Artificial propagation method for black head fish in Tibetan

Abstract

The invention discloses a Tibetan ink head fish artificial propagation method, and belongs to the field of aquaculture. The Tibetan ink head fish artificial propagation method comprises the following steps that male fish parents and female fish parents which are sexually mature are selected, and spawning induction substances are injected into the female fish twice; the male fish injects spawning induction substances at one time when the female fish is injected for the second time; carrying out in-vitro artificial insemination 12 hours after injecting the oxytocic substance to obtain fertilized eggs; hatching the fertilized eggs, increasing the concentration of lysozyme in a hatching system after the membranes of the fish fries are broken in the hatching process, and artificially assisting the membranes to obtain the Tibetan black head fish fries; and feeding the Tibetan ink head fish fries in stages. According to the artificial propagation method for the ink head fish in Tibetan, the fry of the ink head fish in Tibetan is successfully obtained, the quality and the survival rate of the fry are guaranteed, and technical support is provided for artificial propagation of the fry.

Description

Technical Field

[0001] The invention relates to the field of aquaculture, and in particular to an artificial breeding method for Tibetan garfish. Background Art

[0002] The Tibetan garra (Garra tibetana) belongs to the Cypriniformes order, Cyprinidae family, Labeoninae subfamily, and genus Garra. This species is a gregarious species that inhabits small tributaries and mountain streams, particularly under rocks in rapids. Its breeding season is from February to April, with low fecundity. Its eggs are submerged and yellow in color. It primarily feeds on algae that grow on aquatic rocks, primarily diatoms and green algae (Chlorophyta).

[0003] In recent years, due to human factors such as overfishing and water pollution, the wild population of the Tibetan garfish has continued to decline, posing a serious threat to its survival. To protect this rare species, artificial breeding and stocking have become important and effective measures. However, to date, there is no artificial breeding technology for the Tibetan garfish, which provides insufficient technical support for population recovery. Furthermore, the Tibetan garfish's inherently low reproductive capacity, coupled with insufficient research on its reproductive behavior and environmental requirements, means that even if artificial breeding is successful, the survival rate of the fry is likely to be low, making it difficult to meet the needs of large-scale stocking. Furthermore, the Tibetan garfish primarily inhabits rocky areas in rapid-flowing environments, making accurate simulation of its habitat challenging, as existing technology cannot fully replicate its natural habitat.

[0004] In summary, the existing technical system lacks artificial propagation and fry rearing methods for Tibetan garfish, which is a key bottleneck restricting the conservation and recovery of its population. Therefore, it is urgent to develop an artificial propagation and fry rearing method suitable for Tibetan garfish to solve these technical problems and promote the in-depth protection of its population. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for artificially propagating Tibetan garfish to solve the problems existing in the above-mentioned prior art. Through the artificial propagation method of Tibetan garfish provided by the present invention, Tibetan garfish fry are successfully obtained, and the quality and survival rate of the fry are guaranteed, providing technical support for its artificial propagation.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an artificial propagation method for Tibetan garfish, comprising the following steps:

[0008] Select sexually mature male and female fish parents, and inject the female fish with oxytocin twice. The first injection is luteinizing hormone-releasing hormone A2, and the second injection is luteinizing hormone-releasing hormone A2, dioxinone maleate, and human chorionic gonadotropin. The interval between the two injections is 10 hours.

[0009] Male fish received a one-time injection of luteinizing hormone-releasing hormone A2, dioxinone maleate, and human chorionic gonadotropin at the same time as the female fish received the second injection;

[0010] According to the development of female fish, in vitro artificial insemination is performed to obtain fertilized eggs;

[0011] The fertilized eggs are hatched, and when the fry break the membrane during the hatching process, the concentration of the membrane-lytic enzyme in the hatching system is increased to artificially assist the fry to break the membrane, thereby obtaining Tibetan garfish fry;

[0012] The Tibetan garfish fry are fed in stages.

[0013] Optionally, when the female fish is injected with the oxytocin substance for the first time, the injection dose of the luteinizing hormone-releasing hormone A2 is 3 μg / kg.

[0014] Optionally, when the female fish is injected with the oxytocin substance for the second time, the injection dose of the luteinizing hormone-releasing hormone A2 is 10 μg / kg, the injection dose of dioxinone maleate is 10 mg / kg, and the injection dose of the human chorionic gonadotropin is 500 IU / kg.

