Leiocassis crassilabris enhancement and release effect evaluation method

Abstract

The invention belongs to the field of animal molecular biology, and relates to a leiocassis crassilabris enhancement and release effect evaluation method which comprises the following steps: 1) storing a DNA parent of an artificial propagation parent of leiocassis crassilabris; 2) releasing the leiocassis crassilabris artificial propagation offspring; (3) catching field groups of leiocassis crassilabris; 4) extracting DNA of caught field groups of leiocassis crassilabris, and catching; (5) respectively carrying out PCR amplification on the DNA parent obtained in the step (1) and the DNA fishing obtained in the step (4) by utilizing the primer group for identifying the leiocassis crassilabris, and carrying out polyacrylamide gel electrophoresis on PCR products; and 6) analyzing the parent-child relationship between the leiocassis crassilabris parents and the leiocassis crassilabris caught in the field according to the result of the step 5) to obtain the number of the leiocassis crassilabris parents in the caught leiocassis crassilabris, and evaluating the releasing effect according to the number. The invention provides the leiocassis crassilabris enhancement releasing effect evaluation method which can accurately evaluate the leiocassis crassilabris releasing effect and can improve the efficiency of the releasing effect.

Description

Technical Field

[0001] The invention belongs to the field of animal molecular biology and relates to a method for evaluating fish reproduction and release, in particular to a method for evaluating the effect of thick-lipped catfish reproduction and release. Background Art

[0002] Bagridae, with the Latin name Bagridae, is a family of the order Siluriformes. The fish of this family are freshwater nocturnal fish that live in rivers and streams. They are distributed in Africa and Asia from Japan to Borneo. Catfish animals have a dorsal fin with 1 hard spine and 6-7 soft rays; an adipose fin, which is slightly high and long, separated from the tail at the back; and a serrated edge on the pectoral fin hard spines. They mainly live in rivers and streams. They hide in the pores of rocks during the day and only come out to move or forage at dusk or at night. They mainly include the Ussuri catfish, the medium-hipped catfish, the long-snout catfish, the catfish, the striped catfish, the river rat bigfin catfish, the spotted catfish, the thick-lipped catfish, the catfish, the spotted catfish, the bigfin catfish, and the thick-lipped catfish. The body of the thick-lipped catfish is elongated, the head is wide and flat, and it usually has 4 pairs of whiskers, including 2 pairs of snout whiskers and 2 pairs of chin whiskers. The body surface is bare without scales, and the dorsal fin has 1 hard spine and 6-7 soft rays.

[0003] Artificial propagation and release of thick-lipped catfish can effectively increase the amount of thick-lipped catfish resources in the wild, promote the natural reproduction of thick-lipped catfish, and improve the protection of thick-lipped catfish. Every time tens of thousands or hundreds of thousands of thick-lipped catfish are artificially propagated and released, some problems may occur, leading to the problem of high mortality during the artificial propagation and release of thick-lipped catfish. For example, due to the spines on the dorsal and pectoral fins of thick-lipped catfish, thick-lipped catfish often stab each other during the artificial propagation and release, resulting in an increase in the mortality rate of artificial propagation and release; or during the artificial propagation and release of thick-lipped catfish, when the thick-lipped catfish are poured into the river, the speed is very fast, and the impact force generated causes great damage to the thick-lipped catfish to be released.

[0004] The release of thick-lipped catfish is usually combined with artificial breeding technology, through artificial induction of spawning in suitable seasons to improve the survival rate of released fry. The evaluation of the release effect usually involves monitoring the survival rate, growth and impact on the ecosystem of fish after release. Evaluation methods include tag release, fishing surveys and environmental DNA technology, which help to understand the distribution, migration and survival status of released fish. The evaluation results of the release effect are of great significance for guiding future release strategies and improving release technology. When releasing thick-lipped catfish, factors that need to be considered include the genetic diversity of released fish, the selection of release sites, the determination of release time and environmental monitoring after release. In addition, the release activities also need to cooperate with local fishery management departments, scientific research institutions and environmental protection organizations to ensure the success of the release activities and maximize the ecological benefits. However, there is currently no effective evaluation technology for the release effect of thick-lipped catfish, which needs to be solved urgently. Summary of the invention

[0005] In order to solve the above technical problems existing in the background technology, the present invention provides a method for evaluating the release effect of thick-lipped catfish, which can accurately evaluate the release effect of thick-lipped catfish and improve the efficiency of the release effect.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A primer set for identifying thick-lipped catfish, characterized in that: the primer set for identifying thick-lipped catfish comprises 5 sets of double PCR primer combinations, each set of double PCR primer combinations comprises 2 pairs of microsatellite primers, and the sequence information and combination mode of the primer set for identifying thick-lipped catfish are respectively:

[0008]

[0009] Preferably, in the primer set for identifying thick-lipped catfish provided by the present invention, primer CCW1 and primer CCW2 are used in combination at the same time; primer CCW3 and primer CCW4 are used in combination at the same time; primer CCW5 and primer CCW6 are used in combination at the same time; primer CCW7 and primer CCW8 are used in combination at the same time; primer CCW9 and primer CCW10 are used in combination at the same time.

