Artificial breeding method of Tibet barbel

Abstract

The application discloses a Tibet black-head fish artificial breeding method and belongs to the field of aquaculture. The Tibet black-head fish artificial breeding method comprises the following steps: selecting sexually mature male and female parent fishes, injecting a parturition promoting substance into the female fish twice, injecting the parturition promoting substance into the male fish once when the female fish is injected the second time, performing in-vitro artificial insemination 12 hours after the injection of the parturition promoting substance to obtain fertilized eggs, hatching the fertilized eggs, improving the concentration of membrane dissolving enzyme in the hatching system after the hatched larvae break the membrane, and performing artificial membrane breaking assistance to obtain Tibet black-head fish larvae, and feeding the Tibet black-head fish larvae in stages. The Tibet black-head fish artificial breeding method provided by the application successfully obtains Tibet black-head fish larvae and guarantees the quality and survival rate of the larvae, thereby providing technical support for the artificial breeding of the Tibet black-head fish.

Description

Technical Field

[0001] This invention relates to the field of aquaculture, and in particular to a method for the artificial breeding of Tibetan cuttlefish. Background Technology

[0002] The Tibetan tibetana (Garra tibetana) belongs to the order Cypriniformes, family Cyprinidae, subfamily Labeoninae, and genus Garra. This species exhibits a gregarious distribution habit, primarily inhabiting small tributaries and mountain streams, especially favoring the area under rocks in fast-flowing waters. Its breeding season is from February to April each year, with low reproductive rate; the eggs are demersal and yellow. The Tibetan tibetana mainly feeds on algae growing on rocks in the water, primarily diatoms and green algae (Chlorophyta).

[0003] In recent years, due to overfishing, water pollution, and other human factors, the wild population of Tibetan blackfish has continued to decline, facing a serious threat to its survival. Artificial breeding and restocking have become important and effective means to protect this rare species. However, as of now, there is a lack of artificial breeding technology for Tibetan blackfish, which cannot provide technical support for its population recovery. Furthermore, Tibetan blackfish have low reproductive rates, and insufficient research on their reproductive behavior and environmental requirements means that even if artificial breeding is successful, the survival rate of fry may be low, making it difficult to meet the needs of large-scale restocking. In addition, Tibetan blackfish mainly inhabit rocky areas in fast-flowing environments, making accurate simulation of their habitat a significant challenge, and current technologies cannot completely replicate their natural habitat conditions.

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

[0005] The purpose of this invention is to provide a method for the artificial breeding of Tibetan cuttlefish, thereby solving the problems existing in the prior art. Through the artificial breeding method for Tibetan cuttlefish provided by this invention, Tibetan cuttlefish fry have been successfully obtained, and the quality and survival rate of the fry have been guaranteed, providing technical support for their artificial breeding.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for artificial breeding of Tibetan cuttlefish, comprising the following steps:

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

[0009] The male fish was injected with luteinizing hormone-releasing hormone A2, dioxone maleate, and human chorionic gonadotropin in a single injection during the second injection of the female fish.

[0010] In vitro artificial insemination was performed on the female fish according to her developmental status to obtain fertilized eggs;

[0011] The fertilized eggs were incubated. During the incubation process, after the fry broke through the membrane, the concentration of membrane-dissolving enzymes in the incubation system was increased to artificially assist in the membrane breaking, thus obtaining Tibetan blackhead fish fry.

[0012] The Tibetan blackfish fry were fed in stages.

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

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

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

[0016] Optionally, the in vitro artificial insemination specifically involves squeezing out the female fish eggs, then squeezing the male fish semen onto the eggs, adding water and stirring to mix evenly, letting it stand for 2 minutes, washing away the mucus and impurities, and obtaining fertilized eggs.

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

[0018] Optionally, the staged feeding is as follows:

[0019] Phase I: 0-3 days old, do not feed;

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

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

[0022] Stage IV: 21-40 days old. The fry are divided into two batches based on their average body length: those greater than the average body length and those less than or equal to the average body length. They are fed separately, once every 6 hours. The feed consists of a mixture of crushed tilapia feed and algae powder.