[0015] Optionally, when the male fish is injected with oxytocin, the injection dose of the luteinizing hormone-releasing hormone A2 is 5 μg / kg, the injection dose of dioxinone maleate is 5 mg / kg, and the injection dose of the human chorionic gonadotropin is 400 IU / kg.

[0016] Optionally, the in vitro artificial insemination is specifically performed by squeezing out the female fish eggs, squeezing the male fish sperm onto the eggs, adding water and stirring to mix evenly, and then standing for 2 minutes to wash away mucus and impurities to obtain fertilized eggs.

[0017] Optionally, oxygen is continuously introduced during the incubation process, and the fertilized eggs are soaked in a 2% by mass sodium chloride solution at a temperature of 18-20° C. for 30 minutes every other day.

[0018] Optionally, the phased feeding is:

[0019] Stage I: 0-3 days old, no feeding;

[0020] Stage II: 4-6 days old, feed Artemia, once every 2 hours, each feeding amount is 8% of the total fish weight;

[0021] Stage III: 7-20 days old, feed a mixture of Artemia, crushed tilapia feed and algae powder every 3 hours;

[0022] Stage IV: 21-40 days old, the fry are divided into two groups according to their average body length: those larger than the average body length and those smaller than or equal to the average body length. They are fed separately, once every 6 hours, with a mixture of crushed tilapia feed and algae powder;

[0023] At 21-40 days of age, fry show individual differences and tend to cluster together. Larger fry take priority in feeding, and smaller fry will leave after feeding, making it difficult for smaller fry to obtain sufficient food. Feeding them separately in batches ensures that fry of different lengths can obtain sufficient food.

[0024] Stage V: After 40 days of age, the fish are transferred to outdoor ponds for breeding and fed once a day at 8:00 and 20:00 with a mixture of crushed tilapia feed and algae powder. The feed amount is adjusted by weighing once every two weeks.

[0025] Optionally, in the III stage, the mass ratio of the Artemia, the crushed tilapia feed and the algae powder is 1:1:1, and the feeding amount each time is 5% of the total weight of the fish.

[0026] Optionally, in the IV stage, the mass ratio of the crushed tilapia feed to the algae powder is 2:1, and the feeding amount each time is 5% of the total weight of the fish.

[0027] Optionally, in the V stage, the mass ratio of the crushed tilapia feed to the algae powder is 4:1, and the feeding amount each time is 3% of the total weight of the fish.

[0028] The present invention discloses the following technical effects:

[0029] By formulating an oxytocin formula and effective duration for Tibetan murrel, the present invention significantly improves the induced labor rate and fertilization rate, filling the gap in artificial induced labor technology in this field. At the same time, the use of an assisted hatching method during the incubation of fertilized eggs not only improves the hatching rate but also effectively shortens the hatching cycle. In addition, the present invention clarifies the feeding methods for Tibetan murrel fry at different age stages, including the time to change the feeding method, the frequency and method of feeding, the type of bait, and the selection of breeding facilities, thereby significantly improving the survival rate of the fry and reducing individual differences in the same batch of fry.

[0030] Through the artificial breeding method of Tibetan garfish provided by the present invention, Tibetan garfish fry are successfully obtained, and the quality and survival rate of the fry are guaranteed, providing technical support for its artificial breeding. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] In the feeding stage of Tibetan garfish fry in the embodiment of the present invention, the microcapsule material was purchased from Shandong Shengsuo Feed Technology Co., Ltd., the artemia was purchased from Wudi Haiji Aquatic Products Co., Ltd., the tilapia feed was purchased from Jiangmen Xinjian Feed Co., Ltd., and the algae powder (spirulina powder) was purchased from Wudi Haiji Aquatic Products Co., Ltd. Unless otherwise specified, other reagents or materials were purchased conventionally.

[0037] Example 1

[0038] On April 2, 2024, we selected 10 males with distinct head cysts, milky white semen that oozes out when lightly pressed on the lower abdomen and disperses upon contact with water. We also selected 10 females without distinct head cysts, with soft lower abdomens and swollen genital pores. We injected the females with luteinizing hormone-releasing hormone A2 (LARHA2) at 3 μg / kg. Ten hours later, we injected them again with LARHA2 (10 μg / kg), dioxinone maleate (DOM) (10 mg / kg), and human chorionic gonadotropin (HCG) (500 IU / kg). Males received a single injection of LARHA2 (5 μg / kg), DOM (5 mg / kg), and HCG (400 IU / kg) at the same time as the second injection. All injections were performed with 0.9% saline, and the single injection dose was 0.5 mL.