[0010] Preferably, the primer set for identifying thick-lipped catfish provided by the present invention is used in identifying or distinguishing thick-lipped catfish, especially in identifying or distinguishing thick-lipped catfish when they are propagated and released.

[0011] A method for evaluating the effect of thick-lipped catfish proliferation and release, characterized in that the method for evaluating the effect of thick-lipped catfish proliferation and release comprises the following steps:

[0012] 1) Preserving DNA of artificially bred parents of thick-lipped catfish 亲本 ;

[0013] 2) Releasing the artificially bred progeny of the thick-lipped catfish;

[0014] 3) Capture of wild populations of rough-lipped catfish;

[0015] 4) Extraction of DNA from wild populations of rough-lipped catfish 捕捞 ;

[0016] 5) Using the primer set for identifying the thick-lipped catfish described above, the DNA obtained in step 1) was respectively 亲本 and the DNA obtained in step 4) 捕捞 Perform PCR amplification and subject the PCR products to polyacrylamide gel electrophoresis;

[0017] 6) Analyze the parent-offspring relationship between the rough-lipped catfish parent and the rough-lipped catfish caught in the wild according to the result of step 5), obtain the number of the rough-lipped catfish parent in the caught rough-lipped catfish, and evaluate the release effect according to the number.

[0018] Preferably, the reaction system for PCR amplification in step 5) of the present invention is 25 μL, including: 3 μL of 10×PCRBuffer, 2 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / L MgCl2, 1 μL of upstream and downstream primers, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, and 10.5 μL of ultrapure water; the procedure for PCR amplification in step 5) is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 29 s, 35 cycles; extension at 72°C for 10 min; storage at 4°C.

[0019] A device for releasing thick-lipped catfish, characterized in that: the device comprises a fish releasing trough, a connecting box, a releasing hose, a protecting pile and a water inlet pipe; the fish releasing trough is placed at the head end of the connecting box and is connected to the connecting box; there are multiple releasing hoses, which are arranged in parallel at the tail end of the connecting box and are respectively connected to the connecting box; the protecting pile is a 1 / 4 cylinder, which is placed inside the connecting box, and the axial direction of the protecting pile is not parallel to the axial direction of the connecting box; the water inlet pipe comprises a first water inlet pipe and a second water inlet pipe; the first water inlet pipe is connected to the inside of the fish releasing trough; the second water inlet pipe is connected to the connecting box; the water inlet direction of the second water inlet pipe is opposite to the flow direction of the water flow in the connecting box.

[0020] Preferably, the discharge hose provided by the present invention comprises a tube body, on which is provided a water inlet connected to the rear end of the connecting box; a hose baffle is provided on the inner wall of the tube body; the axial direction of the hose baffle is non-parallel to the axial direction of the tube body.

[0021] Preferably, the length of the protection pile provided by the present invention is half the width of the connecting box; the thick-lipped catfish releasing device also includes a detachable anchor connected to the connecting box; the thick-lipped catfish releasing device also includes an electric valve arranged between the fish releasing tank and the connecting box; an overflow hole is arranged on the top of the electric valve.

[0022] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0023] The invention provides a method for evaluating the effect of thick-lipped catfish proliferation and release and a thick-lipped catfish release device, wherein the thick-lipped catfish release device comprises a fish release trough, a connecting box, a release hose, a protection pile and a water inlet pipe; the fish release trough is arranged at the head end of the connecting box and is connected with the connecting box; there are multiple release hoses, which are arranged in parallel at the tail end of the connecting box and are respectively connected with the connecting box; the protection pile is a 1 / 4 cylinder, which is arranged inside the connecting box, and the axial direction of the protection pile is not parallel to the axial direction of the connecting box; the water inlet pipe comprises a first water inlet pipe and a second water inlet pipe; the first water inlet pipe is connected with the inside of the fish release trough; the second water inlet pipe is connected with the connecting box; the water inlet direction of the second water inlet pipe is opposite to the flow direction of the water flow in the connecting box. The present invention solves the problem of a large number of thick-lipped catfish stabbing each other during centralized release and the problem of evaluating the release effect of thick-lipped catfish. The thick-lipped catfish release device can effectively prevent the thick-lipped catfish from stabbing each other during the release process, can reduce the impact force generated by the released fish when entering the river, and reduce the damage to the fish. Different release hoses can be placed in different positions according to local conditions to reduce the phenomenon of thick-lipped catfish gathering during the release process. The structure is simple and has wide practical value. At the same time, the present invention uses a specific primer combination to perform paternity testing on the wild-caught individuals to be identified and the breeding parents, and determines whether the wild-caught individuals are released individuals based on the identification results, which can accurately evaluate the release effect of the thick-lipped catfish, and then determine the release effect, thereby improving the efficiency of the release effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a structural schematic diagram of the thick-lipped catfish releasing device provided by the present invention;

