Artificial breeding method of hemibarbus labeo

Abstract

The application provides an artificial breeding method of Onychostoma wujiangense, and belongs to the technical field of aquatic animal breeding. The artificial breeding method of Onychostoma wujiangense comprises the steps of parent fish classification, parent fish cultivation, artificial parturition induction, natural insemination, artificial insemination, fertilized egg hatching, larva cultivation and juvenile fish cultivation. The parturition induction agent is reasonably matched, the parturition induction injection time is accurate, the parturition induction rate, the fertilization rate and the hatching rate are improved, and the parent fish damage is small. The application also clearly defines the feeding methods at different stages of seed cultivation, improves the survival rate of Onychostoma wujiangense seed cultivation stage, and lays a technical support for the artificial breeding of the endangered species Onychostoma wujiangense.

Description

Technical Field

[0001] This invention relates to the field of aquatic animal breeding technology, and in particular to an artificial breeding method for the Wu's flathead carp. Background Technology

[0002] *Discocheilus wui*, belonging to the order Cypriniformes, family Cyprinidae, and genus *Discocheilus*, is a rare, endangered, and endemic fish species with a narrow distribution, found only in Tian'e County, Fengshan County, and Leye County of Guangxi Province. It inhabits fast-flowing, clear, oxygen-rich gravelly streams and is a benthic scraper, feeding on epiphytic algae and organic detritus. Due to its karst habitat, which is easily destroyed, its wild population has declined significantly. Under natural conditions, *Discocheilus wui* requires specific environmental conditions to successfully reproduce, such as specific water flow speeds, water temperature ranges (usually in spring and summer), clear water, and suitable gravelly spawning grounds. Its reproduction rate under natural conditions is extremely low; therefore, artificial breeding and stock enhancement are urgently needed to protect its population and resources.

[0003] However, the artificial breeding of *Spathipoda wuerensis* faces the following technical bottlenecks: asynchronous or suboptimal gonadal development in male and female broodstock makes it difficult to obtain high-quality broodstock with consistent maturity; due to its unique reproductive physiology, accurately determining gonadal maturity and the optimal timing for spawning induction is extremely challenging; and using spawning-inducing hormones from other fish species (such as HCG, LRH-A2, and DOM) has proven ineffective for *Spathipoda wuerensis*, necessitating the exploration of specific hormone types, dosages, and combinations that are effective for it. Currently, there is no mature, stable, and reproducible fully artificial breeding technique for *Spathipoda wuerensis*. Summary of the Invention

[0004] In view of this, the present invention provides an artificial breeding method for *Siniperca wusiensis* to solve the above problems.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for the artificial breeding of *Siniperca wusiensis*, characterized by the following steps:

[0007] S1. During the breeding season of the Wu's flathead carp, the parent fish are collected and divided into mature parent fish, reserve parent fish and postpartum female fish;

[0008] S2. Induce spawning in mature parent fish, and raise replacement parent fish and postpartum female fish separately until they develop into mature parent fish before inducing spawning again;

[0009] The domestication of replacement broodstock and postpartum females is as follows: After placing the packaged bag in the rearing tank for 25-30 minutes, open the packaged bag and slowly add water to the rearing tank until the water occupies 60-70% of the volume of the packaged bag. Then, slowly put the fish into the rearing tank and add 5-10 ppm of povidone-iodine for disinfection. The stocking density is 120-180 fish / tank. Do not feed for the first 3 days. Start daily rearing on the 4th day. Intensive rearing begins in March each year.

[0010] The daily rearing process involves feeding the fish once a day at 18:00 with crushed grass carp feed, which is approximately 2% to 3% of their body weight; the rearing tank has a diameter of 2m and a depth of 1m.

[0011] The intensive cultivation method is as follows: feeding once at 8:00 and 18:00 every day, with each feeding amount being 2-3% of the total amount of pond culture;

[0012] The induction of labor is as follows:

[0013] Mature female broodstock were injected with LHRH-A2 at a dose of 2.8–3.2 μg / kg, while mature male broodstock were not injected.

[0014] Ten hours later, mature female broodstock were injected with LHRH-A2 14.5–15.5 μg / kg + DOM 4.8–5.2 mg / kg + HCG 1900–2100 IU / kg, while mature male broodstock were injected with LHRH-A2 7–8 μg / kg + DOM 2.3–2.7 mg / kg + HCG 900–1100 IU / kg.