[0023] Between 21 and 40 days old, individual differences appear in the fry. They like to gather in groups, with larger individuals feeding first. After feeding, smaller individuals will also leave, making it difficult for smaller individuals to obtain enough food. Feeding them in batches can ensure that individuals of different body lengths can obtain enough food.

[0024] Phase V: After 40 days, transfer to an outdoor pond for rearing. Feed once a day at 8:00 and 20:00. The feed consists of a mixture of crushed tilapia feed and algae powder. Weigh the fish every two weeks to adjust the feeding amount.

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

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

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

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

[0029] This invention significantly improves the spawning rate and fertilization rate of Tibetan blackfish by formulating an oxytocin-inducing agent and determining its effective time, filling a gap in artificial spawning technology in this field. Simultaneously, the use of assisted hatching during the fertilized egg incubation process not only increases the hatching rate but also effectively shortens the hatching cycle. Furthermore, this invention clarifies the rearing methods for Tibetan blackfish fry at different age stages, including the timing of changes in rearing methods, feeding frequency and methods, feed types, and the selection of aquaculture facilities, thereby greatly improving the fry survival rate and reducing individual differences within the same batch of fry.

[0030] The artificial breeding method for Tibetan cuttlefish provided by this invention successfully obtained Tibetan cuttlefish fry, and ensured the quality and survival rate of the fry, providing technical support for its artificial breeding. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0036] In the feeding stage of Tibetan blackfish fry in this embodiment of the invention, the microencapsulated feed was purchased from Shandong Shengsuo Feed Technology Co., Ltd., the brine shrimp from Wudi Haiji Aquatic Products Co., Ltd., the tilapia feed from Jiangmen Xinjian Feed Co., Ltd., and the algae powder (spirulina concentrate) from Wudi Haiji Aquatic Products Co., Ltd. Unless otherwise specified, other reagents or materials were obtained through conventional purchases.

[0037] Example 1

[0038] On April 2, 2024, ten male broodstock were selected, exhibiting prominent head tubercles and a milky white semen that dissipated upon contact with water when the lower abdomen was gently pressed. Ten female broodstock were also selected, exhibiting no prominent head tubercles, a soft lower abdomen, and swollen genital pores. The females were injected with luteinizing hormone-releasing hormone A2 (LARHA2) at 3 μg / kg. Ten hours later, they were injected again with LARHA2 at 10 μg / kg, dioxin maleate (DOM) at 10 mg / kg, and human chorionic gonadotropin (HCG) at 500 IU / kg. The males were injected once during the second injection of the females, with LARHA2 at 5 μg / kg, DOM at 5 mg / kg, and HCG at 400 IU / kg. All solvents were 0.9% physiological saline, and the single-tail injection dose was 0.5 mL.

[0039] Twelve hours later, the development of the female fish was checked. Artificial insemination was performed on the females who successfully ovulated. This involved squeezing out the female eggs, applying male sperm onto the eggs, adding water, mixing thoroughly, letting it stand for 2 minutes, washing away mucus and impurities, and obtaining fertilized eggs. Females that failed to ovulate were returned to the water, and checked every hour. Ultimately, nine females were successfully induced to spawn, a 90% spawning rate, with a total of approximately 2800 eggs laid.

[0040] Fertilized eggs were placed in an incubation tank at a rate of 100 eggs / L. The flow rate was controlled to keep the fertilized eggs slowly tumbling in the water. Oxygen was continuously introduced during incubation to ensure a dissolved oxygen concentration greater than 6 mg / L. Every other day, the eggs were soaked in a 2% sodium chloride solution for 30 minutes. The water temperature was 18℃. Fry hatching was observed 118 hours after fertilization. Since the egg membrane of the Tibetan blackhead fish is relatively thick, after observing hatching, the oxygen and water intake of the incubation tank were turned off, and half of the water was drained to increase the concentration of membrane-dissolving enzymes in the incubation tank, thus aiding hatching. Embryonic development was observed, with approximately 2400 neurulas developed, representing a fertilization rate of 85.71%. A total of 2100 Tibetan blackhead fish fry hatched, achieving a hatching rate of 87.50%.

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

[0042] Stage I fry, 0-3 days old, do not feed;

[0043] For stage II fry, 4-6 days old, feed them 12 times evenly every 24 hours. Each time, feed them brine shrimp equal to 8% of their total body weight evenly in the pond. Turn off the oxygen before feeding and suck out the uneaten feed and feces 30 minutes after feeding.

