Artificial breeding method of Lancang schizothorax

Abstract

This invention discloses an artificial breeding method for *Schizothorax lancensis*, belonging to the field of aquatic animal breeding technology. The artificial breeding method for *Schizothorax lancensis* provided by this invention includes: intensive three-step cultivation of broodstock, injection of spawning induced agents after cultivation, artificial fertilization, hatching of fertilized eggs, and fry cultivation. Using the artificial breeding method of this invention, the success rate of spawning induction in female *Schizothorax lancensis* is 83.3%, the average fertilization rate is 87.5%, the average hatching rate is 93.0%, and the average fry emergence rate is 91.5%. This achieves large-scale artificial breeding of *Schizothorax lancensis*, laying the foundation for preventing the extinction of *Schizothorax lancensis*, and for carrying out the propagation and release of *Schizothorax lancensis* for industrial utilization.

Description

Technical Field

[0001] This invention relates to the field of aquatic animal breeding and propagation technology, specifically to a method for artificial breeding of Lancang schizothorax. Background Technology

[0002] The Lancang schizothorax (scientific name: *Schizothorax lantsangensis*) is a species of fish belonging to the Cyprinidae family and the *Schizothorax* genus. It is a bottom-dwelling fish found in high-altitude mountainous regions, primarily feeding on benthic invertebrate larvae (chironomid larvae) and aquatic insects, while also consuming diatoms and plant debris. The Lancang schizothorax is distributed in the upper and middle reaches of the Lancang River system in China, including Qinghai, Tibet, and Yunnan provinces. Due to its relatively narrow distribution area and extremely limited natural populations, coupled with its slow growth and late sexual maturity in fast-flowing waters, artificial domestication and breeding are extremely difficult.

[0003] Currently, in the breeding process of Lancang schizothorax, existing technologies typically provide broodstock with abundant feed during the rearing process, but neglect the influence of other living conditions. This results in broodstock becoming very plump, but their gonadal development is somewhat inhibited, which is detrimental to artificial breeding and significantly affects the fertilization rate, hatching rate, and larval emergence rate in the artificial breeding of Lancang schizothorax. Therefore, how to develop a method that can promote the gonadal maturation of Lancang schizothorax broodstock and improve the fertilization rate, hatching rate, and larval emergence rate during artificial breeding has become an urgent technical problem to be solved in this field. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the purpose of this invention is to provide an artificial breeding method for Lancang schizothorax, which can significantly improve the gonadal maturation rate, artificial fertilization rate, hatching rate and seedling emergence rate of Lancang schizothorax parent fish.

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

[0006] This invention provides a method for artificial breeding of Lancang schizothorax, comprising the following steps:

[0007] The parent fish undergo a three-step intensive breeding process: after breeding, they are injected with spawning stimulants, and then artificial insemination, fertilized egg hatching, and fry rearing are carried out.

[0008] The three-step intensive breeding process includes: Step 1: Feed the broodstock mealworms once a day and sturgeon pellet sinking compound feed with a protein content of more than 45% twice a day; Step 2: Feed the broodstock mealworms twice a day and sturgeon pellet sinking compound feed with a protein content of more than 45% once a day; Step 3: Feed the broodstock mealworms three times a day and supplement with light at night.

[0009] Preferably, the flow rate in the enhanced cultivation step one is 25-35 L / min, and the cultivation time is 14-16 days.

[0010] Preferably, the flow rate in the enhanced cultivation step two is 25-35 L / min, and the cultivation time is 28-32 days.

[0011] Preferably, in the enhanced cultivation step three, the water flow rate is 30-45 L / min, the light intensity is 800-1200 LX, the light duration is 2-4 h, and the cultivation time is 15-30 days.

[0012] Preferably, during the "three-step" intensive breeding process, the amount of feed or feed given each time is 3 to 5% of the fish's body weight.

[0013] Preferably, female fish are injected with 4 mg DOM + 8 μg LRH-A2 per kg of body weight as an oxytocin agent.

[0014] Preferably, the injection dose of spawning induced agent in male fish is 30-35% of the injection dose in female fish.

[0015] Preferably, the artificial insemination method includes dry insemination.