[0025] Figure 2 It is a schematic diagram of the structure of the discharge hose used in the present invention;

[0026] Figure 3 It is a schematic diagram of the discharge hose used in the present invention;

[0027] Figure 4 It is a partial enlarged structural schematic diagram of the discharge hose used in the present invention;

[0028] Description of reference numerals:

[0029] 1-fish trough; 2-connecting box; 3-protection pile; 4-discharge hose; 41-water inlet; 42-hose baffle; 5-discharge hole; 6-anchor; 7-water inlet pipe; 8-overflow hole; 9-electric valve. DETAILED DESCRIPTION

[0030] The present invention first provides a primer set for identifying thick-lipped catfish, including 5 sets of double PCR primer combinations, each of which includes 2 pairs of microsatellite primers. The sequence information and combination methods of the primer set for identifying thick-lipped catfish are:

[0031]

[0032]

[0033] It should be noted that, in the primer set for identifying thick-lipped catfish, primer CCW1 is used in combination with primer CCW2; primer CCW3 is used in combination with primer CCW4; primer CCW5 is used in combination with primer CCW6; primer CCW7 is used in combination with primer CCW8; and primer CCW9 is used in combination with primer CCW10.

[0034] The primer set for identifying thick-lipped catfish provided by the present invention is used in identifying or distinguishing thick-lipped catfish, especially in identifying or distinguishing thick-lipped catfish when the thick-lipped catfish is propagated and released.

[0035] Based on the foregoing, the present invention also provides a method for evaluating the effect of thick-lipped catfish proliferation and release, comprising the following steps:

[0036] 1) Preserving DNA of artificially bred parents of thick-lipped catfish 亲本 ;

[0037] 2) Releasing the artificially bred progeny of the thick-lipped catfish;

[0038] 3) Capture of wild populations of rough-lipped catfish;

[0039] 4) Extraction of DNA from wild populations of rough-lipped catfish 捕捞 ;

[0040] 5) Using the aforementioned primer set for identifying the thick-lipped catfish, the DNA obtained in step 1) was respectively 亲本 and the DNA obtained in step 4) 捕捞 PCR amplification was performed, and the PCR product was subjected to polyacrylamide gel electrophoresis; wherein, the reaction system for PCR amplification in step 5) was 25 μL, including: 3 μL of 10×PCR Buffer, 2 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / L MgCl2, 1 μL of upstream and downstream primers, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, and 10.5 μL of ultrapure water. The PCR amplification program was: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 58°C annealing temperature annealing for 30 s, 72°C extension for 29 s, 35 cycles; 72°C extension for 10 min; 4°C storage.

[0041] 6) Analyze the parent-offspring relationship between the rough-lipped catfish parent and the rough-lipped catfish caught in the wild according to the result of step 5), obtain the number of the rough-lipped catfish parent in the caught rough-lipped catfish, and evaluate the release effect according to the number.

[0042] The present invention was used to carry out field fishing for thick-lipped catfish in the downstream river section after release, and a total of 510 thick-lipped catfish individuals were caught. After identification by the method, 85 of them were individuals released this time, and the released individuals accounted for 16.7% of the number of wild groups, indicating that the artificial proliferation and release of thick-lipped catfish significantly increased the number of wild resources.