[0015] S3. A simulated habitat is provided for natural fertilization of broodstock fish awaiting spawning; the operation of the simulated habitat is as follows: male and female broodstock fish are mixed in a 3:2 ratio, the water temperature is controlled at 18-19℃, and the dissolved oxygen is 6-9 mg / L; fertilization is completed after 6-8 hours, and fertilized eggs and spawning broodstock fish are obtained; 16 hours after the second injection, the female broodstock fish are checked to see if they have completed spawning, and broodstock fish that have not spawned are removed;

[0016] S4. Artificial insemination is performed on broodstock that have not yet given birth to obtain fertilized eggs and post-birth broodstock. The artificial insemination is as follows: the female fish is squeezed to extract the eggs into a dry and clean container, and then the male fish is squeezed to extract the semen. The semen is then rinsed onto the eggs with a saline solution of 0.85-0.95% by mass, stirred, and a sperm-egg mixture is obtained. Then, 2-3 times the amount of water is added to activate the sperm. After stirring for 2-3 minutes, the mixture is rinsed 3-5 times with clean water to remove mucus and impurities, and fertilized eggs are obtained.

[0017] S5. Place the fertilized eggs in an incubation box for incubation to obtain fry;

[0018] S6. Raise fry to obtain young fish;

[0019] S7. Raise fry to obtain young fish.

[0020] Preferred criteria for classifying broodstock are as follows: female fish with a concave abdomen and obvious stretch marks on the back of the pectoral fins are postpartum female fish; male fish that can expel sperm and weigh more than 3g and female fish that can expel eggs and weigh more than 5g are mature broodstock; others are broodstock with their backs exposed.

[0021] Preferably, during the rearing stages of broodstock and postpartum female fish, water temperature, dissolved oxygen, pH, ammonia nitrogen, and nitrite levels are monitored daily, and water is changed every 3 days, with a single water change volume of 25-35%, to maintain good water quality.

[0022] Preferably, the domestication method for postpartum broodstock that undergo natural or artificial insemination is the same as the routine rearing conditions in S2.

[0023] Preferably, the incubation conditions are: a fertilized egg density of 0.8 to 10,000 eggs / m². 2 The water is exchanged 10-12 times per day, and the water temperature is 18-20℃; the larvae hatch in 84-90 hours.

[0024] Preferably, the rearing of fry is as follows: no feeding for 0-5 days after hatching; remove dead eggs and fry every morning and evening; overfeed brine shrimp once every 6 hours from 6 to 15 days; transfer the fry to plastic tanks 2m long, 1m wide, and 0.6m deep on 16 days, with independent inlet and outlet, and control the water depth at 0.4m, stocking 10,000 fry in each tank; overfeed brine shrimp once every 8 hours from 16 to 25 days; remove uneaten feed and feces 30 minutes after each feeding, and control the water exchange frequency to 5-7 times / day; juveniles are obtained on 26 days.

[0025] Preferably, the rearing of juvenile fish is as follows: at 27 days, the juvenile fish are transferred to a rearing tank with a diameter of 2m and a depth of 1m, and the water level is controlled at a depth of 0.6-0.7m, with a rearing density of 1500-2000 fish / tank; from 27 to 30 days, a mixture of brine shrimp and grass carp feed is fed once a day at 8:00 and 19:00, with a mixing ratio of 1:1; from 31 to 33 days, a mixture of brine shrimp and grass carp feed is fed once a day at 8:00 and 19:00, with a mixing ratio of 1:2; after 34 days, grass carp feed is fed once a day at 8:00 and 19:00; after 60 days, juvenile fish are obtained and domesticated according to the daily rearing method in S2.

[0026] By adopting the above technical solution, the present invention has the following beneficial effects:

[0027] (1) In this invention, classifying parent fish before artificial breeding can significantly improve breeding efficiency, save costs, and increase spawning rate and hatching rate.

[0028] (2) The spawning induced agent of the present invention is reasonably formulated and has an accurate effect time. When artificial spawning is carried out in Wu's smelt carp using this method, the male fish releases a large amount of sperm, the female fish has a high spawning rate, the produced eggs are of high quality, the fertilization rate is high, and the parent fish suffer little damage.

[0029] (3) This invention clarifies the feeding methods at different stages of seedling cultivation, thereby improving the survival rate of seedlings during the cultivation stage. Attached Figure Description

[0030] Figure 1 This is a flowchart illustrating the artificial breeding process of *Spatholobus suberectus*.

[0031] Figure 2 It is a mature male parent fish.

[0032] Figure 3 These are mature female parent fish.

[0033] Figure 4 This is a female fish after giving birth.

[0034] Figure 5 The Wu's pan-mouthed carp was fertilized naturally.

[0035] Figure 6 This refers to the hatching stage of fertilized eggs in *Symplocos wusiensis*.

[0036] Figure 7 Fry hatched from fertilized eggs of the Wu's flathead carp.

[0037] Figure 8 These are newly hatched fry of the Wu's flat-mouthed carp.