[0016] Preferably, deionized water is added during the artificial insemination process to activate sperm.

[0017] Preferably, the amount of deionized water added is 2 to 4 times the volume of the mixture of fish eggs and fish semen.

[0018] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows:

[0019] This invention employs a three-step enhanced breeding method to gradually increase the nutritional supply to the parent fish, which effectively promotes the maturation of the parent fish's gonads, ensures the quality of eggs and sperm, and improves the fertilization rate during artificial insemination. Furthermore, the resulting fertilized eggs are of high quality, which can significantly improve the hatching and fry emergence rates during the artificial breeding of Lancang schizothorax, thus facilitating the large-scale artificial breeding of Lancang schizothorax. Attached Figure Description

[0020] Figure 1 : Phenomorphic characteristics of embryonic development process in Lancang schizothorax;

[0021] Figure 2 The relationship between the age of Lancang schizothorax larvae and their total length and weight. Detailed Implementation

[0022] This invention provides a method for artificial breeding of Lancang schizothorax, comprising the following steps:

[0023] The parent fish undergo a three-step intensive breeding process: after breeding, they are injected with spawning stimulants, and then artificial insemination, fertilized egg hatching, and fry rearing are carried out.

[0024] The three-step intensive breeding process includes: Step 1: Feed the broodstock mealworms once a day and sturgeon pellet sinking compound feed with a protein content of more than 45% twice a day; Step 2: Feed the broodstock mealworms twice a day and sturgeon pellet sinking compound feed with a protein content of more than 45% once a day; Step 3: Feed the broodstock mealworms three times a day and supplement with light at night.

[0025] In this invention, the broodstock used for the artificial breeding of *Schizothorax laniceps* are preferably wild broodstock obtained from the middle and upper reaches of the Lancang River. As an optional implementation, the broodstock are disinfected with potassium permanganate after capture, and before loading, all surfaces of the fish are coated with miconazole nitrate cream or compound ketoconazole ointment. They are then placed in fish fry bags filled with oxygen and transported to the breeding site, thus becoming the broodstock used for artificial breeding.

[0026] This invention involves temporarily holding the captured broodstock. As an optional implementation method, the broodstock are treated with 4-5 million units / m³ of [unclear - possibly referring to a specific drug or treatment]. 3 After soaking in penicillin for 30-40 minutes, the fish are temporarily placed in a holding tank. The holding tank is preferably pre-cleaned with quicklime and soaked in 25-30 ppm potassium permanganate for one day, then filled with clean water and aerated for 1-2 days. Afterward, water intake is stopped, and the tank is disinfected with 2-4% salt for 2-4 hours. The water temperature in the holding tank is 13-17℃, dissolved oxygen is maintained above 6 mg / L, and the pH is 7-9. After the broodstock are placed in the holding tank, they are disinfected daily with 3% salt, and injured fish are treated with miconazole nitrate cream, compound ketoconazole ointment, or erythromycin ointment. Nutrient-rich feed is provided during this period.

[0027] This invention selects uninjured and robust broodstock from a temporary holding tank and then performs intensive cultivation. Preferably, the cultivation tank in this invention has a fixed feeding platform. This invention employs a three-step intensive cultivation method for the broodstock. During this intensive cultivation, the dissolved oxygen level is maintained above 6 mg / L, and the pH value is 7–9, preferably 8.

[0028] The "three-step" enhanced cultivation method described in this invention includes:

[0029] In the first step of intensive cultivation, mealworms are fed once, and sturgeon pelleted sinking feed with a protein content of over 45% is fed twice; the water flow rate is 25-35 L / min. This intensive cultivation step lasts for 14-16 days (mid-December to the end of December), preferably 15 days; the cultivation water temperature is 13-17℃; the feeding order of mealworms and sturgeon pelleted sinking feed is to feed mealworms in the morning, and feed the feed at noon and in the evening; the preferred water flow rate is 26-30 L / min.

