Artificial breeding method of naked loach

Abstract

This invention relates to an artificial breeding method for *Hemiberlesia lataniae*, comprising the following steps: Step 1: Selecting mature broodstock; Step 2: Timing of spawning induction; Step 3: Using a "low-to-high" dosage method for spawning induction; Step 4: Separating female and male fish after injection; Step 5: Observing the fish before moving them to accurately determine the timing of egg release; Step 6: Using anesthesia followed by egg release to improve the fertilization rate of the eggs and the post-spawning survival rate of the broodstock; Step 7: Inseminating multiple females with two males; Step 8: Hatching. This invention provides an artificial breeding method for *Hemiberlesia lataniae*, breaking through the key technologies for artificial breeding of this species and establishing a preliminary artificial breeding technology system. It has a reference and demonstration effect on the protection of rare and endemic fish species in the upper reaches of the Yangtze River, especially of great significance for the protection of small and rare fish.

Description

Technical Field

[0001] This invention relates to the field of conservation technology for rare and endemic fish species, and in particular to an artificial breeding method for the naked loach. Background Technology

[0002] Naked Stomata ( Xenophysogobio nudicorpa The naked loach (Syngonium nautiloides) is an endemic and important economic fish species mainly distributed in the upper reaches of the Yangtze River, the lower reaches of the Jinsha River, the Min River, and the Yalong River. However, in recent years, due to the impact of human activities, its habitat has gradually shrunk or even disappeared, and its natural reproduction scale has drastically decreased, resulting in a sharp decline in its wild resources. In 2000, the naked loach was listed as a key protected fish species in Sichuan Province. Before the start of the ten-year fishing ban in the Yangtze River, wild resources of the naked loach were already extremely difficult to obtain, becoming an occasional species, and the fish resources were very scarce. This led to the long delay in carrying out ex-situ conservation work such as artificial breeding and artificial population construction. Since the start of the ten-year fishing ban in the Yangtze River, the above situation has been slightly alleviated, but research on the naked loach remains very limited, mainly focusing on morphological characteristics and population genetics.

[0003] Currently, artificial breeding techniques for *Hemiberlesia nautiloides* are still in the research stage. This is mainly because *Hemiberlesia nautiloides* has very demanding requirements for its artificial breeding habitat and conditions, resulting in a high mortality rate during domestication. Conventional methods for inducing spawning also fail to successfully obtain fertilized eggs. Therefore, it is urgent to develop an artificial breeding method for *Hemiberlesia nautiloides* to lay the foundation for establishing a comprehensive artificial breeding technology system. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an artificial breeding method for naked loach, which can successfully carry out artificial breeding of naked loach, successfully obtain fertilized eggs and hatch fry, ensure the survival rate of parent fish after spawning, and lay the foundation for establishing its artificial breeding technology system.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A method for the artificial breeding of the naked loach with a swim bladder, the method comprising the following steps:

[0007] Step 1: Select target female and male fish for induction;

[0008] Step 2: Timing of induced spawning: Artificial induced spawning should begin when the water temperature in the breeding pond is between 16 and 19°C.

[0009] Step 3: The "low-then-high" dosage method for inducing labor:

[0010] Step 4: Separate the female and male fish after injection; the conventional method of inducing spawning is to inject the female and male fish together to promote estrus, but this is not suitable for naked loach. The female and male naked loach must be separated after injection; otherwise, the male will release sperm once it detects the female's spawning behavior, leading to spawning failure.

[0011] Step 5: "Observe before moving the fish" – precisely determine the timing for egg extraction. First, observe whether there are eggs in the egg collection bag. If eggs that have absorbed water are found, prepare to start extracting the eggs; otherwise, do not extract. The conventional way to determine the timing for egg extraction is to grab the fish and squeeze out the eggs. If eggs are ejected from the genital opening, prepare to extract the eggs. However, the naked loach is highly irritable. If the eggs in its ovary are not completely freed and it is grabbed and squeezed by hand, it is very easy to cause dystocia. Therefore, the conventional method is not suitable for the naked loach. This invention uses the "observe before moving the fish" method, which can precisely determine the timing for egg extraction. First, observe whether there are eggs in the egg collection bag. If eggs that have absorbed water are found, prepare to start extracting the eggs; otherwise, do not extract.

[0012] Step 6: Use the "anesthetize first, then squeeze out the eggs" method to improve the fertilization rate of the eggs and the postpartum survival rate of the parent fish;

[0013] Step 7: Multiple females and two males are used for fertilization; male naked loach are difficult to obtain, so multiple females and two males are used for fertilization to ensure the success rate of induced spawning and fertilization.

[0014] Step 8: Incubation

[0015] Artificial breeding of naked loach was completed.