[0038] Figure 9 It is a small, juvenile Wu's pan-mouthed carp. Detailed Implementation

[0039] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example

[0041] From May 4th to May 6th, 2025, at 8:00 PM, fish traps were placed on a tributary of the Hongshui River in Fengshan County, Guangxi Zhuang Autonomous Region. The traps were retrieved at 7:00 AM the following morning, yielding 268 Wu's mullet. The collected Wu's mullet were categorized as follows: 83 females (postpartum) with concave abdomens and obvious stretch marks on the posterior side of their pectoral fins; 22 males weighing over 3g and capable of expelling sperm; 40 mature female broodstock weighing over 5g and capable of expelling eggs; and 123 other reserve broodstock. The Wu's mullet were packaged in 80cm x 40cm fry bags, with 40-60 Wu's mullet per bag. Each bag was filled with 1 / 3 river water and 2 / 3 pure oxygen, with the water changed and oxygen refilled every 24 hours. The Wu's mullet were then transported to the base.

[0042] Mature male and female parent fish were separated and temporarily housed in rectangular tanks measuring 80cm x 60cm x 40cm. The tanks were made of opaque material, continuously filled with oxygen, and not fed. The water used for temporary housing was river water from the original habitat, and the temperature was controlled to be the same as the original habitat (18-19℃). Each tank contained 40-60 mature parent fish of the Wu's Scale.

[0043] Replacement broodstock and postpartum females: Place the packing bag in a round plastic breeding tank with a diameter of 2m and a depth of 1m. Drain water from the center of the breeding tank and introduce water from the side. Control the water depth between 0.6 and 0.7m. After about 30 minutes, open the packing bag and slowly add water into the breeding tank until the packing bag is about 2 / 3 full. Then slowly put the fish into the breeding tank and add 8ppm povidone-iodine for disinfection. Breed in two breeding tanks, one with 100 fish and the other with 106 fish.

[0044] Routine rearing: Do not feed for the first 3 days. Starting from the 4th day, feed the fish once a day at 18:00 with grass carp feed crushed feed, which is about 3% of the fish's body weight. Monitor water temperature, dissolved oxygen, pH, ammonia nitrogen, nitrite and other relevant indicators daily. Change the water once every 3 days, with a single water change of about 1 / 3, to maintain good water quality.

[0045] Intensive rearing: Starting March 1, 2025, intensive rearing will be carried out on the replacement broodstock and post-spawning broodstock collected in 2024. Feeding will be conducted twice daily, at 8:00 AM and 6:00 PM, with each feeding amount being 3% of the total pond stock. Water will be flushed once daily at 5:00 PM. Water temperature, dissolved oxygen, pH, ammonia nitrogen, nitrite, and other relevant indicators will be monitored daily. Water will be changed every 3 days, with approximately 1 / 3 of the water replaced each time, to maintain good water quality.

[0046] (1) On May 7, 2025, five female and three male broodstock were selected from the collected mature broodstock for artificial spawning. First, the mature female broodstock were injected with LHRH-A2 3 μg / kg, while the mature male broodstock were not injected. Ten hours later, the mature female broodstock were injected with LHRH-A2 15 μg / kg + DOM 5 mg / kg + HCG 2000 IU / kg, while the mature male broodstock were injected with LHRH-A2 7.5 μg / kg + DOM 2.5 mg / kg + HCG 1000 IU / kg. The solvent was physiological saline, and the injection dose was 10 mL / kg. After injection, the male and female broodstock were placed in a dark-colored bucket with a diameter of 40cm and a depth of 40cm, with a water depth of 30cm. The bucket was covered with a damp towel to prevent the broodstock from jumping out. Oxygen was continuously introduced, and the water temperature was controlled at 18-19℃ and the dissolved oxygen level was 6-9mg / L. After about 6 hours, the female broodstock completed natural fertilization. After changing the water, the fertilized eggs were removed. The induced spawning rate was 100%, and 1100 fertilized eggs were obtained.

[0047] The fertilized eggs were evenly spread in a rectangular plastic frame 60cm long, 40cm wide, and 20cm deep. The plastic frame had 60-mesh holes on the sides and bottom, and the spreading density was 0.8 million eggs / m². 2 The water was exchanged 10 times a day, the water temperature was 19-20℃, the hatching time was 86 hours, the fertilization rate was 90.91%, the hatching rate was 90%, and 900 newly hatched fry were obtained.

[0048] Seedling cultivation was carried out. No feeding was given for 0-5 days after hatching. Dead eggs and fry were removed using a plastic straw every morning and evening. From 6 to 15 days, brine shrimp were overfed once every 6 hours. On 16 days, the fry were transferred to plastic tanks 2m long, 1m wide, and 0.6m deep, with independent and opposite inlets and outlets, and a water depth of 0.4m. 10,000 fry were stocked in each tank. From 16 to 25 days, brine shrimp were overfed once every 8 hours. Uneaten feed and feces were removed 30 minutes after each feeding. Water exchange was controlled at 6 times per day, with routine disinfection and monitoring. Fry were obtained on 26 days.