[0030] In the second step of intensive cultivation, mealworms are fed twice, and sturgeon pelleted sinking feed with a protein content of over 45% is fed once; the water flow rate is 25-35 L / min. This intensive cultivation step lasts 28-32 days (early January to the beginning of spring), preferably 29-31 days, more preferably 30 days, with a cultivation water temperature of 13-17℃. The preferred feeding order for mealworms and sturgeon pelleted sinking feed is: mealworms in the morning and evening, and feed at noon; the preferred water flow rate is 30-32 L / min. During this intensive cultivation step, the pond is inspected 2-3 times daily, and each time the water temperature, pH, and dissolved oxygen are measured, and the water quality, fish swimming, and feeding behavior are observed.

[0031] In the third step of the intensive cultivation process, the feed is mealworms, which are fed three times a day: in the morning, at noon, and in the evening. The water flow rate is 30–45 L / min, the light intensity is 800–1200 LX, and the light duration is 2–4 hours at night. This intensive cultivation step lasts for 15–30 days. The preferred water flow rate is 45 L / min, the preferred light intensity is 900–1000 LX, and the preferred light duration is 3 hours.

[0032] In the enhanced breeding process of this invention, the amount of feed given each time is 3-5% of the fish's body weight, preferably 4%. This invention does not limit the source of mealworms and sturgeon pelleted sinking compound feed with a protein content of more than 45%.

[0033] This invention employs a three-step enhanced breeding method, gradually increasing the feeding of nutrients and the intensity of flowing water stimulation. This effectively promotes the maturation of the gonads of the parent fish, ensures the quality of eggs and sperm, and improves the fertilization rate during artificial insemination. Consequently, it can increase the hatching and fry emergence rates during the artificial breeding of Lancang schizothorax.

[0034] When the male fish's genital opening is pointed and a milky white, viscous semen flows out when the abdomen is gently pressed, and the female fish's abdomen is swollen and soft with a large genital opening, artificial spawning is induced in this invention. The preferred method of spawning induction is to use an artificial intraperitoneal injection of a spawning stimulant. After injection, the parent fish are placed in the same spawning pond with water flow stimulation to wait for spawning. The water flow rate is preferably 44-46 L / min, more preferably 45 L / min.

[0035] In this invention, female fish are injected intraperitoneally with 4 mg DOM + 8 μg LRH-A2 per kg of body weight, while the male fish receive 30-35% of the female's dose of oxytocin, preferably one-third of the female's dose. By controlling the ratio of the male to female oxytocin injections, this invention ensures that the oxytocin effect time is consistent for both fish, thereby effectively improving the fertilization rate. Preferably, the oxytocin injection is administered after 6:00 PM.

[0036] The present invention uses physiological saline to prepare the above-mentioned spawning agent, wherein the amount of physiological saline is 5-7‰ of the fish's body weight, preferably 6‰.

[0037] This invention involves examining the parent fish within 24-48 hours of induced spawning. When the female fish's abdomen is gently pressed and eggs are easily extruded, appearing plump, uniform in size, and dispersed, artificial fertilization is performed in a dark and spacious location. The preferred method of artificial fertilization is dry insemination. During dry insemination, the female fish is dried, wrapped in a pre-sterilized damp towel with the abdomen exposed, and the eggs are squeezed into a pre-sterilized container. The same procedure is performed to squeeze the male fish's semen into the container containing the eggs. The female-to-male ratio of the parent fish is 1:2-4, preferably 1:3. After the eggs and semen are squeezed into the container, they are rapidly mixed with a feather within 30 seconds.

[0038] This invention involves adding deionized water to activate sperm during artificial insemination. The fish eggs and semen are rapidly mixed with a feather, and then deionized water is directly added to activate the sperm. The amount of deionized water added is 2 to 4 times the volume of the fish egg and semen mixture, preferably 3 times. After adding the deionized water, stirring continues for 2 to 3 minutes to allow the sperm and eggs to fully contact and fuse. Then, the mixture is rinsed 4 to 5 times with deionized water to remove broken eggs and impurities. The mixture is then allowed to stand for 12 to 20 minutes, and the number of fertilized eggs is counted using a volumetric method. This invention activates sperm with deionized water, which contains no substances other than water and does not affect the osmotic pressure of the sperm, thus improving the fertilization rate.