[0016] Preferably, in step 1, naked *Heterobalostoma garut* with a body length ≥ 8 cm, body width ≥ 1.8 cm, body height ≥ 2.0 cm, and head width ≥ 1.3 cm are selected as target females for induction; naked *Heterobalostoma garut* with a body length ≥ 7.5 cm, body width approximately 1.0~1.2 cm, body height approximately 1.4~1.6 cm, and head width approximately 1.0~1.2 cm are selected as target males for induction. Mature female and male naked *Heterobalostoma garut* do not have obvious secondary sexual characteristics; sex and maturity are determined by external biological characteristics.

[0017] Preferably, in step 3, the female fish is given a three-injection method with a "low-then-high" dosage of oxytocin. The first two injections have low dosages, and the third injection has a dosage 8-12 times that of the second injection. The male fish is given two injections with the same dosage as the female fish's first two injections. The first injection of oxytocin is LHRH-A2, and the second and third injections of oxytocin are LHRH-A2 (luteinizing hormone-releasing hormone A2), HCG (human chorionic gonadotropin), and DOM (demorton maleate).

[0018] Preferably, in step 3, the first injection is 0.3-0.6 ug LHRH-A2; the second injection is 1.5-2.5 ug LHRH-A2 + 45-55 IU HCG + 0.04-0.06 mg DOM; and the third injection is 18-22 ug LHRH-A2 + 480-520 IU HCG + 0.4-0.6 mg DOM.

[0019] Preferably, in step 6, the fish are anesthetized with MS-222 (ethyl m-aminobenzoate methanesulfonate), and the surface of the fish is wiped dry with a towel before the eggs are extracted. The fish are then revived after extraction. Mature naked loaches are very small, with smooth, scaleless bodies, making them difficult to handle. Forcing them can lead to injury and death. Furthermore, their small size means that water from handling can drip into the egg collection basin, causing unfertilized eggs to come into contact with water and become spoiled. This invention employs an "anesthetize first, then extract" method, using MS-222 to anesthetize the fish, wiping the surface of the fish with a dry towel before extracting the eggs, and then reviving the fish after extraction. This method improves the fertilization rate of the eggs and the post-spawning survival rate of the parent fish.

[0020] Preferably, in step 8, the hatching method is direct hatching in the rearing pond. An aeration device is installed in the rearing pond, the water inlet pipe is turned on, and the pond is filled with water for later use. After fertilization, the eggs are first poured into a basin with a diameter of 50-70cm, stirred until they absorb water, and then poured into the rearing pond for hatching. Conventional hatching methods involve incubating in an incubator, where the fry are transferred out after hatching, which often results in significant damage and death of the fry. This invention uses a direct hatching method in the rearing pond, improving the survival rate of newly hatched fry. First, an aeration device is installed in the rearing pond, the water inlet pipe is turned on, and the pond is filled with water for later use. After fertilization, the eggs are first poured into a basin with a diameter of 50-70cm, stirred until they absorb water, and then poured into the rearing pond for hatching.

[0021] The present invention provides an artificial breeding method for the naked loach, which has the following beneficial effects:

[0022] 1. This invention has successfully broken through the key technology for artificial breeding of naked loach, and has initially established an artificial breeding technology system for naked loach. It has a reference and demonstration effect on the protection of rare and endemic fish species in the upper reaches of the Yangtze River, and is of great significance for the protection of small and rare fish species.

[0023] 2. Mature female and male naked loach have no obvious secondary sexual characteristics. This invention provides a standard for mature female and male naked loach. This invention also provides a method for inducing spawning in naked loach by using a "low-to-high" dosage, which can successfully induce spawning, while conventional methods all fail.

[0024] 3. This invention adopts the method of "observing the fish first and then moving the fish" to accurately grasp the timing of egg release, avoiding the dystocia of naked loach that occurs when using conventional methods; this invention adopts the method of "anesthetizing before releasing eggs" to significantly improve the fertilization rate of eggs and the postpartum survival rate of parent fish.

[0025] 4. Male naked loach are difficult to obtain. This invention uses a multi-female, two-male insemination method to ensure the success rate of induced spawning and fertilization. Conventional incubation methods involve incubating in an incubator and then transferring the fry out of the incubator after they hatch. This transfer process causes a large number of fry to be damaged and die. This invention uses a direct incubation method in the breeding pond, which significantly improves the survival rate of newly hatched fry. Detailed Implementation

[0026] Example 1

[0027] On April 23, 2023, the artificial breeding of naked loach (Sinonovacula nautiloides) was carried out at the Wudongde Stocking and Release Station of the Chinese Sturgeon Research Institute of China Three Gorges Corporation using the method provided in this invention. The specific steps included:

[0028] Step 1: Selecting mature parent fish. Mature females and males of the naked loach (Gnaphalium affineum) do not exhibit obvious secondary sexual characteristics; sex and maturity are determined by external biological features. Thirty-four naked loach (Gnaphalium affineum) with a body length of over 8cm, a body width of over 1.8cm, a body height of over 2.0cm, and a head width of over 1.3cm were selected as target females for induction. Twelve naked loach (Gnaphalium affineum) with a body length of over 7.5cm, a body width of approximately 1.0-1.2cm, a body height of approximately 1.4-1.6cm, and a head width of approximately 1.0-1.2cm were selected as target males for induction.