[0049] At 27 days, all fry were transferred to circular plastic rearing tanks with a diameter of 2m and a depth of 1m. The water level was maintained between 0.6m and 0.7m, with drainage from the bottom center and water intake from the sides. From 27 to 30 days, the fry were fed a mixture of brine shrimp and crushed grass carp feed at 8:00 AM and 7:00 PM daily, in a 1:1 ratio. From 31 to 33 days, the fry were fed a mixture of brine shrimp and crushed grass carp feed at 8:00 AM and 7:00 PM daily, in a 1:2 ratio. After 34 days, the fry were fed crushed grass carp feed at 8:00 AM and 7:00 PM daily. The survival rate of the fry was 83.33%.

[0050] (2) On May 10, 2025, 35 female and 19 male broodstock were selected from the collected mature broodstock for artificial spawning. First, mature female broodstock were injected with LHRH-A2 3 μg / kg, while mature male broodstock were not injected. 10 hours later, mature female broodstock were injected with LHRH-A2 15 μg / kg + DOM 5 mg / kg + HCG 2000 IU / kg, while mature male broodstock were injected with LHRH-A2 7.5 μg / kg + DOM 2.5 mg / kg + HCG 1000 IU / kg. The solvent was physiological saline, and the injection dose was 10 mL / kg. After injection, place the male and female broodstock in a dark-colored bucket with a diameter of 40cm and a depth of 40cm, with 5 fish in each bucket, a male-to-female ratio of 3:2, and a water depth of 30cm. Cover the bucket with a damp towel to prevent the broodstock from jumping out. Continuously circulate oxygen, control the water temperature at 18-19℃, and the dissolved oxygen level at 6-9mg / L. After about 6-8 hours, the female broodstock will complete natural fertilization. Then change the water and remove the fertilized eggs, obtaining 6000 fertilized eggs.

[0051] Seven female broodstock fish that had not yet laid eggs were examined. Artificial insemination was then performed 16 hours after the second injection. The female broodstock fish were dried with a towel, and the eggs were squeezed into a dry plastic basin. The male fish was then squeezed, and the semen was rinsed onto the eggs with 0.9% saline solution. The sperm and eggs were stirred evenly with a feather, and approximately three times the volume of water was added to activate the sperm. After stirring for 2 minutes, the mixture was rinsed five times with water to remove mucus and impurities, yielding 1500 fertilized eggs. The spawning induction rate was 100%, and a total of 7500 fertilized eggs were obtained.

[0052] The fertilized eggs were evenly spread in a rectangular plastic frame 60cm long, 40cm wide, and 20cm deep. The plastic frame had 60-mesh holes on the sides and bottom, and the spreading density was 0.8 million eggs / m². 2 The water was exchanged 12 times a day, the water temperature was 18-19℃, the hatching time was 84-86 hours, the fertilization rate was 93.33%, the hatching rate was 92.86%, and 6500 newly hatched fry were obtained.

[0053] Seedling cultivation was carried out. No feeding was given for 0-5 days after hatching. Dead eggs and fry were removed using a plastic straw every morning and evening. From 6 to 15 days, brine shrimp were overfed once every 6 hours. On 16 days, the fry were transferred to plastic tanks 2m long, 1m wide, and 0.6m deep, with independent and opposite inlets and outlets, and a water depth of 0.4m. 10,000 fry were stocked in each tank. From 16 to 25 days, brine shrimp were overfed once every 8 hours. Uneaten feed and feces were removed 30 minutes after each feeding. Water exchange was controlled at 6 times per day, with routine disinfection and monitoring. Fry were obtained on 26 days.

[0054] At 27 days, the fry were transferred to circular plastic rearing tanks with a diameter of 2m and a depth of 1m. The water level was maintained at 0.6-0.7m, with drainage from the bottom center and side intake. The stocking density of fry in each tank varied from 1500 to 2000 fry per tank. From 27 to 30 days, the fry were fed a mixture of brine shrimp and crushed grass carp feed at 8:00 and 19:00 daily, with a mixing ratio of 1:1. From 31 to 33 days, the fry were fed a mixture of brine shrimp and crushed grass carp feed at 8:00 and 19:00 daily, with a mixing ratio of 1:2. After 34 days, the fry were fed crushed grass carp feed at 8:00 and 19:00 daily. The survival rate of the fry was 95.38%.

[0055] (3) On May 13, 2025, 18 male fish weighing more than 3g and able to expel sperm and 19 female fish weighing more than 5g and able to expel eggs were selected from domesticated Wu's mullet collected in 2024 and artificially induced to spawn. First, the mature female broodstock were injected with LHRH-A2 3μg / kg, while the mature male broodstock were not injected. 10 hours later, the mature female broodstock were injected with LHRH-A2 15μg / kg + DOM 5mg / kg + HCG 2000IU / kg, while the mature male broodstock were injected with LHRH-A2 7.5μg / kg + DOM 2.5mg / kg + HCG 1000IU / kg. The solvent was physiological saline and the injection dose was 10mL / kg. After injection, place the male and female broodstock in a dark-colored bucket with a diameter of 40cm and a depth of 40cm, with 5 fish in each bucket, a male-to-female ratio of 3:2, and a water depth of 30cm. Cover the bucket with a damp towel to prevent the broodstock from jumping out. Continuously circulate oxygen, control the water temperature at 18-19℃, and the dissolved oxygen level at 6-9mg / L. After about 6-8 hours, the female broodstock will complete natural fertilization. After changing the water, remove the fertilized eggs, obtaining 3500 fertilized eggs.