[0039] After 12 hours, the females were checked for development. Those that successfully ovulated underwent artificial insemination. This involved squeezing out the female eggs, then applying male sperm to the eggs. Water was added, the mixture was stirred, and the mixture was left to rest for two minutes to wash away mucus and impurities. Fertilized eggs were obtained. Females that failed to ovulate were returned to the water and checked every hour. Ultimately, nine females were successfully induced to ovulate, with a 90% induced ovulation rate and a cumulative 2,800 eggs laid.

[0040] Fertilized eggs were placed in an incubation bucket at a rate of 100 eggs / L. The flow rate was controlled to maintain a slow tumbling of the eggs in the water. Oxygen was continuously introduced during the incubation process to maintain a dissolved oxygen concentration above 6 mg / L. The eggs were then immersed in a 2% sodium chloride solution for 30 minutes every other day. The water temperature was 18°C. 118 hours after fertilization, the membranes of the fry were observed to break. Tibetan garrulella eggs have thicker membranes. Once these fry were observed, the oxygen and water supply to the incubation bucket were turned off, half the water was drained, and the membrane-dissolving enzyme concentration in the bucket was increased to assist in membrane emergence. Embryonic development was observed, and approximately 2,400 neural embryos developed, with a fertilization rate of 85.71%. Ultimately, 2,100 Tibetan garrulella fry hatched, with a hatch rate of 87.50%.

[0041] Raise Tibetan garfish fry according to their developmental stages:

[0042] Stage I fry, 0-3 days old, no feeding;

[0043] Stage II fry, 4-6 days old, are fed evenly 12 times a day over 24 hours. Each time, 8% of the total weight of the fish is evenly fed to the entire pond. The oxygen is turned off before feeding, and the leftover bait and feces are sucked out 30 minutes after feeding.

[0044] Stage III fry, 7-20 days old, are fed evenly eight times a day, with 5% of the total fish weight being fed at a fixed point each time. The feed is a mixture of Artemia, crushed tilapia feed, and algae powder in a mass ratio of 1:1:1. Oxygen is turned off before feeding, and residual bait and feces are sucked out 30 minutes after feeding.

[0045] Stage IV fry, 21-40 days old, had their average total length measured at 21 days of age. The fry were divided into two groups and raised separately based on their length relative to the average body length to observe developmental changes and prevent disease from spreading to one group, leading to complete death. The first group consisted of larger individuals with a body length greater than the average, while the second group consisted of smaller individuals with a body length less than or equal to the average. They were fed separately; the fry were fed four times a day, with 5% of their total body weight of bait being mixed with crushed tilapia feed and algae meal in a mass ratio of 2:1. The oxygen supply was turned off before feeding, and any remaining bait and feces were aspirated 30 minutes after the feeding ended.

[0046] After 40 days of age, stage V fry are transferred to outdoor ponds for breeding and fed once a day at 8:00 and 20:00. Each time, 3% of the total weight of the fish body weight is fed at a fixed point. The bait is crushed tilapia feed and algae powder in a mass ratio of 4:1. The feed amount is adjusted by weighing once every two weeks.

[0047] The size differences between individual fry were small, and they grew evenly and rapidly. Ultimately, 2,000 Tibetan garfish fry were obtained, with a fry survival rate of 95.24%.

[0048] Example 2

[0049] On April 8, 2024, 15 males with distinct pearl stars on their heads and milky white semen that oozes out when lightly pressed on their lower abdomen and disperses upon contact with water were selected. Fifteen females without distinct pearl stars on their heads, with soft lower abdomens and swollen genital openings were selected. The females were injected with luteinizing hormone-releasing hormone A2 (LARHA2) at 3 μg / kg. Ten hours later, they were again injected with LARHA2 at 10 μg / kg, dioxinone maleate (DOM) at 10 mg / kg, and human chorionic gonadotropin (HCG) at 500 IU / kg. Males received a single injection of LARHA at 25 μg / kg, DOM at 5 mg / kg, and HCG at 400 IU / kg during the second injection. The solvent used was 0.9% saline, and the injection dose per fish was 0.5 mL.

[0050] After 12 hours, the females were checked for development. Those that successfully ovulated underwent artificial insemination. This involved squeezing out the female eggs, then applying male sperm to the eggs. Water was added, the mixture was stirred, and the mixture was left to rest for two minutes to wash away mucus and impurities. Fertilized eggs were obtained. Females that failed to ovulate were returned to the water and checked every hour. Ultimately, 14 females were successfully induced to ovulate, with an induction rate of 93.33%. A total of approximately 4,800 eggs were laid.