[0029] Step 2: Timing of induced spawning. Artificial spawning is initiated when the water temperature in the lower reaches of the Jinsha River reaches 16℃ and the water temperature in the aquaculture pond reaches 17℃.

[0030] Step 3: "Low-to-high" dosage induction method. Female fish are given a three-injection regimen using a "low-to-high" dosage of oxytocin. The first two injections are low doses, and the third injection is 10 times the dose of the second. The first injection is given to both male and female fish at 8:00 PM on April 23rd, the second at 8:00 AM on April 24th, and the third at 8:00 PM on April 24th. Male fish are not injected. Male fish receive two injections, with the same dosage as the female's first two injections. First injection: 0.5ug LHRH-A2; Second injection: 2ug LHRH-A2 + 50 IU HCG + 0.05 mg DOM; Third injection: 20ug LHRH-A2 + 500 IU HCG + 0.5 mg DOM.

[0031] Step 4: After injection, the female and male fish are placed separately in different spawning tanks.

[0032] Step 5: "Observe the fish before moving it" – precisely determine the timing for egg extraction. The conventional method for determining the timing of egg extraction is to catch the fish and squeeze out the eggs. If eggs are ejected from the genital opening, then preparation for egg extraction begins. However, the naked loach is highly irritable. If the eggs in its ovary are not completely freed and it is caught and squeezed by hand, it is very likely to cause dystocia (difficult birth). Therefore, the conventional method is not suitable for the naked loach. This invention uses the "observe the fish before moving it" method, which allows for precise timing of egg extraction. First, observe whether there are eggs in the egg collection bag. If eggs that have absorbed water are found, then preparation for egg extraction begins; otherwise, do not extract. At 9:00 AM on April 25th, eggs were found in the egg collection bag, and egg extraction began.

[0033] Step 6: Employing anesthesia followed by egg extraction improves fertilization rate and post-spawning survival rate of broodstock. Mature naked loaches are very small, with smooth, scaleless bodies, making them difficult to handle. Forcing them can lead to injury and death. Furthermore, their small size means water from handling can drip into the egg collection basin, resulting in unfertilized eggs. This invention uses anesthesia followed by egg extraction. The fish are anesthetized with 100mg / L MS-222, and the surface water is wiped clean with a dry towel before egg extraction. After extraction, the fish are revived. Of 34 females, 28 successfully spawned, achieving an induced spawning rate of 82.35%. All males produced sperm, achieving a 100% induced spawning rate. Over 20,000 fertilized eggs were obtained, with a fertilization rate exceeding 90%. The post-spawning survival rate of the broodstock was 91.30%.

[0034] Step 7: Insemination by multiple females and two males. Male naked loach are difficult to obtain. To ensure the success rate of induced spawning and fertilization, insemination is carried out by using a method of 6 females and 2-3 males. That is, after squeezing the eggs of 6 females into the same egg-collecting basin, the sperm of 2-3 males is squeezed into the basin, and then water is added and stirred for insemination.

[0035] Step 8: Hatching. Conventional hatching methods involve incubating in an incubator, then transferring the fry out of the incubator after hatching. This transfer process often results in significant damage and death of the fry. This invention uses a direct hatching method in the rearing pond, improving the survival rate of newly hatched fry. First, an aeration device and escape-prevention net are installed in the rearing pond, and the water inlet pipe is opened and filled with water for later use. After fertilization, the eggs are poured into a 60cm diameter basin, stirred until they absorb water, and then more than 20,000 fertilized eggs are poured into the rearing pond for hatching. Hatching is carried out at 19.0~19.8℃ for 130 hours until the fry hatch.

[0036] Example 2

[0037] In April 2023, four spawning induction experiments were conducted in groups. Except for the different dosages of the spawning induced agent, all other steps were carried out according to the method in Example 1. The experimental results are shown in Table 3. The results showed that the spawning induction effect was best when female fish were treated with a three-shot injection method with a "low-then-high" dosage of spawning induced agent (the first two injections were low dosages, and the third injection was 10 times the dosage of the second injection).