[0056] Five female broodstock fish that had not yet laid eggs were examined. Artificial insemination was then performed 16 hours after the second injection. The female broodstock fish were dried with a towel, and the eggs were squeezed into a dry plastic basin. The male fish was then squeezed, and the semen was rinsed onto the eggs with 0.9% saline solution. The sperm and eggs were stirred evenly with a feather, and approximately three times the volume of water was added to activate the sperm. After stirring for 2 minutes, the mixture was rinsed five times with water to remove mucus and impurities, yielding 1200 fertilized eggs. The spawning induction rate was 100%, and a total of 4700 fertilized eggs were obtained.

[0057] The fertilized eggs were evenly spread in a rectangular plastic frame 60cm long, 40cm wide, and 20cm deep. The plastic frame had 60-mesh holes on the sides and bottom, and the spreading density was 0.8 million eggs / m². 2 The water was exchanged 12 times a day, the water temperature was 18-19℃, the hatching time was 84-87 hours, the fertilization rate was 95.74%, the hatching rate was 88.89%, and 4000 newly hatched fry were obtained.

[0058] Seedling cultivation was carried out. No feeding was given for 0-5 days after hatching. Dead eggs and fry were removed using a plastic straw every morning and evening. From 6 to 15 days, brine shrimp were overfed once every 6 hours. On 16 days, the fry were transferred to plastic tanks 2m long, 1m wide, and 0.6m deep, with independent and opposite inlets and outlets, and a water depth of 0.4m. 10,000 fry were stocked in each tank. From 16 to 25 days, brine shrimp were overfed once every 8 hours. Uneaten feed and feces were removed 30 minutes after each feeding. Water exchange was controlled at 6 times per day, with routine disinfection and monitoring. Fry were obtained on 26 days.

[0059] At 27 days, the fry were transferred to circular plastic rearing tanks with a diameter of 2m and a depth of 1m. The water level was maintained at 0.6-0.7m, with drainage from the bottom center and side inlet. The stocking density of fry in each tank varied from 1500 to 2000 fry per tank. From 27 to 30 days, the fry were fed a mixture of brine shrimp and crushed grass carp feed at 8:00 and 19:00 daily, with a mixing ratio of 1:1. From 31 to 33 days, the fry were fed a mixture of brine shrimp and crushed grass carp feed at 8:00 and 19:00 daily, with a mixing ratio of 1:2. After 34 days, the fry were fed crushed grass carp feed at 8:00 and 19:00 daily. The survival rate of the fry was 95%.

[0060] Comparative Example

[0061] On May 7, 2024, at 8:00 PM, fish traps were placed on a tributary of the Hongshui River in Fengshan County, Guangxi Zhuang Autonomous Region. The traps were retrieved at 7:00 AM on May 8, yielding 50 Wu's flathead carp. The collected Wu's flathead carp were categorized as follows: 0 females with concave abdomens and obvious stretch marks behind their pectoral fins (postpartum); 13 males weighing over 3g and capable of expelling sperm; 19 mature females weighing over 5g and capable of expelling eggs; and 18 other potential broodstock. The fish were packaged in 80cm x 40cm fry bags, filled with 1 / 3 river water and 2 / 3 pure oxygen, with water changed and oxygen replenished every 24 hours. The Wu's flathead carp were then transported to the base.

[0062] After separating the mature male and female parent fish, they were temporarily housed in two rectangular tanks with dimensions of 80cm x 60cm x 40cm. The tanks were made of opaque material and continuously filled with oxygen to ensure a dissolved oxygen level of 6-9 mg / L. No feeding was provided. The water used for temporary housing was river water from the original habitat, and the temperature was controlled to be the same as that of the original habitat (18-19℃).

[0063] For replacement broodstock and postpartum females: Place the packaged bag in a circular plastic rearing tank with a diameter of 2m and a depth of 1m. Drain water from the center of the tank and allow water to enter from the side, maintaining a water depth of 0.6-0.7m. After about 25 minutes, open the packaged bag and slowly add water to the rearing tank until the bag is about 2 / 3 full. Then, slowly place the fish into the rearing tank. Add 7ppm povidone-iodine to the tank for disinfection. Do not feed for the first 3 days. Starting from the 4th day, feed the fish once a day at 18:00 with crushed grass carp feed, which is about 2%-3% of their body weight. Monitor water temperature, dissolved oxygen, pH, ammonia nitrogen, nitrite, and other relevant indicators daily. Change the water every 3 days, replacing about 1 / 3 of the water each time, to maintain good water quality.