[0044] Stage III fry, 7-20 days old, are fed 8 times a day evenly, with each feeding at a fixed point accounting for 5% of the total fish weight. The feed is a mixture of brine shrimp, crushed tilapia feed and algae powder in a mass ratio of 1:1:1. The oxygen is turned off before feeding, and the uneaten feed and feces are sucked out 30 minutes after feeding.

[0045] Stage IV fry, 21-40 days old. At 21 days old, the average total length is measured. The fry are divided into two batches according to their length relative to the average body length and raised separately to observe the differences in development and to prevent the spread of disease and complete death in only one batch. The first batch consists of large individuals with a body length greater than the average body length, and the second batch consists of small individuals with a body length less than or equal to the average body length. They are fed separately. They are fed evenly 4 times a day, with 5% of the total body weight of the fish being fed at a fixed point each time. The feed is a mixture of crushed tilapia feed and algae powder in a 2:1 mass ratio. The oxygen is turned off before feeding, and the uneaten feed and feces are removed 30 minutes after feeding.

[0046] Stage V fry, after 40 days of age, are transferred to outdoor ponds for rearing. They are fed once a day at 8:00 and 20:00, with each feeding being 3% of the total body weight of the fish. The feed consists of crushed tilapia feed and algae powder in a mass ratio of 4:1. The feeding amount is adjusted by weighing the fish every two weeks.

[0047] The fish fry showed little size variation and grew uniformly and rapidly, ultimately yielding 2,000 Tibetan blackfish fry with a survival rate of 95.24%.

[0048] Example 2

[0049] On April 8, 2024, 15 male parent fish with prominent head tubercles and milky white semen flowing out when the lower abdomen was gently pressed, which dispersed upon contact with water, were selected. 15 female parent fish without prominent head tubercles, with soft lower abdomens and swollen genital pores were also selected. The females were injected with luteinizing hormone-releasing hormone A2 (LARHA2) 3 μg / kg, and 10 hours later, they were injected again with LARHA2 10 μg / kg, dioxin maleate (DOM) 10 mg / kg, and human chorionic gonadotropin (HCG) 500 IU / kg. The males were injected with LARHA2 25 μg / kg, DOM 5 mg / kg, and HCG 400 IU / kg during the second injection of the females. All solvents were 0.9% physiological saline, and the single-fish injection dose was 0.5 mL.

[0050] Twelve hours later, the development of the female fish was checked. Artificial insemination was performed on the females who successfully ovulated. This involved squeezing out the female eggs, applying male sperm onto them, adding water, mixing thoroughly, letting it stand for 2 minutes, washing away mucus and impurities, and obtaining fertilized eggs. Females that failed to ovulate were returned to the water, and checked every hour. Ultimately, 14 females were successfully induced to spawn, a spawning rate of 93.33%, with a total of approximately 4800 eggs laid.

[0051] Fertilized eggs were placed in an incubation tank at a rate of 100 eggs / L. The flow rate was controlled to keep the fertilized eggs slowly tumbling in the water. Oxygen was continuously introduced during incubation, and the eggs were soaked in a 2% sodium chloride solution for 30 minutes every other day. The water temperature was 20℃. Fry hatching was observed 118 hours after fertilization. Since the egg membrane of the Tibetan blackhead fish is relatively thick, after observing hatching, the oxygen and water intake of the incubation tank were turned off, and half of the water was drained to increase the concentration of membrane-dissolving enzymes in the incubation tank, thus aiding hatching. Embryonic development was observed, with approximately 4500 neurulas developed, resulting in a fertilization rate of 93.75%. A total of 4000 Tibetan blackhead fish fry hatched, achieving a hatching rate of 88.89%.

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

[0053] Stage I fry, 0-3 days old, do not feed;

[0054] For stage II fry, 4-6 days old, feed them 12 times evenly over 24 hours a day. Each time, feed them brine shrimp equal to 8% of their total body weight. Turn off the oxygen before feeding and remove any uneaten feed and feces 30 minutes after feeding.

