Eleuciscus waleckii embryo hatching liquid, preparation method thereof and method for improving embryo hatching rate of leuciscus waleckii

Abstract

The invention discloses a leuciscus waleckii embryo hatching solution, a preparation method thereof and a method for improving the hatching rate of leuciscus waleckii embryos, and the leuciscus waleckii embryo hatching solution is a hatching mother solution formed by dissolving 170-180g of sodium chloride, 20-30g of potassium chloride, 4-6g of calcium chloride and 0.4-0.6 g of methylene blue in distilled water and fixing the volume to 1L; the invention further specifically discloses a preparation method of the leuciscus waleckii embryo hatching liquid and application of the leuciscus waleckii embryo hatching liquid in increasing the hatching rate of leuciscus waleckii embryos. According to the hatching working solution, the hatching mother solution is prepared and used immediately, water pollution is avoided, water dissolved oxygen is increased, the embryo development environment is stabilized, invasion of saprolegniasis spores and other bacteria can be inhibited under the low-temperature condition, saprolegniasis outbreak is inhibited, the hatching rate and the survival rate of leuciscus waleckii embryos are increased, and the survival rate of leuciscus waleckii embryos is increased. And a reliable technical scheme is provided for realizing large-scale and standardized artificial propagation of the leuciscus waleckii.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, and relates to the embryo hatching technology of Leuciscus var. var., specifically to a Leuciscus var. var. embryo hatching solution and its preparation method, and a method for improving the hatching rate of Leuciscus var. var. embryos. Background Technology

[0002] Leuciscus waleckii, belonging to the Cyprinidae family and Leucisinae subfamily, is a cold-water fish with important ecological and economic value in northern my country. It usually inhabits slow-moving, clear, still water areas, the upper and middle layers of mountain tributaries, and brackish lakes. Its flesh is delicious and it has excellent characteristics such as salt and alkali tolerance and strong adaptability, making it a promising fish in the market.

[0003] Under natural breeding conditions, the hatching of fertilized eggs in *Leuciscus vannamei* is severely limited by multiple environmental factors, including water temperature, water flow, and spawning grounds, leading to a decline in natural resources. Artificial breeding of *Leuciscus vannamei* commonly suffers from problems such as asynchronous gonadal development, difficulty in natural spawning, and insensitivity to spawning-inducing drugs. Currently, artificial spawning induction in *Leuciscus vannamei* often follows methods used in other cyprinid fish, such as using human chorionic gonadotropin (hCG) or a combination with ineffective spawning-inducing agents. This method suffers from defects such as inaccurate dosage, improper timing, and non-standard procedures. Furthermore, traditional hatching methods often involve directly placing fertilized eggs obtained from the parent fish into an incubator for flow-through hatching. However, the egg membranes of fertilized eggs are thin and extremely sensitive to fluctuations in external aquatic environmental factors such as pH, ammonia nitrogen, and mechanical vibration. Additionally, eggs produced from the parent fish may carry coelomic fluid, unwashed semen, and organic debris. These substances gradually decompose during hatching, consuming oxygen and promoting the growth of harmful microorganisms, creating stress during the most vulnerable early stages of embryonic development, leading to developmental arrest or malformation.

[0004] The current artificial breeding technology of Leuciscus var. wilfordii has the following problems: (1) The breeding water temperature is low, the fertilized eggs develop slowly, the hatching time is long, and the hatching rate is low; (2) The low water temperature is prone to the growth of water mold, which causes the embryos to suffocate during the hatching process and cannot hatch, resulting in a low hatching rate. Although some farms use chemical agents such as malachite green to disinfect fertilized eggs in production, they have high residues and risks of teratogenicity and carcinogenicity. At present, it is necessary to develop a pretreatment method that can effectively remove harmful substances from the egg surface, enhance embryo viability, and stabilize the hatching environment, based on the physiological characteristics of Leuciscus var. wilfordii embryos. This is of great significance for improving the hatching rate of Leuciscus var. wilfordii fertilized eggs. Summary of the Invention

[0005] To address the technical problem of low hatching rate of artificially bred Leuciscus var. var. embryos, this invention provides a Leuciscus var. var. embryo hatching solution, its preparation method, and a method for improving the hatching rate of Leuciscus var. var. embryos.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a hatching solution for the embryo of the wrasse, which is a hatching mother liquor formed by dissolving 170-180g of sodium chloride, 20-30g of potassium chloride, 4-6g of calcium chloride and 0.4-0.6g of methylene blue in distilled water and making up to 1L.