[0051] Fertilized eggs were placed in an incubator at a rate of 100 per liter. The flow rate was controlled to maintain a slow tumbling of the eggs in the water. Oxygen was continuously introduced during the incubation process, and the eggs were immersed in a 2% sodium chloride solution for 30 minutes every other day. The water temperature was 20°C. 118 hours after fertilization, the membranes of the fry were observed to break. Tibetan garfish eggs have thicker membranes. Once these fry were observed, the oxygen and water supply to the incubator were turned off, half the water was drained, and the membrane-lytic enzyme concentration in the incubator was increased to assist in membrane emergence. Embryonic development was observed, and approximately 4,500 neural embryos developed, with a fertilization rate of 93.75%. 4,000 Tibetan garfish fry were eventually hatched, with a hatch rate of 88.89%.

[0052] Raise Tibetan garfish fry according to their developmental stages:

[0053] Stage I fry, 0-3 days old, no feeding;

[0054] Stage II fry, 4-6 days old, are fed evenly 12 times a day over 24 hours. Each time, 8% of the total weight of the fish is evenly fed to the entire pond. The oxygen is turned off before feeding, and the leftover bait and feces are sucked out 30 minutes after feeding.

[0055] Stage III fry, 7-20 days old, are fed evenly eight times a day, with 5% of the total fish weight being fed at a fixed point each time. The feed is a mixture of Artemia, crushed tilapia feed, and algae powder in a mass ratio of 1:1:1. Oxygen is turned off before feeding, and residual bait and feces are sucked out 30 minutes after feeding.

[0056] Stage IV fry, 21-40 days old, had their average total length measured at 21 days of age. The fry were divided into two groups based on their length relative to the average body length: the first group consisted of larger individuals with a body length greater than the average, and the second group consisted of smaller individuals with a body length less than or equal to the average. These fry were fed separately; the fry were fed four times a day, with 5% of their total body weight of bait being a mixture of crushed tilapia feed and algae meal in a mass ratio of 2:1. The oxygen supply was turned off before feeding, and any remaining bait and feces were removed 30 minutes after the feeding period.

[0057] After 40 days of age, stage V fry are transferred to outdoor ponds for breeding and fed once a day at 8:00 and 20:00. Each time, 3% of the total weight of the fish body weight is fed at a fixed point. The bait is crushed tilapia feed and algae powder in a mass ratio of 4:1. The feed amount is adjusted by weighing once every two weeks.

[0058] The size differences between individual fry were small, and they grew evenly and rapidly. Ultimately, 3,800 Tibetan garfish fry were obtained, with a fry survival rate of 95%.

[0059] Comparative Example 1

[0060] On March 5, 2024, we selected 10 males with distinct pearl stars on their heads and milky white semen that oozes out when lightly pressed on their lower abdomen and disperses upon contact with water. We also selected 10 females without distinct pearl stars on their heads, with soft lower abdomens and swollen genital openings. We injected the females with a single dose of luteinizing hormone-releasing hormone A2 (LARHA2) at 10 μg / kg, dioxinone maleate (DOM) at 5 mg / kg, and human chorionic gonadotropin (HCG) at 500 IU / kg. The injection dose for males was half that for females. The solvent used was 0.9% saline, and the single injection dose was 0.5 mL.

[0061] After 12 hours, the females were checked for development. Those that successfully ovulated underwent artificial insemination. This involved squeezing out the female eggs, then applying male sperm to the eggs. Water was added, the mixture was stirred, and the mixture was left to rest for two minutes to wash away mucus and impurities. Fertilized eggs were obtained. Females that failed to ovulate were returned to the water and checked every hour. Ultimately, three females were successfully induced to ovulate, with a 30% induced ovulation rate and a cumulative egg production of approximately 1,000.

[0062] Fertilized eggs were placed in an incubation bucket at a rate of 100 eggs / L. The flow rate was controlled to maintain a slow tumbling of the eggs in the water. Oxygen was continuously introduced during the incubation process. The eggs were immersed in a 2% sodium chloride solution for 30 minutes every other day. The water temperature was 18-20°C. Fry were observed to break the membrane 120 hours after fertilization. Embryonic development was observed, and approximately 200 neural embryos developed, with a fertilization rate of 20%. Finally, 60 Tibetan garfish fry were hatched, with a hatch rate of 30%. During the incubation process, the fry were observed to have difficulty breaking the membrane.