[0064] (1) On May 9, 2024, five female fish and five male fish were selected from the collected mature parent fish for artificial spawning. A single injection of LHRH-A into mature female broodstock 2 10 μg / kg + DOM 5 mg / kg, simultaneously inject mature male broodstock with LHRH- A 2 5μg / kg + DOM 2.5mg / kgThe solvent was physiological saline, and the injection dose was 10 mL / kg. After injection, the male and female broodstock were placed in two separate dark-colored buckets, each 40 cm in diameter and 40 cm deep, with a water depth of 30 cm. The buckets were covered with damp towels to prevent the broodstock from jumping out. Oxygen was continuously supplied. After approximately 12 hours, if the female broodstock had not yet laid eggs, artificial dry insemination was performed. The female broodstock was dried with a dry towel, and the eggs were squeezed into a dry plastic basin. The male broodstock was then squeezed, and the semen was rinsed onto the eggs with 0.9% physiological saline. The sperm and eggs were stirred evenly with a feather, and approximately twice the volume of water was added to activate the sperm. The mixture was stirred continuously for 3 minutes, and then rinsed three times with clean water to remove mucus and impurities. Two female broodstock fish laid eggs, totaling 500 eggs, none of which were hatched. Sperm; 0 females survived giving birth, 2 males survived. .

[0065] Analysis: During this artificial breeding process, it was found that the female fish had difficulty ovulating, and the eggs produced were of uneven size and poor quality, resulting in unfertilized eggs. This was due to their low sensitivity to spawning stimulants, insufficient dosage of spawning stimulants, and inappropriate formulation of spawning stimulants.

[0066] (2) On May 10, 2024, 7 female fish and 5 male fish were selected from the collected mature parent fish for artificial spawning. A single injection of LHRH-A into mature female broodstock 2 15 μg / kg + DOM 8 mg / kg + HCG 2000 IU / kg, simultaneously affecting mature males LHRH-A injection in broodstock 2 7.5μg / kg+DOM4mg / kg+HCG1000IU / kg The solvent was physiological saline, and the injection dose was 10 mL / kg. After injection, both male and female broodstock were placed in a dark-colored bucket (40 cm in diameter, 40 cm deep, 30 cm deep) with a wet towel covering the bucket to prevent them from jumping out. Oxygen was continuously supplied. Artificial insemination was performed approximately 12 hours later. The female broodstock was dried with a dry towel, and the eggs were squeezed into a dry plastic basin. The male was then squeezed, and the semen was rinsed onto the eggs with 0.9% physiological saline. The sperm and eggs were stirred evenly with a feather, and approximately twice the volume of water was added to activate the sperm. After stirring for 3 minutes, the mixture was rinsed three times with water to remove mucus and impurities. A total of 5 fish spawned, yielding 1000 fertilized eggs. The fertilized eggs were evenly spread in a rectangular plastic frame (60 cm long, 40 cm wide, 20 cm deep) with 60-mesh holes on the sides and bottom, at a density of 10,000 eggs / m². 2 The water exchange frequency is 10 times per day, of which The fertilized eggs died one after another 12 hours later, and no newborn fry were produced. Zero female fish survived giving birth, while two male fish survived. .

[0067] Analysis: It is possible that the water temperature at the breeding base (27-28℃) was much higher than that in the original habitat (18-19℃), which led to the termination of embryonic development.

[0068] (3) On May 13, 2024, 7 female fish and 3 male fish were selected from the collected mature parent fish for artificial spawning. A single injection of LHRH-A into mature female broodstock 2 15 μg / kg + DOM 10 mg / kg + HCG 4000 IU / kg, simultaneously affecting mature males LHRH-A injection in broodstock2 7.5μg / kg+DOM5mg / kg+HCG2000IU / kg The solvent was physiological saline, and the injection dose was 10 mL / kg. After injection, both male and female broodstock were placed in a dark-colored bucket with a diameter of 40 cm and a depth of 40 cm, with a water depth of 30 cm. The bucket was covered with a damp towel to prevent the broodstock from jumping out. Oxygen was continuously supplied, and the water temperature was controlled at 18-19℃. After about 7 hours, the broodstock completed natural fertilization, yielding 600 fertilized eggs. Four female broodstock were found not to have laid eggs. Artificial insemination was carried out 16 hours after the second injection. The female broodstock were dried with a dry towel, and the eggs were squeezed into a dry plastic basin. Then, the male was squeezed, and the semen was rinsed onto the eggs with 0.9% physiological saline. The sperm and eggs were stirred evenly with a feather, and about three times the volume of water was added to activate the sperm. After stirring continuously for 2 minutes, the mixture was rinsed 5 times with water to remove mucus and impurities, yielding 800 fertilized eggs, with a 100% induced spawning rate. The fertilized eggs were evenly spread in a rectangular plastic frame 60cm long, 40cm wide, and 20cm deep. The plastic frame had 60-mesh holes on the sides and bottom, and the spreading density was 0.8 million eggs / m². 2 The water exchange frequency is 12 times / day, and the water temperature is controlled at 18-19℃. 500 seeds did not absorb water and swelled. After 90 hours, they hatched, with a fertilization rate of 16.66% and a hatching rate of 66.67%, yielding newly hatched eggs. 100 fry were born. Four females and two males survived after giving birth. .