[0007] Furthermore, the incubation solution is an incubation stock solution formed by dissolving 175g sodium chloride, 25g potassium chloride, 5g calcium chloride and 0.5g methylene blue in distilled water and bringing the volume to 1L.

[0008] The preparation method of the embryo hatching solution of the var. valieri described in this invention includes the following specific preparation steps: dissolving sodium chloride in 300 mL of distilled water according to the above weight ratio to obtain solution A; dissolving potassium chloride, calcium chloride and methylene blue in 500 mL of distilled water according to the above weight ratio to obtain solution B; stirring and mixing solution A and solution B evenly, and then adding distilled water to make up to 1 L to obtain the hatching mother liquor.

[0009] Furthermore, during the preparation of solution A, the stirring speed is 120 rpm and the stirring time is 30 minutes; during the preparation of solution B, the stirring speed is 90 rpm and the stirring time is 30 minutes; and during the preparation of the incubation mother liquor, the stirring speed is 90 rpm and the stirring time is 20 minutes.

[0010] The application of the *Leuciscus vallar* embryo incubation solution described in this invention in improving the hatching rate of *Leuciscus vallar* embryos is as follows: The *Leuciscus vallar* embryo incubation solution and aerated water are mixed evenly at a volume ratio of 1:40~60 to obtain the incubation working solution. The incubation working solution is then poured into an incubation tank, and *Leuciscus vallar* deadhesive fertilized eggs are added. The water temperature is maintained at 14~16℃ for incubation. Every 24 hours, 1 / 3 of the incubation working solution in the incubation tank is released, and an equal amount of new incubation working solution is added for incubation. When the embryos are incubated for 10~14 days, the embryos can be seen rotating inside the egg membrane, which is the pre-embryo hatching stage. At this time, the use of the incubation working solution is stopped, and fresh aerated water is added until the membrane hatches. Finally, the hatching rate of *Leuciscus vallar* embryos is as high as 86.69%.

[0011] Furthermore, the dissolved oxygen content of the incubation working solution is higher than 8 mg / L, and the pH value is 7.5~8.0.

[0012] Furthermore, the specific preparation steps for the deadhesive fertilized eggs of the *Leuciscus vallis* are as follows: Step S1: Collect parent fish, aged 2-3 winters, with a total length greater than 25cm, a weight greater than 250g, intact scales, symmetrical body shape, no deformities, and no diseases. For female fish, select individuals with a swollen and rounded abdomen and a clear ovarian outline. For male fish, select individuals with rough scales all over their bodies, a rough feel to the touch, obvious white granular "stars" on their heads, and white semen flowing out when the abdomen is gently pressed. The ratio of female to male parent fish should be 3:1 to 4:1. Step S2: Artificial spawning induction. For female fish, inject 10 μg of luteinizing hormone-releasing hormone analogue 2 (LHR-A2) and 8 mg / kg of domperidone maleate (DOM) per kilogram of body weight. The dosage for male fish is halved. Dissolve the spawning-inducing drugs in 0.9 wt% physiological saline solution, preparing the solution immediately before use. The dosage is calculated at 0.5 mL per broodstock, using a single injection method, injecting into the body cavity from the base of the pectoral fin or into the muscle from the base of the dorsal fin. After the hormone injection, place the female broodstock in separate cement ponds or net cages to await spawning, with a stocking density not exceeding 10 kg / m². 3 The effect time is 48-50 hours when the water temperature is 10-14℃ and 22-24 hours when the water temperature is 16-18℃. Step S3: When the effect time is reached, squeeze the abdomen of the female fish to collect mature eggs in a clean and smooth porcelain bowl. Collect the sperm of the male fish, squeeze it into the eggs, stir it evenly with a clean feather, add aerated water and stir gently to perform artificial insemination. Step S4: Place the fertilized eggs into the pre-prepared talcum powder and stir gently to remove the adhesive. Then wash them three times with clean water to obtain the de-adhesive fertilized eggs of the Wagyu fish.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects: the calcium chloride dissolves slowly during the preparation of the incubation mother solution, and the calcium chloride solution is prepared first to avoid direct preparation that could lead to solution saturation and precipitation; the incubation working solution is prepared and used immediately to avoid water pollution, increase dissolved oxygen in the water, stabilize the embryonic development environment, and inhibit the invasion of water mold spores and other bacteria under low temperature conditions, thereby inhibiting the outbreak of water mold disease and improving the hatching rate and survival rate of Leuciscus vallis embryos, providing a reliable technical solution for the large-scale and standardized artificial breeding of Leuciscus vallis. Detailed Implementation