[0063] Raise Tibetan garfish fry according to their developmental stages:

[0064] 0-3 days old, at this time they are not yet able to swim horizontally, and all nutrition is provided by the yolk sac, so no feeding is required;

[0065] Starting from the 4th day of age, the fry were fed with microencapsulated feed at 5% of their body weight. The fry had poor feeding and lacked nutrition. After 15 days, all of them died, and the fry survival rate was 0%.

[0066] Comparative Example 2

[0067] On March 10, 2024, we selected 10 males with distinct pearl stars on their heads and milky white semen that oozes out when lightly pressed on their lower abdomen and disperses upon contact with water. We also selected 10 females without distinct pearl stars on their heads, with soft lower abdomens and swollen genital openings. We injected the females with a single dose of luteinizing hormone-releasing hormone A2 (LARHA2) at 10 μg / kg, dioxinone maleate (DOM) at 5 mg / kg, and human chorionic gonadotropin (HCG) at 500 IU / kg. The injection dose for males was half that for females. The solvent used was 0.9% saline, and the single injection dose was 0.5 mL.

[0068] After 12 hours, the females were checked for development. Those that successfully ovulated underwent artificial insemination. This involved squeezing out the eggs, then applying male sperm to them. Water was added, the mixture was stirred, and the mixture was left to rest for two minutes to wash away mucus and impurities. Fertilized eggs were obtained. Females that failed to ovulate were returned to the water and checked every hour. Ultimately, four females were successfully induced to ovulate, with a 40% induced ovulation rate and a cumulative 1,200 eggs laid.

[0069] Fertilized eggs were placed in an incubator at a rate of 100 eggs / L. The flow rate was controlled to maintain a slow tumbling of the eggs in the water. Oxygen was continuously introduced during the incubation process, and the eggs were immersed in a 2% sodium chloride solution for 30 minutes every other day. The water temperature was maintained at 18-20°C. 118 hours after fertilization, the membranes of the fry were observed to break. Tibetan garfish eggs have thick membranes. Once the membranes were observed to break, the oxygen and water supply to the incubator were turned off, half of the water was drained, and the membrane lytic enzyme concentration in the incubator was increased to assist in membrane emergence. Embryonic development was observed, and approximately 300 neural embryos developed, with a fertilization rate of 25%. Ultimately, 260 Tibetan garfish fry hatched, with a hatch rate of 86.67%.

[0070] Raise Tibetan garfish fry according to their developmental stages:

[0071] 0-3 days old, at this time they are not yet able to swim horizontally, and all nutrition is provided by the yolk sac, so no feeding is required;

[0072] From 4 to 39 days old, feed evenly three times a day over 24 hours. Each time, feed the entire pond with an excess of crushed tilapia feed (10% of the total weight of the fry). Turn off the oxygen before feeding, and remove the remaining bait and feces 30 minutes after feeding.

[0073] After 40 days of age, the fish were fed once daily at 8:00 AM and 8:00 PM, with a feed consisting of crushed tilapia feed (3% of their total body weight) delivered at a designated location. The feed was weighed and adjusted every two weeks. The fry varied significantly in size and were deficient in some nutrients. Ultimately, 120 Tibetan garfish fry were harvested, with a fry survival rate of 46.15%.

[0074] Comparative Example 3

[0075] On March 15, 2024, we selected 10 males with a distinct pearly star on their heads and milky white semen that oozes out when lightly pressed on their lower abdomen and disperses upon contact with water. We also selected 10 females without a distinct pearly star on their heads, with a soft lower abdomen and a red and swollen genital pore. We injected the females with luteinizing hormone-releasing hormone A2 (LARHA2) at 3 μg / kg. Ten hours later, we injected them again with LARHA2 at 10 μg / kg, dioxinone maleate (DOM) at 10 mg / kg, and human chorionic gonadotropin (HCG) at 500 IU / kg. Males received a single injection of LARHA2 at 25 μg / kg, DOM at 5 mg / kg, and HCG at 400 IU / kg during the second injection. All injections were made with 0.9% saline, and the single injection dose was 0.5 mL.

[0076] After 12 hours, the females were checked for development. Those that successfully ovulated underwent artificial insemination. This involved squeezing out the female eggs, then applying male sperm to the eggs. Water was added, the mixture was stirred, and the mixture was left to rest for two minutes to wash away mucus and impurities. Fertilized eggs were obtained. Females that failed to ovulate were returned to the water and checked every hour. Ultimately, nine females were successfully induced to ovulate, with a 90% induced ovulation rate and a cumulative 2,500 eggs laid.