[0039] This invention relates to the incubation of fertilized eggs. As an optional implementation, the fertilized eggs are slowly and systematically poured into a Yushchenko incubator for incubation. Two days before using the Yushchenko incubator, the incubation frames are soaked in a 100ml / L potassium permanganate solution for 11-13 hours, followed by soaking in a 5-10% salt solution for 11-13 hours. Then, they are dried in an oven or exposed to sunlight for one day. During incubation, each incubation frame contains 10,000-15,000 fertilized eggs. A slight flow of water is maintained at a depth of 18-22cm and a flow rate of 24-26L / min. The Yushchenko incubator automatically turns the eggs once every 5 minutes. The water temperature is maintained at 14-16℃, and the pH value at 8.0-9.0. During incubation, dead eggs, moldy eggs, and debris are promptly removed from the incubation frames, and the number of dead eggs is counted to calculate the fertilization rate. The fertilized eggs begin to hatch after 7-15 days of incubation.

[0040] This invention relates to the cultivation of fish fry. After hatching, the fry continue to be cultivated in incubation frames, maintaining a continuous flow of water at a depth of 14–16 cm, a flow rate of 18–22 L / min, and a water temperature of 14–16 °C. No feed is provided; the fry rest on the bottom of the incubation frames of the Yusenko incubator, fully absorbing and utilizing the pale yellow yolk sac. Six to eight days after hatching, the fry begin to swim horizontally and start feeding. The preferred initial feed is live food with high protein content. They are then transferred to fry rearing ponds with a diameter of 280–320 cm, a water depth of 28–32 cm, a water temperature of 15–16 °C, and a pH of 8–9 at a stocking density of 10,000 fry per pond.

[0041] During the fry rearing process, the water flow rate at the inlet of the rearing pond should be 24-26 L / min. Feed the fry with brine nauplius or rotifers disinfected with salt water three times a day, with each pond receiving 50-80 mL of feed daily. Once the fry reach 2 cm in length, start feeding them with Type 48 feed specifically for fish and shrimp fry rearing, three times a day, at a rate of 5-8% of the fry's body weight. Observe the condition of the fry by patrolling the pond every morning. If any abnormalities are found, immediately examine the fish under a microscope. If any fish disease is found, isolate and treat them promptly.

[0042] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0043] Unless otherwise specified, the following embodiments are all conventional methods.

[0044] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0045] In a specific embodiment of the present invention, the parent fish were caught in the Tu'e-Zhongpai section of the Lancang River; the yellow mealworms were purchased from the Yellow Mealworm Ecological Farm in Dezhou, Shandong; the sturgeon pellet sinking compound feed with a protein content of more than 45% was purchased from Tongwei Feed Co., Ltd.; DOM and LRH-A2 were purchased from Alayunyu Fishery Materials Business Department in Kunming Economic and Technological Development Zone; and Tianbang Fish and Shrimp Seedling Breeding Special Type 48 Feed was purchased from Tianbang Aquatic Feed Co., Ltd.

[0046] Example 1

[0047] This embodiment provides a method for artificial breeding of Lancang schizothorax:

[0048] 1. Harvesting and Temporary Holding of Broodstock: Wild broodstock were obtained from the middle and upper reaches of the Lancang River in November to early December. After harvesting, the broodstock were disinfected with 20 ppm potassium permanganate for 15 minutes. Before loading, all fish were coated with miconazole nitrate cream, placed in fish fry bags filled with oxygen, and transported back to the experimental base. Upon arrival at the base, the fish were treated with 4.5 million units / m³ of oxygen. 3 After soaking in penicillin for 35 minutes, the animals were temporarily kept in a holding tank.

[0049] The temporary holding tank was cleaned with quicklime and soaked in 28ppm potassium permanganate for 1 day beforehand. After filling it with clean water, it was aerated for 1 day. Then, the water intake was stopped, and the tank was disinfected with 3% salt for 3 hours. The water temperature in the temporary holding tank was 15℃, the dissolved oxygen was maintained above 6mg / L, and the pH value was 8. After the fish were placed in the temporary holding tank, the tank was disinfected with 3% salt every day, and the injured fish were treated with miconazole nitrate cream. During this period, the fish were fed with mealworms as a nutritious food.