[0014] The specific technical solutions of the present invention will be described in detail with reference to specific embodiments. Example 1

[0015] Prepare 5L of incubation stock solution: Weigh 25g of calcium chloride and add it to 1.5L of distilled water. Stir at 120 rpm for 25 minutes until the calcium chloride dissolves. Weigh 875g of sodium chloride, 125g of potassium chloride, and 2.5g of methylene blue and add them to 2.5L of distilled water. Stir at 90 rpm for 10 minutes until completely dissolved. Mix the two solutions and add distilled water to bring the volume to 5L to obtain the incubation stock solution. Take 2L of the incubation stock solution and add it to 98L of aeration water to prepare 100L of incubation working solution.

[0016] Two to three-year-old broodstock were collected, including 20 females and 8 males. Artificial spawning was induced by a single injection. The spawning-inducing drugs were dissolved and mixed with 0.9 wt% physiological saline and prepared fresh for each use. The dosage for females was 10 μg / kg (LHR-A2) and 8 mg / kg (DOM), while the dosage for males was halved. Each broodstock was injected with 0.5 mL.

[0017] At a water temperature of 10-12℃, after 50 hours, the female fish's abdomen was squeezed to collect the eggs. The 20 female fish were divided into three groups of 6, 6, and 7 fish for artificial insemination. The eggs of 6 female fish were collected in a porcelain bowl that had been pre-ice-bathed. The semen of 2 male fish was squeezed into the porcelain bowl and gently stirred with a feather for 2 minutes until the semen evenly coated the eggs. Aerated water was added to activate the fertilized eggs. After removing the stickiness with talcum powder and rinsing with clean water 3 times, the eggs were poured into an incubation tank containing working solution for incubation.

[0018] The incubation temperature was 14℃, the dissolved oxygen content of the incubation working solution was higher than 8mg / L, the pH value was 7.5~8.0, the incubation time was 14 days, and the hatching rate of the fertilized eggs of Leuciscus var. var. var. was 86.63%. Example 2

[0019] Prepare 20L of incubation stock solution: Weigh 100g of calcium chloride and add 5L of distilled water, stirring at 120 rpm for 30 minutes until the calcium chloride dissolves; weigh 3.5kg of sodium chloride, 500g of potassium chloride, and 10g of methylene blue and add 10L of distilled water, stirring at 90 rpm for 10 minutes until completely dissolved; mix the two solutions and add distilled water to bring the volume to 20L to obtain the incubation stock solution. Take 2L of the incubation stock solution and add 98L of aeration water to prepare 100L of incubation working solution.

[0020] Two to three-year-old broodstock, 18 females and 8 males, were collected. Artificial spawning was induced by a single injection. The spawning-inducing drugs were dissolved and mixed with 0.9 wt% physiological saline and prepared fresh for each use. The dosage for females was 10 μg / kg (LHR-A2) and 8 mg / kg (DOM), while the dosage for males was halved. Each broodstock was injected with 0.5 mL.