[0077] Fertilized eggs were placed in an incubator at a rate of 100 per liter. The flow rate was controlled to maintain a slow tumbling of the eggs in the water. Oxygen was continuously introduced during the incubation process, and the eggs were immersed in a 2% sodium chloride solution for 30 minutes every other day. The water temperature was maintained at 18-20°C. 118 hours after fertilization, the membranes of the fry were observed to break. Tibetan garfish eggs have thick membranes. Once these fry were observed, the oxygen and water supply to the incubator were turned off, half the water was drained, and the membrane lytic enzyme concentration in the incubator was increased to assist in membrane emergence. Embryonic development was observed, and approximately 2,000 neural embryos developed, with a fertilization rate of 80%. Ultimately, 1,700 Tibetan garfish fry hatched, with a hatch rate of 85%.

[0078] Raise Tibetan garfish fry according to their developmental stages:

[0079] 0-3 days old, at this time they are not yet able to swim horizontally, and all nutrition is provided by the yolk sac, so no feeding is required;

[0080] Starting from the 4th day of age, fry were fed algae at 5% of their body weight, but the feeding situation was poor and the fry survival rate was 32.15%.

[0081] Comparative Example 4

[0082] On March 20, 2024, we selected 10 males with a distinct pearly star on their heads, milky white semen that leaked out when lightly pressed on their lower abdomen and dispersed upon contact with water. We also selected 10 females without a distinct pearly star on their heads, with a soft lower abdomen and a red and swollen genital pore. We injected the females with luteinizing hormone-releasing hormone A2 (LARHA2) at 3 μg / kg. Ten hours later, we injected them again with LARHA2 at 10 μg / kg, dioxinone maleate (DOM) at 10 mg / kg, and human chorionic gonadotropin (HCG) at 500 IU / kg. Males received a single injection of LARHA2 at 25 μg / kg, DOM at 5 mg / kg, and HCG at 400 IU / kg during the second injection. All injections were made with 0.9% saline, and the single injection dose was 0.5 mL.

[0083] After 12 hours, the females were checked for development. Those that successfully ovulated underwent artificial insemination. This involved squeezing out the eggs, then applying male sperm to them. Water was added, the mixture was stirred, and the mixture was left to rest for two minutes to wash away mucus and impurities. Fertilized eggs were obtained. Females that failed to ovulate were returned to the water and checked every hour. Ultimately, eight females were successfully induced to ovulate, with an 80% induced ovulation rate and a cumulative 2,400 eggs laid.

[0084] Fertilized eggs were placed in an incubator at a rate of 100 per liter. The flow rate was controlled to maintain a slow tumbling of the eggs in the water. Oxygen was continuously introduced during the incubation process, and the eggs were immersed in a 2% sodium chloride solution for 30 minutes every other day. The water temperature was maintained at 18-20°C. 118 hours after fertilization, the membranes of the fry were observed to break. Tibetan garfish eggs have thick membranes. Once these fry were observed, the oxygen and water supply to the incubator were turned off, half the water was drained, and the membrane-lytic enzyme concentration in the incubator was increased to assist in membrane emergence. Embryonic development was observed, and approximately 1,700 neural embryos developed, with a fertilization rate of 70.8%. Ultimately, 1,500 Tibetan garfish fry hatched, with a hatch rate of 88.24%.

[0085] Raise Tibetan garfish fry according to their developmental stages:

[0086] 0-3 days old, at this time they are not yet able to swim horizontally, and all nutrition is provided by the yolk sac, so no feeding is required;

[0087] For fish aged 4-6 days, feed them evenly 12 times a day, 24 hours a day. Each time, feed the whole pond evenly with Artemia worms at a concentration of 8% of the total fish weight. Turn off the oxygen before feeding, and vacuum out the remaining bait and feces 30 minutes after feeding.

[0088] Feed fish evenly eight times a day for 7-20 days of age. Each time, feed 5% of the total fish weight with a mixture of Artemia and crushed tilapia feed in a mass ratio of 1:1. Turn off the oxygen supply before feeding, and remove any remaining bait and feces 30 minutes after feeding.

[0089] From 21 to 40 days of age, the fish were fed four times daily, each time at a designated location, with 5% of their total body weight of crushed tilapia feed. Oxygen was turned off before feeding, and residual feed and feces were aspirated 30 minutes after feeding. After 40 days of age, the fish were fed once daily at 8:00 AM and 8:00 PM, each time at a designated location, with 3% of their total body weight of crushed tilapia feed. The fish were weighed and fed every two weeks to adjust the feed amount. The fry varied significantly in size and were deficient in some nutrients. Ultimately, 1,000 Tibetan garfish fry were obtained, with a fry survival rate of 66.67%.