[0069] Analysis: Due to the short duration of the spawning-inducing effect from a single injection, the eggs produced were not mature, resulting in a low fertilization rate in artificial breeding. Secondly, because the broodstock of *Siniperca wuscheriensis* are relatively small, averaging about 6 cm in length, the artificial insemination process caused significant damage to the broodstock, leading to a low survival rate after spawning.

[0070] Seedling cultivation was carried out. No feeding was given for 0-5 days after hatching. Dead eggs and fry were removed daily morning and evening using a plastic straw. From 6 to 15 days, the fish were overfed with brine shrimp every 6 hours. On 16 days, the fry were transferred to plastic tanks 2m long, 1m wide, and 0.6m deep, with independent and opposite inlets and outlets, maintaining a water depth of 0.4m. 10,000 fish were stocked in each tank. From 16 to 25 days, the fish were overfed with brine shrimp every 8 hours. Uneaten food and feces were removed 30 minutes after each feeding. Water exchange was maintained at 6 times per day, with routine disinfection and monitoring. The seedling survival rate was 63%, and 63 juveniles were obtained after 26 days.

[0071] At 27 days, all juveniles were transferred to circular plastic rearing tanks with a diameter of 2m and a depth of 1m. The water level was maintained between 0.6m and 0.7m, with drainage from the bottom center and water intake from the sides. From 27 to 30 days, the fish were fed a mixture of brine shrimp and crushed grass carp feed at 8:00 AM and 7:00 PM daily, in a 1:1 ratio. From 31 to 33 days, the fish were fed a mixture of brine shrimp and crushed grass carp feed at 8:00 AM and 7:00 PM daily, in a 1:2 ratio. After 34 days, the fish were fed crushed grass carp feed at 8:00 AM and 7:00 PM daily. After 60 days, 63 juvenile fish were obtained, and they were subsequently raised using the same methods as the broodstock.

[0072] Table 1. Comparative Examples and Implementation Examples: Results of Artificial Propagation and Seedling Cultivation of *Spatholobus suberectus*

[0073]

[0074] Comparative Examples 1, 2, and 3 show that increasing the dosage of the spawning induced agent can improve the spawning rate. The comparative examples and implementation examples also show that using a second injection of spawning induced agent can improve the quality of spawning in female fish and increase the fertilization rate. Comparative Examples 2 and 3 show that in Comparative Example 2, the water temperature was too high, causing the fertilized egg development to terminate. In Comparative Example 3, by controlling the water temperature to be the same as the habitat water temperature, the complete development of the fertilized egg embryo was ensured, and the fertilized egg hatched. Furthermore, the comparative examples and implementation examples show that using only natural fertilization results in incomplete spawning in the female fish awaiting spawning. Since *Litopenaeus vannamei* is relatively small, artificial insemination alone is very harmful to the parent fish. Therefore, this invention combines natural and artificial insemination, and specifies the timing, which not only improves the spawning rate but also reduces the loss of parent fish. Examples 2 and 3 illustrate the artificial breeding method for *Litopenaeus vannamei* proposed in this invention, which combines natural and artificial insemination. This method enables the artificial breeding of *Litopenaeus vannamei*, can guide large-scale production, and provides technical guidance for the conservation of this species.

[0075] The embodiments of the present invention improve the quality of produced eggs and the physical condition of larvae by optimizing and improving the compatibility and effect time of spawning agents. The fertilization rate and fry survival rate are significantly higher than those of the comparative example.

[0076] As can be seen from the above embodiments, the present invention provides an artificial breeding method for *Spathithecus wuys*. The method of the present invention significantly improves the spawning rate and hatching rate of *Spathithecus wuys*.