[0043] In order to improve the release effect, that is, to reduce the mortality rate during the release process, the present invention also provides a device for releasing thick-lipped catfish, see Figure 1 The thick-lipped catfish release device provided by the present invention includes a fish release trough 1, a connecting box 2, a release hose 4, a protection pile 3 and a water inlet pipe 7; the fish release trough 1 is placed at the head end of the connecting box 2 and is connected to the connecting box 2; there are multiple release hoses 4, and the multiple release hoses 4 are arranged in parallel at the tail end of the connecting box 2 and are respectively connected to the connecting box 2; the protection pile 3 is a 1 / 4 cylinder, and the protection pile 3 is placed inside the connecting box 2, and the axial direction of the protection pile 3 is not parallel to the axial direction of the connecting box 2; the water inlet pipe 7 includes a first water inlet pipe and a second water inlet pipe; the first water inlet pipe is connected to the inside of the fish release trough 1; the second water inlet pipe is connected to the connecting box 2; the water inlet direction of the second water inlet pipe is opposite to the flow direction of the water flow in the connecting box 2. The fish release trough 1 is a semicircular structure as a whole. There are multiple protection piles 3, and they are placed in a way that the arc surface of the 1 / 4 cylinder faces the direction of liquid flow in the connecting box 2.

[0044] See also Figure 2 as well as Figure 3 The discharge hose 4 includes a tube body, on which a water inlet 41 connected to the rear end of the connecting box 2 is arranged; a hose baffle 42 is arranged on the inner wall of the tube body; the axial direction of the hose baffle 42 is not parallel to the axial direction of the tube body. The discharge hose 4 is a double-layer hose, and the double-layer hose is directly provided with a hose baffle 42. The hose baffle 42 can be arranged on the inner tube, illustratively, extending from the inner tube wall to the outer tube wall (such as Figure 4 As shown); it can also be set on the outer tube, extending from the outer tube wall to the inner tube; of course, it can also be set on the inner tube wall (extending to the outer tube direction) and the outer tube wall (extending to the inner tube direction), and the hose baffles 42 on each layer of hose are staggered, and the hose baffles 42 face the water inlet 41. Figure 4 , a schematic diagram of the enlarged structure of the hose baffle 42 used in the present invention, the structure is that the hose baffle 42 is set on the inner tube, that is, extending from the inner tube wall to the outer tube. Figure 4In the figure, the direction of the hose baffle 42 is at an angle to the axial direction of the inner tube. At the same time, two flow holes are opened on the inner tube, and the two flow holes are placed on both sides of the hose baffle 42. The liquid (in the direction indicated by the small arrow) enters the space between the inner tube and the outer tube through the flow holes, and flows back to the inside of the inner tube along the axial direction of the hose baffle 42, collides with the liquid flow inside the inner tube (in the direction of the large arrow), and forms a flow barrier. The structure and function of the hose baffle 42 used in the present invention are similar to those of the Tesla valve, and will not be repeated here. Since multiple hose baffles 42 are provided on the inner tube and the outer tube, they can play a good buffering role for the release of the thick-lipped catfish, further improving the survival rate of the thick-lipped catfish after release.

[0045] The length of the protection pile 3 is half the width of the connecting box 2; the thick-lipped catfish release device also includes a detachable anchor 6 connected to the connecting box 2; the thick-lipped catfish release device also includes an electric valve 9 arranged between the fish release tank 1 and the connecting box 2; a plurality of overflow holes 8 are arranged on the top of the electric valve 9. It should be noted that a discharge hole 5 is arranged at the tail end of the connecting box 2, and the number of the discharge holes 5 matches the number of the discharge hoses 4, that is, each discharge hole 5 is connected to a discharge hose 4.

[0046] In May 2023, an artificial propagation and release activity of thick-lipped catfish was held in Chengdu, with a release number of 50,000. Four thick-lipped catfish release devices provided by the present invention participated in this release activity. When using this device, open the water inlet of all water inlet pipes 7. The tail end of the release hose 4 is placed in different positions according to the release terrain, and it is determined whether the anchor 6 needs to be connected according to the terrain requirements. When using this device for release, fill the fish release tank 1 with water, put the fish to be released into the fish release tank 1, open the electric valve 9, and the fish to be released enter the connecting box 2. The water inlet and water flow directions of the water inlet pipe 7 in the connecting box 2 are opposite, which can slow down the flow speed of the water body. The protective pile 3 in the connecting box can slow down the flow of the fish to be released to the tail end of the connecting box.

[0047] After the fish to be released enters the release hose 4, the unique setting of the juvenile fish release hose 4 can slow down the speed of the water flow, slow down the flow rate of the fish to be released in the release hose 4, reduce the speed of the fish to be released entering the river, avoid the damage to the fish to be released caused by the impact force, and complete the release activity. The thick-lipped catfish is transported to the release site by a fish transporter. By using this device, the drainage speed at the tail end of the release hose 4 is reduced from 1-2m / s of the traditional release device to 0.5-1m / s, avoiding the damage caused by the impact force of the thick-lipped catfish entering the river to be released. During the use of this device, the protective pile 3 can effectively avoid the damage caused by the thick-lipped catfish gathering at the tail end of the connection box. In this release, the injury rate of the thick-lipped catfish was reduced from 25% of the traditional device to 5%, increasing the success rate of the artificial propagation and release of the thick-lipped catfish.