[0050] 2. Selection of broodstock and three-step intensive rearing: In mid-December, select healthy and well-proportioned broodstock from the temporary holding tank for intensive rearing. The water temperature, dissolved oxygen, and pH value of the rearing tank are the same as in step 1; a fixed feeding platform is maintained in the rearing tank.

[0051] Intensive rearing 1: From mid-December to the end of December, a total of 15 days, the selected parent fish were fed 3 times a day, once with mealworms and twice with sturgeon pellet sinking compound feed with a protein content of more than 45%. The feeding order was: feed mealworms at 8:30 am, feed feed at 12:30 pm and 6:00 pm, and feed feed at 4% of the fish's body weight each time. At the same time, the fish were stimulated with flowing water at a flow rate of 35 L / min.

[0052] Intensive Cultivation 2: Starting in early January, for a total of 30 days, change the feeding method. Feed mealworms twice a day and sturgeon pellet sinking compound feed with a protein content of more than 45% once a day. Feed mealworms at 8:30 am and 6:00 pm, and feed at 12:30 pm. The amount of feed is 4% of the fish's body weight. Patrol the pond 2-3 times a day, measuring water temperature, pH value and dissolved oxygen each time, and observing water quality, fish swimming and feeding. At the same time, stimulate with flowing water at a flow rate of 35L / min.

[0053] Enhanced Cultivation 3: Cultivate for 15-30 days after the beginning of spring, strengthen the feeding of nutritious feed, feed yellow mealworms 3 times a day, the amount of feed is 4% of the fish's body weight, the feeding time is 8:30 am, 12:30 pm and 6:00 pm respectively, while ensuring nutrition, increase the water flow stimulation, the flow rate is 45L / min, and add 3 hours of light stimulation every night, the light intensity is 1000LX. During this period, the pond temperature needs to be kept constant at 15-16℃ to promote the development of the broodstock's gonads.

[0054] 3. Artificial spawning induction: When the male fish's genital opening is pointed and a milky white, viscous semen flows out when the abdomen is gently pressed, and the female fish's abdomen is swollen and soft with a large genital opening, artificial spawning induction is performed. Spawning induction is performed by intraperitoneal injection of spawning stimulant at around 6:30 pm, with a water temperature of 15-16℃. For female fish, inject 4mg DOM + 8μg LRH-A2 intraperitoneally per kg of body weight. For male fish, reduce the injection dose to 1 / 3. Prepare the above spawning stimulant with physiological saline, with the amount of physiological saline being 6‰ of the fish's body weight. After injection, place the parent fish in the same spawning pond with water flow stimulation, with a flow rate of 45L / min, to await spawning.

[0055] 4. Artificial Insemination: Within 24-48 hours of induced spawning, examine the parent fish. When the female fish can easily expel eggs by gently pressing her abdomen, and the eggs are plump, uniform in size, and dispersed, choose a dark and spacious place for artificial dry insemination. During artificial dry insemination, dry the female fish's body, wrap her body with a pre-sterilized damp towel, leaving her abdomen exposed, and squeeze the eggs into a pre-sterilized container. Perform the same operation to squeeze the male fish's sperm into the container containing the eggs, with a female-to-male ratio of 1:3. Within 30 seconds, quickly stir the eggs and sperm with a feather to mix them thoroughly. Then, activate the sperm with deionized water. The volume ratio of the mixture (eggs and sperm) to deionized water is 1:3. Continue stirring for 2 minutes to allow the sperm and eggs to fully contact and fuse. Then, rinse 5 times with deionized water to remove broken eggs and debris. Let it stand for 12-20 minutes and count the fertilized eggs using the volumetric method.

