fを一層安静させた状態で保持して、供試魚Fに対するダメージを一層大幅に抑制することができる。例えば、下溝部位3cの深さは、5~25mmの範囲内であることが望ましい。下溝部位3cの深さが5mm未満の場合には、下溝部位3cに対する供試魚Fの進入が困難になることがある。一方、下溝部位3cの深さが25mm超の場合には、下溝部位3cに進入した供試魚Fが、進入した際の鉛直姿勢のまま保持されて水平姿勢とすることが困難になることがある。また、下溝部位3cの横幅は、長手方向のいずれの位置でも供試魚Fの横幅より広くなっている。
また、上述の如く上部位12の横幅が狭く形成されているため、下部位13を、上方に曲げるように変形し易くなっている。したがって、中間保持部位1dは、下部位13を上方に折り曲げつつ上部位12を幅方向に圧縮させるようにして、丸めるように変形させ易くなっている。これによって、下溝部位3cを下方側および横幅方向の両側から圧縮して供試魚Fを保持することができるとともに、上溝部位3bを横幅方向に圧縮して塞ぐことが可能となっている。
本実施形態の供試魚Fの処置方法は、これまで説明してきた供試魚処置設備を使用して、保持具1に保持された供試魚Fに対して所定の処置を施す方法として構成されている。
このような供試魚Fへの処理方法では、上述した供試魚処置設備を使用するので、供試魚Fを保持するに際して、供試魚Fに対するダメージを極力抑制して所定の姿勢で確実に保持することができる。そして、その状態で、保持具1に保持された供試魚Fに対して所定の処置を施すので、当該処置を容易、確実に行うことができる。また、保持具1において供試魚Fを保湿しながら安静させた状態で保持することができるので、供試魚Fに対するダメージを大幅に抑制しながら、無麻酔で反復して供試魚Fに対して経口投与や非経口投与などの所定の処置を行うことができる。
上記供試魚Fへの処理方法の具体例としては、上記所定の処置として、図5に示すように、保持具1の姿勢維持用溝3で所定の姿勢に維持された供試魚Fの口腔に挿入される経口ゾンデ8を通じて被験物質を当該供試魚Fに直接経口投与する経口投与を行う経口投与方法と、図14に示すように、上記所定の処置として、保持具1の姿勢維持用溝3で所定の姿勢に維持された供試魚Fへの注射により被験物質を当該供試魚Fに投与する非経口投与を行う非経口投与方法とがある。以下、これら経口投与方法並びに非経口投与方法について説明を加える。
本実施形態の保持具1を採用した供試魚処置設備を使用して、供試魚Fへ直接経口投与を行う経口投与方法について説明する。
まず、供試魚処置設備を図10および図12に示すように設置し、漏斗状の漏斗部4(図1参照)に水と一緒に供試魚Fを入れる。すると、水がパイプ部材2cの内部を下方へ向けて流れるため、供試魚Fは、その習性にしたがって頭を下方に向けて泳いで行き、流出部5のスリット5aから所定の姿勢、つまり、頭を先端側に向けた状態で保持具1の姿勢維持用溝3へ入り込み、頭が前保持部位1cに接した状態で停止する。その状態で保持具1の中間保持部位1dを前保持部位1cおよび後保持部位1eから分離して供試魚Fを保持すれば、供試魚Fの口あるいは頭が中間保持部位1dの先端近くに位置するので、その供試魚Fに経口ゾンデ8を備えたシリンジ7により直接経口投与を行うことができる。
また、供試魚Fが所定の姿勢とは逆の姿勢、つまり、尾ひれを先端側に向けた状態で姿勢維持用溝3へ入り込む場合があるが、この場合では、尾ひれが前保持部位1cに接した状態で停止する。その状態で保持具1の中間保持部位1dを前保持部位1cおよび後保持部位1eから分離して供試魚Fを保持すれば、供試魚Fの口あるいは頭が中間保持部位1dの後端近くに位置するので、その供試魚Fに経口ゾンデ8を備えたシリンジ7により直接経口投与を行うことができる。
本実施形態の保持具1を採用した供試魚処理設備を使用して、注射により被験物質を当該供試魚Fに投与する非経口投与方法について説明する。
まず、供試魚処置設備を図10および図12に示すように設置し、上述の経口投与方法と同様に、保持具1の中間保持部位1dの姿勢維持用溝3に供試魚Fを保持する。
次に、図14に示すように、注射9の注射針9aを刺す注射部位が供試魚Fの腹部Faである場合には、供試魚Fを直接触れることなく供試魚Fに負担をかけないようにしながら、保持具1の姿勢維持用溝3の左右両側の上部位12を交互に動かすなどして供試魚Fを軸周りに回転させることで、姿勢維持用溝3の上方側に供試魚Fの腹部Faを露出させることができる。そして、このように姿勢維持用溝3において所定の姿勢に維持された供試魚Fの腹部Faに注射9の注射針9aを刺して被験物質を投与する所謂腹腔内投与を行うことができる。また、腹腔内投与以外にも、供試魚Fの静脈に注射9の注射針9aを刺して静脈内投与を行うこともできる。