[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for the artificial breeding of *Spathithecus wusiensis*, characterized in that, Includes the following steps: S1. During the breeding season of the Wu's flathead carp, the parent fish are collected and divided into mature parent fish, reserve parent fish and postpartum female fish; S2. Induce spawning in mature parent fish, and raise replacement parent fish and postpartum female fish separately until they develop into mature parent fish before inducing spawning again; The domestication of replacement broodstock and postpartum females is as follows: After placing the packaged bag in the rearing tank for 25-30 minutes, open the packaged bag and slowly add water to the rearing tank until the water occupies 60-70% of the volume of the packaged bag. Then, slowly put the fish into the rearing tank and add 5-10 ppm of povidone-iodine for disinfection. The stocking density is 120-180 fish / tank. Do not feed for the first 3 days. Start daily rearing on the 4th day. Intensive rearing begins in March each year. The daily rearing process involves feeding the fish once a day at 18:00 with crushed grass carp feed equal to 2% to 3% of their body weight; the rearing tank has a diameter of 2m and a depth of 1m. The enhanced cultivation method is as follows: feeding once at 8:00 and 18:00 every day, with each feeding amount being 2-3% of the total amount of pond culture; The induced spawning process involves injecting mature female broodstock with LHRH-A2 at 2.8~3.2 μg / kg, while mature male broodstock are not injected. Ten hours later, mature female broodstock were injected with LHRH-A2 14.5~15.5μg / kg + DOM 4.8~5.2mg / kg + HCG 1900~2100IU / kg, while mature male broodstock were injected with LHRH-A2 7~8μg / kg + DOM 2.3~2.7mg / kg + HCG 900~1100IU / kg. S3. Simulated habitat for natural fertilization of broodstock fish awaiting spawning; the operation of the simulated habitat is as follows: male and female broodstock fish are mixed in a 3:2 ratio, the water temperature is controlled at 18~19℃, and the dissolved oxygen is 6~9mg / L; fertilization is completed after 6~8 hours, and fertilized eggs and post-spawning broodstock fish are obtained; Sixteen hours after the second injection, check whether the female parent fish have completed spawning and remove any that have not yet spawned. S4. Artificial insemination is performed on broodstock that have not yet given birth to obtain fertilized eggs and post-birth broodstock. The artificial insemination is performed as follows: the female fish is squeezed to extract the eggs into a dry and clean container, and then the male fish is squeezed to extract the semen. The semen is then rinsed onto the eggs with a saline solution of 0.85-0.95% by mass, stirred, and a sperm-egg mixture is obtained. Then, 2-3 times the amount of water is added to activate the sperm. After stirring for 2-3 minutes, the mixture is rinsed 3-5 times with clean water to remove mucus and impurities, and fertilized eggs are obtained. S5. Place the fertilized eggs in an incubation box for incubation to obtain fry; S6. Raise fry to obtain young fish; S7. Raise fry to obtain young fish; The incubation conditions are as follows: the density of fertilized eggs is 0.800 to 10,000 per square meter. 2 The water is exchanged 10-12 times per day, and the water temperature is 18-20℃; the larvae hatch in 84-90 hours.

2. The artificial breeding method according to claim 1, characterized in that, The classification criteria for broodstock are as follows: female fish with a concave abdomen and obvious stretch marks on the back of the pectoral fins are postpartum female fish; male fish that can squeeze out sperm and weigh more than 3g and female fish that can squeeze out eggs and weigh more than 5g are mature broodstock; others are broodstock with their backs exposed.

3. The artificial breeding method according to claim 1, characterized in that, During the rearing of broodstock and postpartum females, water temperature, dissolved oxygen, pH, ammonia nitrogen, and nitrite levels are monitored daily. Water is changed every 3 days, with a single water change volume of 25-35%, to maintain good water quality.

4. The artificial breeding method according to claim 1, characterized in that, The domestication methods for postpartum broodstock that undergo natural or artificial insemination are the same as the routine rearing conditions in S2.

5. The artificial breeding method according to claim 1, characterized in that, The rearing process for larvae is as follows: no feeding for 0-5 days after hatching; remove dead eggs and fry every morning and evening; overfeed brine shrimp once every 6 hours from 6 to 15 days; transfer the larvae to plastic tanks 2m long, 1m wide, and 0.6m deep on day 16, with independent inlet and outlet, and maintain a water depth of 0.4m, stocking 10,000 larvae in each tank; overfeed brine shrimp once every 8 hours from day 16 to 25; remove uneaten feed and feces 30 minutes after each feeding, and maintain a water exchange frequency of 5-7 times per day; juveniles are obtained on day 26.

6. The artificial breeding method according to claim 1, characterized in that, The juvenile fish were raised as follows: at 27 days, the juveniles were transferred to rearing tanks with a diameter of 2m and a depth of 1m, with the water level controlled at a depth of 0.6-0.7m and a stocking density of 1500-2000 fish per tank; from 27 to 30 days, they were fed a mixture of brine shrimp and crushed grass carp feed at 8:00 and 19:00 daily, with a mixing ratio of 1:1; from 31 to 33 days, they were fed a mixture of brine shrimp and crushed grass carp feed at 8:00 and 19:00 daily, with a mixing ratio of 1:2; after 34 days, they were fed crushed grass carp feed at 8:00 and 19:00 daily; after 60 days, the fry were obtained and domesticated according to the daily rearing method of S2.

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