[0021] At a water temperature of 14℃, after 48 hours, the female fish's abdomen was squeezed to collect the eggs. The 18 female fish were divided into three groups for artificial insemination. The eggs of 6 female fish were collected in a porcelain bowl that had been pre-ice-bathed. The semen of 2 male fish was squeezed into the porcelain bowl and gently stirred with a feather for 2 minutes until the semen evenly coated the eggs. Aerated water was added to activate the fertilized eggs. After being de-adhesive with talcum powder and rinsed three times with clean water, the eggs were poured into an incubation tank containing working solution for incubation.

[0022] The incubation temperature was 16℃, the dissolved oxygen content of the incubation working solution was higher than 8mg / L, the pH value was 7.5~8.0, the incubation time was 10 days, and the hatching rate of the fertilized eggs of Leuciscus var. var. var. was 86.69%. Example 3

[0023] Prepare 20L of hatching stock solution. Weigh 100g of calcium chloride and add 5L of distilled water, stirring at 120 rpm for 30 minutes until the calcium chloride dissolves. Weigh 3.5kg of sodium chloride, 500g of potassium chloride, and 10g of methylene blue and add 10L of distilled water, stirring at 90 rpm for 10 minutes until completely dissolved. Mix the two solutions and add distilled water to bring the volume to 20L to obtain the hatching stock solution. Take 2L of the hatching stock solution and add 98L of aeration water to prepare 100L of hatching working solution. Collect 2-3 year old broodstock, including 18 females and 6 males. Artificial spawning temperature is 18-20℃, and artificial insemination is performed after 22 hours of effect. The incubation temperature is 16℃, the dissolved oxygen content of the hatching working solution is higher than 8mg / L, the pH value is 7.5-8.0, and the incubation time is 10 days. Other conditions are the same as in Example 1. The hatching rate of fertilized eggs of *Leuciscus vallis* is 85.76%.

[0024] Comparative Example 1 Two to three-year-old parent fish were collected, including 18 females and 6 males. Artificial spawning was induced at a temperature of 10-12℃, and artificial insemination was performed after an effect time of 50 hours. The incubation temperature was 14℃, the incubation solution was aerated water with dissolved oxygen content above 8 mg / L and pH value of 7.5-8.0, and the incubation time was 14 days. Other conditions were the same as in Example 1. The hatching rate of fertilized eggs of Leuciscus var. ...

[0025] Comparative Example 2 Two to three-year-old parent fish were collected, including 18 females and 8 males. Artificial spawning was induced at a temperature of 14°C, and artificial insemination was performed after an effect time of 48 hours. The incubation temperature was 16°C, the incubation solution was aerated water with dissolved oxygen content above 8 mg / L and pH value of 7.5-8.0, and the incubation time was 10 days. Other conditions were the same as in Example 1. The hatching rate of fertilized eggs of Leuciscus var. ...

[0026] Comparative Example 3 Two to three-year-old parent fish were collected, including 18 females and 8 males. Artificial spawning was induced at a temperature of 18-20°C, and artificial insemination was performed after 22 hours of induced spawning. The incubation temperature was 16°C, the incubation solution was aerated water with dissolved oxygen content above 8 mg / L and pH value of 7.5-8.0, and the incubation time was 10 days. Other conditions were the same as in Example 1. The hatching rate of fertilized eggs of Leuciscus var. ...

[0027] Compared with Comparative Examples 1, 2, and 3, Examples 1, 2, and 3 used a hatching working solution during the hatching process of Leuciscus var. ...

[0028] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A hatching solution for *Leuciscus var. vara* embryos, characterized in that: The incubation solution is an incubation mother liquor formed by dissolving 170-180g sodium chloride, 20-30g potassium chloride, 4-6g calcium chloride and 0.4-0.6g methylene blue in distilled water and making up to 1L.