[0090] Comparative Example 5

[0091] On March 20, 2024, we selected 10 males with a distinct pearly star on their heads, milky white semen that leaked out when lightly pressed on their lower abdomen and dispersed upon contact with water. We also selected 10 females without a distinct pearly star on their heads, with a soft lower abdomen and a red and swollen genital pore. We injected the females with luteinizing hormone-releasing hormone A2 (LARHA2) at 3 μg / kg. Ten hours later, we injected them again with LARHA2 at 10 μg / kg, dioxinone maleate (DOM) at 10 mg / kg, and human chorionic gonadotropin (HCG) at 500 IU / kg. Males received a single injection of LARHA2 at 25 μg / kg, DOM at 5 mg / kg, and HCG at 400 IU / kg during the second injection. All injections were made with 0.9% saline, and the single injection dose was 0.5 mL.

[0092] After 12 hours, the females were checked for development. Those that successfully ovulated underwent artificial insemination. This involved squeezing out the female eggs, then applying male sperm to the eggs. Water was added, the mixture was stirred, and the mixture was left to rest for two minutes to wash away mucus and impurities. Fertilized eggs were obtained. Females that failed to ovulate were returned to the water and checked every hour. Ultimately, nine females were successfully induced to ovulate, with a 90% induced ovulation rate and a cumulative 2,500 eggs laid.

[0093] Fertilized eggs were placed in an incubator at a rate of 100 eggs / L. The flow rate was controlled to maintain a slow tumbling of the eggs in the water. Oxygen was continuously introduced during the incubation process, and the eggs were immersed in a 2% sodium chloride solution for 30 minutes every other day. The water temperature was maintained at 18-20°C. 118 hours after fertilization, the membranes of the fry were observed to break. The membranes of Tibetan garfish eggs are thick. Once these fry were observed, the oxygen and water supply to the incubator were turned off, half of the water was drained, and the membrane lytic enzyme concentration in the incubator was increased to assist in membrane emergence. Embryonic development was observed, and approximately 2,100 neural embryos developed, with a fertilization rate of 84.0%. Ultimately, 1,800 Tibetan garfish fry hatched, with a hatch rate of 85.71%.

[0094] Raise Tibetan garfish fry according to their developmental stages:

[0095] 0-3 days old, no feeding;

[0096] For fish aged 4-6 days, feed them evenly 12 times a day, 24 hours a day. Each time, feed the whole pond evenly with Artemia worms at a concentration of 8% of the total fish weight. Turn off the oxygen before feeding, and vacuum out the remaining bait and feces 30 minutes after feeding.

[0097] Between 7 and 20 days of age, feed evenly eight times a day, with 5% of the fish's total body weight of bait mixed with algae powder and crushed tilapia feed at a mass ratio of 4:1. Turn off the oxygen supply before feeding, and remove any remaining bait and feces 30 minutes after feeding.

[0098] Between 21 and 40 days of age, feed the fish four times a day, feeding them 5% of their total body weight with a mixture of algae meal and crushed tilapia feed at a mass ratio of 4:1. Oxygen should be turned off before feeding, and any remaining bait and feces should be vacuumed out 30 minutes after feeding.

[0099] After 40 days of age, the fish were fed once daily at 8:00 AM and 8:00 PM. A 4:1 mixture of algae meal and crushed tilapia feed (3% of their total body weight) was added to the fry at designated locations. The fry were weighed and adjusted every two weeks. The fry showed significant size variation and nutritional deficiencies. Ultimately, 800 Tibetan garfish fry were harvested, with a fry survival rate of 44.44%.

[0100] The statistical results of the induced labor rate, fertilization rate, hatching rate and seedling survival rate of Examples 1-2 and Comparative Examples 1-5 are shown in Table 1.

[0101] Table 1

[0102]

[0103] It can be seen from the data in Table 1 that, compared with the comparative example, the method for inducing labor, insemination and hatching in the embodiment of the present invention has high labor induction rate, fertilization rate and hatching rate and stable effect. The labor induction injection method in the embodiment of the present invention can significantly improve the labor induction rate and hatching rate; assisted hatching can significantly increase the hatching rate of Tibetan garfish; in the seedling cultivation process of the embodiment, by optimizing the feeding method, feeding the fry in batches in the IV stage, and adding algae powder to the feed, the seedling survival rate can be further significantly improved to more than 90%.