[0038] Table 1

[0039]

[0040] Example 3

[0041] In April 2023, experiments were conducted in groups, with male and female fish being placed separately and together after injection. All other steps were performed according to the method in Example 1. The results showed that in the experimental group where male and female fish were placed together after injection, male fish were successfully induced to spawn, but by the time the effect period of the third injection on the female fish arrived, the male fish had already ejaculated. In the experimental group where male and female fish were placed separately after injection, male fish were successfully induced to spawn, but by the time the effect period of the third injection on the female fish arrived, the male fish had not ejaculated.

[0042] Table 2

[0043]

[0044] Example 4

[0045] In April 2023, two groups were formed to determine the timing of egg release in female fish: the "observe first, then move the fish" group and the conventional method group. All other steps were carried out according to the method in Example 1. The results showed that all female fish in the "observe first, then move the fish" group gave birth smoothly, while one female fish in the conventional method group gave birth smoothly, and the rest gave birth dystocia.

[0046] Table 3

[0047]

[0048] Example 5

[0049] In April 2023, a control experiment was conducted in groups to compare "egg extraction after anesthesia" and "egg extraction directly without anesthesia". All other steps were carried out according to the method in Example 1. The results showed that the fertilization rate and the survival rate of the broodstock after spawning were significantly higher in the "egg extraction directly without anesthesia" group than in the "egg extraction directly without anesthesia" group.

[0050] Table 4

[0051]

[0052] The above are merely specific embodiments of the present invention, but the technical features of the present invention are not limited thereto. Any equivalent changes, substitutions, or modifications made based on the present invention to solve essentially the same technical problems and achieve essentially the same technical effects should still fall within the patent coverage of the present invention.

Claims

1. A method for the artificial breeding of *Hemiberlesia lataniae*, characterized in that: The method includes the following steps: Step 1: Select target female and male fish for induction; Step 2: Timing of induced spawning: Artificial induced spawning should begin when the water temperature in the breeding pond is between 16 and 19°C. Step 3: The "low-to-high" dosage method for spawning induction: For female fish, a three-injection method using a "low-to-high" dosage of spawning induced agent is employed. The first two injections use low doses, while the third injection uses 8-12 times the dose of the second injection. For male fish, two injections are used, with the same dosage as the female's first two injections. The first injection uses LHRH-A2, the second uses a combination of LHRH-A2, HCG, and DOM, and the third injection uses the same combination of LHRH-A2, HCG, and DOM. Step 4: Separate the female and male fish after injection; Step 5: "Observe the fish before moving it". Precisely grasp the timing of squeezing the eggs. First observe whether there are eggs in the egg collection bag. If you find eggs that have absorbed water, then prepare to squeeze the eggs. Otherwise, do not squeeze. Step 6: Use the "anesthetize first, then squeeze out the eggs" method to improve the fertilization rate of the eggs and the postpartum survival rate of the parent fish; Step 7: Multiple females and two males are used for insemination; male naked loach are difficult to obtain, so multiple females and two males are used for insemination to ensure the success rate of induced spawning and fertilization. Step 8: Hatching; Artificial breeding of naked loach was completed.

2. The artificial breeding method for *Gnaphalium affine* according to claim 1, characterized in that, In step 1, naked loach with a body length ≥8cm, body width ≥1.8cm, body height ≥2.0cm, and head width ≥1.3cm are selected as female fish to be induced; naked loach with a body length ≥7.5cm, body width between 1.0 and 1.2cm, body height between 1.4 and 1.6cm, and head width between 1.0 and 1.2cm are selected as male fish to be induced.

3. The artificial breeding method for *Gnaphalium affine* according to claim 1, characterized in that, In step 3, the first injection is 0.3-0.6 ug LHRH-A2; the second injection is 1.5-2.5 ug LHRH-A2 + 45-55 IU HCG + 0.04-0.06 mg DOM; and the third injection is 18-22 ug LHRH-A2 + 480-520 IU HCG + 0.4-0.6 mg DOM.

4. The artificial breeding method for *Gnaphalium affine* according to claim 1, characterized in that, In step 6, the fish are anesthetized with MS-222, the water on the surface of the fish is wiped off with a dry towel, and the eggs are squeezed out. The fish are then revived after the eggs are squeezed out.

5. The artificial breeding method for *Gnaphalium affine* according to claim 1, characterized in that, In step 8, the incubation method adopted is to directly incubate in the breeding pond. An aeration device is installed in the breeding pond, the water inlet pipe is turned on, and the water is filled and ready for use. After the eggs are fertilized, they are first poured into a basin with a diameter of 50-70cm, stirred until they absorb water, and then poured into the breeding pond for incubation.

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