Claims

1. A primer set for identifying Ctenopharyngodon serratus, characterized in that: The primer set for identifying the thick-lipped catfish includes 5 sets of dual PCR primer combinations, each of which includes 2 pairs of microsatellite primers. The sequence information and combination methods of the primer set for identifying the thick-lipped catfish are:

2. The primer set for identifying the thick-lipped catfish according to claim 1, characterized in that: In the primer set for identifying the thick-lipped catfish, primer CCW1 and primer CCW2 are used together; Primer CCW3 and primer CCW4 are used together; Primer CCW5 and primer CCW6 are used together; Primer CCW7 and primer CCW8 are used together; Primer CCW9 and primer CCW10 are used together.

3. Use of the primer set for identifying thick-lipped catfish according to claim 1 or 2 in identifying or distinguishing thick-lipped catfish.

4. Use of the primer set for identifying thick-lipped catfish according to claim 3 in identifying or distinguishing thick-lipped catfish when the thick-lipped catfish is propagated and released.

5. A method for evaluating the effect of thick-lipped catfish proliferation and release, characterized in that: The method for evaluating the effect of thick-lipped catfish proliferation and release comprises the following steps: 1) Preserving DNA of artificially bred parents of thick-lipped catfish 亲本 ; 2) Releasing the artificially bred progeny of the thick-lipped catfish; 3) Capture of wild populations of rough-lipped catfish; 4) Extraction of DNA from wild populations of rough-lipped catfish 捕捞 ; 5) using the primer set for identifying the thick-lipped catfish according to claim 1 or 2 to respectively detect the DNA obtained in step 1) 亲本 and the DNA obtained in step 4) 捕捞 Perform PCR amplification and subject the PCR products to polyacrylamide gel electrophoresis; 6) Analyze the parent-offspring relationship between the rough-lipped catfish parent and the rough-lipped catfish caught in the wild according to the result of step 5), obtain the number of the rough-lipped catfish parent in the caught rough-lipped catfish, and evaluate the release effect according to the number.

6. The method for evaluating the effect of the proliferation and release of rough-lipped catfish according to claim 5, characterized in that: The reaction system of PCR amplification in step 5) is 25 μL, including: 3 μL of 10×PCR Buffer, 2 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / LMgCl2, 1 μL of upstream and downstream primers, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, and 10.5 μL of ultrapure water.

7. The method for evaluating the effect of the proliferation and release of rough-lipped catfish according to claim 6, characterized in that: The procedure of PCR amplification in step 5) is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 29 s, 35 cycles; extension at 72°C for 10 min; and storage at 4°C.

8. A device for releasing rough-lipped catfish, characterized in that: The thick-lipped catfish release device comprises a fish release trough (1), a connecting box (2), a release hose (4), a protection pile (3) and a water inlet pipe (7); the fish release trough (1) is placed at the head end of the connecting box (2) and is connected to the connecting box (2); the release hose (4) is a plurality of hoses, which are arranged in parallel at the tail end of the connecting box (2) and are respectively connected to the connecting box (2); the protection pile (3) is a 1 / 4 cylinder, the protection pile (3) is placed inside the connecting box (2), and the axial direction of the protection pile (3) is not parallel to the axial direction of the connecting box (2); the water inlet pipe (7) comprises a first water inlet pipe and a second water inlet pipe; the first water inlet pipe is connected to the inside of the fish release trough (1); the second water inlet pipe is connected to the connecting box (2); the water inlet direction of the second water inlet pipe is opposite to the flow direction of the water flow in the connecting box (2).

9. The device for releasing rough-lipped catfish according to claim 8, characterized in that: The discharge hose (4) comprises a tube body, on which is provided a water inlet (41) connected to the rear end of the connecting box (2); on the inner wall of the tube body is provided a hose baffle (42); the axial direction of the hose baffle (42) is not parallel to the axial direction of the tube body.

10. The device for releasing rough-lipped catfish according to claim 8 or 9, characterized in that: The length of the protection pile (3) is half the width of the connecting box (2); the thick-lipped catfish releasing device further comprises a detachable anchor (6) connected to the connecting box (2); the thick-lipped catfish releasing device further comprises an electric valve (9) arranged between the fish releasing tank (1) and the connecting box (2); an overflow hole (8) is arranged at the top of the electric valve (9).

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