[0056] 5. Incubation of Fertilized Eggs: Slowly and systematically pour the fertilized eggs into the Yushchenko incubator for incubation. Two days before using the Yushchenko incubator, soak the incubation frames in a 100ml / L potassium permanganate solution for 12 hours, then soak them in an 8% salt solution for 12 hours, and then dry them in an oven or expose them to sunlight for one day. During the incubation process, place approximately 12,000 fertilized eggs in each incubation frame. Maintain a slight flow of water at a depth of 20cm and a flow rate of 25L / min. The Yushchenko incubator will automatically turn the eggs once every 5 minutes. Maintain the water temperature at 15℃ and the pH value at 8.5. During incubation, promptly remove dead eggs, moldy eggs, and debris from the incubation frames, and count the number of dead eggs to calculate the fertilization rate. Fertilized eggs begin to hatch after 7–15 days of incubation.

[0057] 6. Fry Rearing: Immediately after hatching, the fry have a large, pale yellow yolk sac and need to continue rearing in the hatching frame. During this period, maintain a flowing water environment with a depth of 15cm, a flow rate of 20L / min, and a water temperature of 14-16℃. Do not feed them; allow them to lie on the bottom of the hatching frame of the Yusenko incubator to fully absorb and utilize the pale yellow yolk sac. Six to eight days after hatching, the fry begin to swim horizontally and start feeding. At a stocking density of 10,000 fry per pond, transfer them to fry rearing ponds with a diameter of 300cm, a water depth of 30cm, a water temperature of 15-16℃, and a pH of 8.5. During the rearing process, the water flow rate at the inlet of the rearing pond is 25L / min. Artemia nauplii are fed three times a day, with each pond receiving 60ml of Artemia eggs hatched into nauplii daily. Once the fish reach 2cm in length, they are fed Tianbang Fish and Shrimp Fry Rearing Special Type 48 Feed three times a day, at a rate of 6% of the fry's body weight. The ponds are inspected every morning to observe the condition of the fry. If any abnormalities are found, the fish are immediately examined under a microscope. If any fish disease is found, isolation and treatment are promptly implemented.

[0058] Comparative Example 1

[0059] The only difference between this comparative example and Example 1 is that mealworms were not fed during the two stages of intensive broodstock rearing one and intensive broodstock rearing two, and sturgeon pellet sinking compound feed with more than 45% was fed three times a day, morning, noon and evening.

[0060] Comparative Example 2

[0061] The only difference between this comparative example and Example 1 is that in the broodstock intensive cultivation II and broodstock intensive cultivation III, the fish were fed three times a day: once with mealworms and twice with a sturgeon pellet sinking compound feed with a protein content of more than 45%. The feeding order was: mealworms at 8:30 am, feed at 12:30 pm and 6:00 pm, and the amount of feed each time was 4% of the fish's body weight.

[0062] Comparing the gonadal development of broodstock in Example 1 and Comparative Examples 1-2, it can be seen that in Comparative Example 1, the broodstock rapidly fattened up in the early stage of cultivation, becoming very plump, but their gonadal development was slow. After the beginning of spring, their gonads were not yet mature, with most broodstock still in stage IV, making artificial spawning induction impossible. In Comparative Example 2, the broodstock did not show significant fattening, but their gonadal development was also slow; the lack of gonadal development after the beginning of spring was mainly due to insufficient nutrition. In Example 1, the broodstock reached gonadal maturity after the beginning of spring, at stage V. It was estimated that approximately 70% of the females laid eggs, and 80% of the males could produce sperm. This indicates that the "three-step" intensive cultivation method for broodstock described in this invention can effectively promote gonadal maturation in broodstock.

[0063] Example 2

[0064] This embodiment investigates the effects of different flow rates and light conditions on gonadal development in Lancang schizothorax during the third step of intensive breeding of broodstock through a single-factor controlled experiment.

[0065] (1) The conditions for strengthening the cultivation of the three streams are shown in Table 1. The other experimental conditions are the same as in Example 1:

[0066] Table 1. Effects of water flow stimulation on gonadal development

[0067]

[0068] Table 1 shows that after the beginning of spring (intensive cultivation step three), increased water flow is needed to stimulate gonad development in the broodstock. Insufficient water flow is detrimental to gonad development, while excessive flow hinders spawning and sperm extraction. At a flow rate of 45 L / min, the number of female spawning fish is highest, and the number of males capable of producing sperm is also highest. This indicates that a water flow rate of 45 L / min is optimal for gonad development in the broodstock during intensive cultivation step three.