2. The embryo incubation solution for *Leuciscus vallar* according to claim 1, characterized in that: The incubation solution is an incubation stock solution formed by dissolving 175g sodium chloride, 25g potassium chloride, 5g calcium chloride and 0.5g methylene blue in distilled water and bringing the volume to 1L.

3. A method for preparing the embryo incubation solution for *Leuciscus vallar* as described in claim 1 or 2, characterized in that... The specific preparation steps are as follows: Dissolve sodium chloride in 300 mL of distilled water to obtain solution A; dissolve potassium chloride, calcium chloride and methylene blue in 500 mL of distilled water to obtain solution B; stir and mix solution A and solution B evenly, and then add distilled water to make up to 1 L to obtain the incubation mother liquor.

4. The method for preparing the embryo incubation solution for *Leuciscus vallar* according to claim 3, characterized in that: The stirring speed during the preparation of solution A is 120 rpm and the stirring time is 30 minutes; the stirring speed during the preparation of solution B is 90 rpm and the stirring time is 30 minutes; the stirring speed during the preparation of the incubation mother liquor is 90 rpm and the stirring time is 20 minutes.

5. The application of the embryo incubation solution of *Leptochloa valgosa* as described in claim 1 or 2 in improving the hatching rate of *Leptochloa valgosa* embryos.

6. The application according to claim 5, characterized in that... The specific application process is as follows: Mix the hatching solution of *Leuciscus valens* embryos with aerated water at a volume ratio of 1:40~60 to obtain the hatching working solution. Pour the hatching working solution into the hatching tank and add the deadhesive fertilized eggs of *Leuciscus valens*. Maintain a water temperature of 14~16℃ for hatching. Every 24 hours, release 1 / 3 of the hatching working solution in the hatching tank and add an equal amount of new hatching working solution for hatching. When the embryos are 10~14 days old and the embryos can be seen rotating inside the egg membrane (i.e., the pre-embryo hatching stage), stop using the hatching working solution and add fresh aerated water until the membrane hatches. The final hatching rate of *Leuciscus valens* embryos is as high as 86.69%.

7. The application according to claim 6, characterized in that: The dissolved oxygen content of the incubation working solution is higher than 8 mg / L, and the pH value is 7.5~8.

0.

8. The application according to claim 6, characterized in that: The specific steps for preparing the deadhesive fertilized eggs of the Leuciscus var. vara are as follows: Step S1: Collect parent fish, aged 2-3 winters, with a total length greater than 25cm, a weight greater than 250g, intact scales, symmetrical body shape, no deformities, and no diseases. For female fish, select individuals with a swollen and rounded abdomen and a clear ovarian outline. For male fish, select individuals with rough scales all over their bodies, a rough feel, obvious white granular "breathing stars" on their heads, and white semen flowing out when the abdomen is gently pressed. The ratio of female to male parent fish should be 3:1 to 4:

1. Step S2: Artificial spawning induction. For female fish, inject 10 μg of luteinizing hormone-releasing hormone analogue 2 (LHR-A2) and 8 mg / kg of domperidone maleate (DOM) per kilogram of body weight. The dosage for male fish is halved. Dissolve the spawning-inducing drugs in 0.9 wt% physiological saline solution, preparing the solution immediately before use. The dosage is calculated at 0.5 mL per broodstock, using a single injection method, injecting into the body cavity from the base of the pectoral fin or into the muscle from the base of the dorsal fin. After the hormone injection, place the female broodstock in separate cement ponds or net cages to await spawning, with a stocking density not exceeding 10 kg / m². 3 The effect time is 48-50 hours when the water temperature is 10-14℃ and 22-24 hours when the water temperature is 16-18℃. Step S3: When the effect time is reached, squeeze the abdomen of the female fish to collect mature eggs in a clean and smooth porcelain bowl. Collect the sperm of the male fish, squeeze it into the eggs, stir it evenly with a clean feather, add aerated water and stir gently to perform artificial insemination. Step S4: Place the fertilized eggs into the pre-prepared talcum powder and stir gently to remove the adhesive. Then wash them three times with clean water to obtain the de-adhesive fertilized eggs of the Wagyu fish.