[0104] The present invention elaborates in detail the fish parent selection, oxytocin compatibility, effect time, fertilization method, hatching method and fry rearing method in the process of artificial propagation of Tibetan tarpaulin, and the oxytocin rate, fertilization rate, hatching rate and fry survival rate are high. By referring to the method of the present invention, the artificial propagation and fry rearing of Tibetan tarpaulin can be completed, and the artificial propagation, large-scale production and proliferation and release of Tibetan tarpaulin can be realized, which is of great significance to the protection, development and utilization of Tibetan tarpaulin resources.

[0105] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for artificial propagation of Tibetan garfish, characterized in that: The following steps are involved: Select sexually mature male and female fish parents, and inject the female fish with oxytocin twice. The first injection is luteinizing hormone-releasing hormone A2, and the second injection is luteinizing hormone-releasing hormone A2, dioxinone maleate, and human chorionic gonadotropin. The interval between the two injections is 10 hours. Male fish received a one-time injection of luteinizing hormone-releasing hormone A2, dioxinone maleate, and human chorionic gonadotropin at the same time as the female fish received the second injection; According to the development of female fish, in vitro artificial insemination is performed to obtain fertilized eggs; The fertilized eggs are hatched, and when the fry break the membrane during the hatching process, the concentration of the membrane-lytic enzyme in the hatching system is increased to artificially assist the fry to break the membrane, thereby obtaining Tibetan garfish fry; The Tibetan garfish fry are fed in stages.

2. The artificial propagation method of Tibetan garfish according to claim 1, wherein: When the female fish is injected with the oxytocin substance for the first time, the injection dose of the luteinizing hormone-releasing hormone A2 is 3 μg / kg.

3. The artificial propagation method of Tibetan garfish according to claim 1, wherein: When the female fish is injected with the oxytocin substance for the second time, the injection dose of the luteinizing hormone-releasing hormone A2 is 10 μg / kg, the injection dose of dioxinone maleate is 10 mg / kg, and the injection dose of the human chorionic gonadotropin is 500 IU / kg.

4. The artificial propagation method of Tibetan garfish according to claim 1, wherein: When the male fish is injected with oxytocin, the injection dose of the luteinizing hormone-releasing hormone A2 is 5 μg / kg, the injection dose of dioxinone maleate is 5 mg / kg, and the injection dose of the human chorionic gonadotropin is 400 IU / kg.

5. The artificial propagation method of Tibetan garfish according to claim 1, characterized in that: The in vitro artificial insemination specifically comprises squeezing out the female fish eggs, squeezing the male fish sperm onto the eggs, adding water and stirring to mix evenly, and then standing for 2 minutes to wash away mucus and impurities to obtain fertilized eggs.

6. The artificial propagation method of Tibetan garfish according to claim 1, characterized in that: During the incubation process, oxygen is continuously introduced, and the fertilized eggs are soaked in a 2% sodium chloride solution at a temperature of 18-20° C. for 30 minutes every other day.

7. The artificial propagation method of Tibetan garfish according to claim 1, characterized in that: The phased feeding is: Stage I: 0-3 days old, no feeding; Stage II: 4-6 days old, feed Artemia, once every 2 hours, each feeding amount is 8% of the total fish weight; Stage III: 7-20 days old, feed with a mixture of Artemia, crushed tilapia feed and algae meal, every 3 hours; Stage IV: 21-40 days old, the fry are divided into two groups according to their average body length: those larger than the average body length and those smaller than or equal to the average body length. They are fed separately, once every 6 hours, with a mixture of crushed tilapia feed and algae powder; Stage V: After 40 days of age, the fish are transferred to outdoor ponds for breeding and fed once a day at 8:00 and 20:00 with a mixture of crushed tilapia feed and algae powder. The feed amount is adjusted by weighing once every two weeks.

8. The artificial propagation method of Tibetan garfish according to claim 7, characterized in that: In the III stage, the mass ratio of the Artemia, the crushed tilapia feed and the algae powder is 1:1:1, and the feeding amount each time is 5% of the total weight of the fish.

9. The artificial propagation method of Tibetan garfish according to claim 7, characterized in that: In the IV stage, the mass ratio of the crushed tilapia feed to the algae powder is 2:1, and the feeding amount each time is 5% of the total weight of the fish.

10. The artificial propagation method of Tibetan garfish according to claim 7, characterized in that: In the V stage, the mass ratio of the crushed tilapia feed to the algae powder is 4:1, and the feeding amount each time is 3% of the total weight of the fish.

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