図15に、これまで説明してきた保持具1及び誘導手段2を他の設備構成と共に一体化した供試魚処理設備100全体の構成を示す。
かかる供試魚処理設備100は、誘導手段2の投入口2aへの投入前の供試魚Fを収容する第1水槽21と、保持具1による保持から開放された供試魚Fを収容する第2水槽30とを備える。このことで、第1水槽21に収容されている供試魚Fを水と共に取り出して直ぐに、誘導手段2の投入口2aに投入することができる。また、保持具1による保持から開放された供試魚Fを直ぐに第2水槽30に収容することができる。
また、排水口26aを水受け部25の底面よりも若干上に位置させる形態で、水受け部25の貯水水位を所定の設定水位に維持する水位維持手段を構築することができる。このような水位維持手段を設けることにより、水受け部25に設置される保持具1の濡れ状態が一定状態に維持されて、当該保持具1に保持される供試魚Fの保湿状態が適切な状態に保たれる。
このような供試魚処理設備100は、コンタミの侵入防止のために全体をカバーで囲うことができる。更に、このカバーに囲われた空間に対してクリーンエアーを供給することもできる。
(1)先の実施形態では、保持具1の全体がスポンジで構成された例を示したが、柔軟性、透水性、および、保水性を備えた他の材料、例えば、不織布などで構成することもできる。更に、柔軟性さえ備えていれば、スポンジや不織布以外の材料、例えば、ソフトなゴムや合成の布などで構成することもできる。その場合、保持具1の全体を、柔軟性などを有する材料で構成するのはもちろんのこと、例えば、保持具1を柔軟性のない発砲スチロールで構成し、保持具1の姿勢維持用溝3の内面に柔軟性などを有する材料を貼着して実施することもできる。
また、保持具1の姿勢維持用溝3の形状に関しては、供試魚Fの種類や習性などに応じて適宜変更可能であり、平面視で先端側ほど先細りに、また、その底3aを側面視で先端側ほど下方に傾斜するように構成する必要はなく、特に、姿勢維持用溝3の横幅に関しては、生体観察や生体実験に適応するように変更するのが好ましい。例えば、保持具1の姿勢維持用溝3の横幅については、平面視において先端側ほど先広がりとなる形状としたり、平面視において基端側と先端側とで変化しない形状とすることができる。また、保持具1の姿勢維持用溝3の底については、先端側ほど上方に傾斜する形状としたり、基端側と先端側との間で水平となる形状とすることができる。
更に、先の実施形態では、保持具1の中間保持部位1dにおいて、下溝部位3cの長手方向視の形状を円形状としたが、図18に示すように三角形状にしたり、図19に示すように楕円形状にしたり、図20に示すように幅広の下溝部位3c(図8、図9、図16、図17参照)を省略した細溝形状にすることもできる。
例えば、後端部が最大横幅W1で先端部が最小横幅W2となるように、平面視において先端側ほど徐々に先細りにし、先端側保持部位1bの姿勢維持用溝3で供試魚Fを所定の姿勢に維持した状態で、供試魚Fの尾ひれのみが先端側保持部位1bから後端側へ突出するように設定することもでき、その場合には、供試魚Fの尾ひれの観察などを容易、確実に行うことができる。
ただし、保持具1は必ずしも二分割にする必要はなく、保持具1を全体として単体に構成することも、また、三分割することも可能である。
また、誘導手段2であるパイプ部材2cの投入口2aに漏斗状の漏斗部4が接続され、流出口2bにパイプ状の流出部5が接続された例を示したが、これら漏斗部4と流出部5については、必要不可欠なものではなく、これら漏斗部4と流出部5をなくして実施することもできる。
なお、本発明による供試魚処置設備は、供試魚への直接経口投与の際に限らず、供試魚に対する各種生体観察や採血の際など、種々の目的に使用することができる。
1a 後端側保持部位
1b 先端側保持部位
2 誘導手段
2a 投入口
2b 流出口
2c パイプ部材
3 姿勢維持用溝
3a 姿勢維持用溝の底
4 漏斗部
5 流出部
5a スリット
7 シリンジ
8 経口ゾンデ(経口チューブ)
9 注射
15 給水管(水流形成手段)
21 第1水槽
25 水受け部
30 第2水槽
100 供試魚処理設備
F 供試魚
SW 給水
WF 水流
Claims (18)
- 供試魚を保持する保持具を備え、
前記保持具が、供試魚を所定の姿勢に維持する姿勢維持用溝を備えて、その姿勢維持用溝の少なくとも内面が柔軟性を有する材料で構成されている供試魚処置設備。 - 前記保持具が、柔軟性、透水性、および、保水性を有する材料で構成されている請求項1に記載の供試魚処置設備。
- 前記姿勢維持用溝が、先端側ほど先細りに構成されている請求項1または2に記載の供試魚処置設備。
- 前記姿勢維持用溝が、先端側から後端側に亘って同じ幅で形成され、上方に開口する上溝部位と、その下方に連続し当該上溝部位よりも幅が広い下溝部位とからなる請求項1または2に記載の供試魚処置設備。