[0069] (2) The light conditions for strengthening cultivation are shown in Table 2. The other experimental conditions are the same as in Example 1:

[0070] Table 2 Effects of light conditions on gonadal development in broodstock

[0071]

[0072] As shown in Table 2, increasing light exposure at night can promote gonadal development. It was found that when the light intensity was increased by 3 hours every night, the spawning situation was the best when the light intensity was 1000 LX. Increasing or decreasing the light intensity would affect the spawning effect of female fish. Female fish without increased light stimulation had the worst spawning situation.

[0073] Example 3

[0074] This embodiment investigates the effect of male fish spawning stimulant injection dosage on male fish sperm motility using a single-factor controlled experiment. The injection dosage conditions for male fish spawning stimulant are shown in Table 3, and the remaining experimental conditions are the same as in Embodiment 1.

[0075] Table 3. Sperm motility of male fish under different oxytocin dosages

[0076]

[0077]

[0078] Sperm motility was tested in each group of male fish: a small amount of semen was collected using the tip of a 1mL microsyringe and placed on a counting plate pre-dropped with deionized water to activate the sperm. The sperm motility was then observed under a digital microscope in various hypotonic solutions. Sperm motility was determined by the percentage of rapidly motile sperm after activation. "++" indicates approximately 100% rapid motility; "++" indicates approximately 80% rapid motility; "+" indicates approximately 50% rapid motility; "+-" indicates 10-30% rapid motility; and "-" indicates a very small number of sperm vibrating in place or all sperm dead. The results are shown in Table 3.

[0079] As shown in Table 3, sperm motility is optimal when the injection dose of oxytocin is 1 / 3 of that of the female fish, with almost 100% of the sperm being able to move rapidly. When the injection dose of oxytocin is too high or too low, sperm motility will decrease.

[0080] Example 4

[0081] This embodiment investigates the fertilization rate of artificial insemination in Lancang schizothorax by using different proportions of a mixture (sperm and eggs) and deionized water. The proportions are shown in Table 4, and the remaining experimental conditions are the same as in Example 1.

[0082] Table 4 Comparison of different proportions of mixed fluid (sperm and egg) and deionized water

[0083]

[0084] As shown in Table 4, the fertilization rate, hatching rate and emergence rate are highest when the ratio of the mixed solution (sperm and eggs) to deionized water is 1:3. The fertilization rate can reach 98%, the hatching rate can reach 95%, and the emergence rate can reach 91%.

[0085] Example 5

[0086] Artificial propagation and release were carried out using the artificial breeding method for Lancang schizothorax described in Example 1.

[0087] (1) Spawning induction of broodstock. From March to April 2017, artificial spawning was induced in three batches of Lancang schizothorax. A total of 27 fish were induced to spawn, including 12 females and 15 males. Ten females spawned, achieving a success rate of 83.3%. The effect lasted 24–72 hours, with a total of 80,000 eggs laid, averaging 8,400 eggs per fish. All males were successfully induced. See Table 5 for details.

[0088] Table 5. Artificial spawning in Lancang schizothorax

[0089]

[0090] (2) Artificial insemination and hatching. From March to April 2017, artificial insemination and hatching of fertilized eggs were carried out 6 times in the artificial breeding of Lancang schizothorax, yielding a total of 80,000 eggs. After removing unfertilized eggs, 71,000 fertilized eggs were obtained, with an average fertilization rate of 87.5%. The fertilized eggs began to hatch after 210 hours at a water temperature of around 14.0℃, and reached the hatching stage after 264 hours. 66,000 Lancang schizothorax larvae hatched, with a hatching rate of 93.0%. 65,000 fry emerged, with a fry emergence rate of 91.5%. See Table 6 for details.

[0091] Table 6. Artificial insemination and hatching of fertilized eggs in Lancang schizothorax

[0092]

[0093] (3) Embryonic Development. From March 20th to 31st, 2017, the embryonic development process and morphological characteristics of *Schizothorax lancensis* were observed. The fertilized eggs were spherical and golden yellow, with an average diameter of 2.77 mm immediately after fertilization and 3.82 mm after absorbing water and swelling. Hatching began after 217 hours in a Yushchenko incubator with a water depth of 15 cm, a flow rate of 20 L / min, a water temperature of 13.0–14.0℃, and a pH of 7.8–8.5, and reached the hatching stage after 265 hours. See Table 7 for details. Figure 1 .

[0094] Table 7. Embryonic development process and morphological characteristics of *Schizothorax lancica*

[0095]

[0096] Figure 1 In the images, A represents the fertilized egg stage; B represents the embryonic disc development stage; C represents the two-cell stage; D represents the four-cell stage; E represents the eight-cell stage; F represents the morula stage; G represents the early blastocyst stage; H represents the late blastocyst stage; I represents the early gastrulation stage; J represents the mid-gastrulation stage; K represents the neurula stage; L represents the myosarcota emergence stage; M represents the tail bud emergence stage; N represents the muscle effect stage; O represents the pre-emergence stage; and P represents the emergence stage.

[0097] (4) Larval growth. Observations showed that the average total length of 1-day-old Lancang schizothorax larvae was 9.0 mm / fish, and the average weight was 32 mg / fish; the average total length of 5-day-old larvae was 12.2 mm / fish, and the average weight was 44 mg / fish; the average total length of 10-day-old larvae was 13.8 mm / fish, and the average weight was 49 mg / fish; the average total length of 20-day-old larvae was 18.3 mm / fish, and the average weight was 62 mg / fish; the average total length of 30-day-old larvae was 22.7 mm / fish, and the average weight was 99 mg / fish; the average total length of 40-day-old larvae was 27.0 mm / fish, and the average weight was 167 mg / fish; and the average total length of 50-day-old larvae was 31.6 mm / fish, and the average weight was 241 mg / fish. See Table 8 for details. Figure 2 .

[0098] Table 8. Relationship between age and total length and body weight of Lancang schizothorax

[0099]

[0100] (5) Release situation

[0101] From August 25 to 28, 2017, 20,000 Lancang schizothorax fry (greater than 3cm / tail) were provided for fish breeding and release activities held at Huangdeng Hydropower Station, Dahuaqiao Hydropower Station, Lidi Hydropower Station and Wunonglong Hydropower Station on the upper reaches of the Lancang River. This totaled 40,000 fry. This marked the first artificial breeding and release of Lancang schizothorax fry. The release was notarized by the Weixi Lisu Autonomous County Notary Office of Yunnan Province, People's Republic of China, and a notarized certificate was obtained.

[0102] 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 artificial breeding of Schizothorax grahami, characterized in that, The method comprises the following steps: The parent fish is subjected to "three-step" intensive cultivation, and after cultivation, a laboratorial oviposition accelerator is injected to perform artificial insemination, fertilized egg hatching and fry cultivation; The "three-step" intensive cultivation comprises: step one, the parent fish is fed with Tenebrio molitor once a day and fed with sturgeon granular sinking compound feed with a protein content of 45% twice a day; step two, the parent fish is fed with Tenebrio molitor twice a day and fed with sturgeon granular sinking compound feed with a protein content of 45% once a day; and step three, the parent fish is fed with Tenebrio molitor three times a day and supplemented with light at night. In the first step of the intensive cultivation, the water flow is 25-35 L / min, and the cultivation time is 14-16 days. In the second step of the intensive cultivation, the water flow is 25-35 L / min, and the cultivation time is 28-32 days. In the third step of the intensive cultivation, the water flow is 45 L / min, the light intensity is 800-1200 LX, the light time is 2-4 h, and the cultivation time is 15-30 days. In the "three-step" intensive cultivation, the feeding amount of each bait or feed is 3-5% of the fish body weight. The female fish is injected with 4 mg DOM+8 μg LRH-A2 per 1 kg of body weight. The injection dose of the laboratorial oviposition accelerator for the male fish is 30-35% of that for the female fish.

2. The method of claim 1, wherein, The artificial insemination is performed by a dry method.

3. The method of claim 1, wherein, Deionized water is added to activate the sperm during the artificial insemination.

4. The method of claim 3, wherein, The added amount of the deionized water is 2-4 times the volume of the mixture of the fish eggs and the fish milt.

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