- 前記姿勢維持用溝の深さが、当該姿勢維持用溝に保持される供試魚の高さよりも大きいものに設定されている請求項1~4のいずれか1項に記載の供試魚処置設備。
- 前記保持具が、前記姿勢維持用溝を長手方向に二分する後端側保持部位と先端側保持部位を備え、それら両保持部位が分離自在に構成されている請求項1~5のいずれか1項に記載の供試魚処置設備。
- 前記保持具が、前記姿勢維持用溝の長手方向に沿って並ぶ前保持部位と中間保持部位と後保持部位とを前記中間保持部位が前記前保持部位と前記後保持部位との間に位置する状態で備え、それら3つの保持部位が分離自在に構成され、
前記姿勢維持用溝が、その先端が前記前保持部位と前記中間保持部位との境界箇所に位置するように、前記前保持部位と前記中間保持部位とのうちの前記中間保持部位のみに備えられている請求項1~5のいずれか1項に記載の供試魚処置設備。 - 供試魚を水と一緒に前記保持具にまで誘導する誘導手段を備え、
前記誘導手段が、その一端に供試魚が水と一緒に投入される投入口を備え、他端に供試魚を水と一緒に前記姿勢維持用溝に流出させる流出口を備えて構成されている請求項1~7のいずれか1項に記載の供試魚処置設備。 - 前記誘導手段が、その一端に前記投入口を有し、他端に前記流出口を有するパイプ部材により構成されており、
前記姿勢維持用溝の上方に隣接して配置されるパイプ状の流出部が、前記流出口に接続され、その流出部が、前記姿勢維持用溝に沿って対面するスリットを備え、そのスリットから水と一緒に供試魚を前記姿勢維持用溝に所定の姿勢で流出する請求項8に記載の供試魚処置設備。 - 前記投入口への投入前の供試魚を収容する第1水槽と、前記保持具による保持から開放された供試魚を収容する第2水槽とを備えた請求項8または9に記載の供試魚処置設備。
- 所定の処置が施される供試魚を水と一緒に所定の処置部にまで誘導する誘導手段を備え、
前記誘導手段が、その一端に供試魚が水と一緒に投入される投入口を備え、他端に供試魚を水と一緒に前記処置部に流出させる流出口を備えて構成されている供試魚処置設備。 - 前記誘導手段が、その一端に前記投入口を有し、他端に前記流出口を有するパイプ部材により構成されている請求項8~11のいずれか1項に記載の供試魚処置設備。
- 前記投入口に給水して前記パイプ部材内に水流を形成する水流形成手段を備える請求項12に記載の供試魚処置設備。
- 前記投入口に水と一緒に供試魚を導く漏斗状の漏斗部が、前記投入口に接続されている請求項8~13のいずれか1項に記載の供試魚処置設備。
- 前記処置部が設置されて前記流出口から排出される水を受ける水受け部を備える請求項8~14のいずれか1項に記載の供試魚処置設備。
- 上記請求項1~10のいずれか1項に記載の供試魚処置設備を使用して、前記保持具に保持された供試魚に対して所定の処置を施すことを特徴とする供試魚の処置方法。
- 前記所定の処置として、前記保持具の姿勢維持用溝で所定の姿勢に維持された供試魚の口腔に挿入される経口チューブを通じて被験物質を当該供試魚に直接経口投与する経口投与を行うことを特徴とする請求項16に記載の供試魚の処置方法。
- 前記所定の処置として、前記保持具の姿勢維持用溝で所定の姿勢に維持された供試魚への注射により被験物質を当該供試魚に投与する非経口投与を行うことを特徴とする請求項16に記載の供試魚の処置方法。
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WO2024080129A1 (ja) * | 2022-10-14 | 2024-04-18 | 株式会社神鋼環境ソリューション | 供試魚保持装置、及び供試魚の処置方法 |
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2022
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JPH0372201A (ja) * | 1989-08-03 | 1991-03-27 | Bilchingr H F | 魚の数量と寸法を測定し類別するための方法及び装置 |
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JPH072322U (ja) * | 1991-05-02 | 1995-01-13 | 但商株式会社 | アユ等の雌雄選別用バケット |
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JP7488869B2 (ja) | 2022-10-14 | 2024-05-22 | 株式会社神鋼環境ソリューション | 供試魚保持装置、及び供試魚の処置方法 |
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