[0055] Stage III fry, 7-20 days old, are fed 8 times a day evenly, with each feeding at a fixed point accounting for 5% of the total fish weight. The feed is a mixture of brine shrimp, crushed tilapia feed and algae powder in a mass ratio of 1:1:1. The oxygen is turned off before feeding, and the uneaten feed and feces are sucked out 30 minutes after feeding.

[0056] Stage IV fry, 21-40 days old. At 21 days old, the average total length is measured. The fry are divided into two groups according to their length relative to the average body length. The first group consists of large individuals with a body length greater than the average body length, and the second group consists of small individuals with a body length less than or equal to the average body length. They are fed separately. They are fed evenly 4 times a day, with 5% of the total body weight of the fish being fed at a fixed location each time. The feed is a mixture of crushed tilapia feed and algae powder in a 2:1 mass ratio. The oxygen is turned off before feeding, and the uneaten feed and feces are removed 30 minutes after feeding.

[0057] Stage V fry, after 40 days of age, are transferred to outdoor ponds for rearing. They are fed once a day at 8:00 and 20:00, with each feeding being 3% of the total body weight of the fish. The feed consists of crushed tilapia feed and algae powder in a mass ratio of 4:1. The feeding amount is adjusted by weighing the fish every two weeks.

[0058] The fish fry showed little size difference among individuals, grew uniformly and rapidly, and ultimately yielded 3,800 Tibetan blackhead fish fry with a survival rate of 95%.

[0059] Comparative Example 1

[0060] On March 5, 2024, 10 male parent fish with prominent head tubercles and milky white semen flowing out when the lower abdomen is gently pressed, which disperses upon contact with water, were selected. Ten female parent fish were also selected, exhibiting no prominent head tubercles, a soft lower abdomen, and red and swollen genital pores. The females were given a single injection of 10 μg / kg of luteinizing hormone-releasing hormone A2 (LARHA2), 5 mg / kg of domperidone maleate (DOM), and 500 IU / kg of human chorionic gonadotropin (HCG). The males received half the dosage of the females. Both were treated with 0.9% physiological saline, and the single-fish injection dose was 0.5 mL.

[0061] Twelve hours later, the development of the female fish was checked. Artificial insemination was performed on the females who successfully ovulated. This involved squeezing out the female eggs, applying male sperm onto the eggs, adding water and mixing thoroughly, letting it stand for two minutes, washing away mucus and impurities, and obtaining fertilized eggs. Females that failed to ovulate were returned to the water, and checked every hour. Ultimately, three females were successfully induced to spawn, a spawning rate of 30%, with a total of approximately 1000 eggs laid.

[0062] Fertilized eggs were placed in an incubation tank at a rate of 100 eggs / L. The flow rate was controlled to keep the fertilized eggs slowly tumbling in the water. Oxygen was continuously introduced during incubation. Every other day, the eggs were soaked in a 2% sodium chloride solution for 30 minutes. The water temperature was maintained at 18-20℃. Fry hatching was observed 120 hours after fertilization. Embryonic development was monitored, with approximately 200 neurites developing, resulting in a fertilization rate of 20%. Ultimately, 60 Tibetan blackhead fish fry hatched, achieving a hatching rate of 30%. Observing fry hatching during incubation was relatively difficult.

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

[0064] From 0 to 3 days old, the eggs are not yet swimming horizontally and receive all nutrition from the yolk sac; no feeding is required.

[0065] Starting from 4 days old, the fish were fed microencapsulated feed at 5% of their body weight. The fish had poor feeding and were malnourished. All of them died after 15 days, with a survival rate of 0%.

[0066] Comparative Example 2

[0067] On March 10, 2024, 10 male parent fish with prominent head tubercles and milky white semen flowing out when the lower abdomen was gently pressed, which dispersed upon contact with water, were selected; and 10 female parent fish without prominent head tubercles, with soft lower abdomens and swollen genital pores were selected. The female fish were given a single injection of 10 μg / kg of luteinizing hormone-releasing hormone A2 (LARHA2), 5 mg / kg of domperidone maleate (DOM), and 500 IU / kg of human chorionic gonadotropin (HCG). The male fish received half the injection dose as the female fish. The solvent for both was 0.9% physiological saline, and the injection dose per fish was 0.5 mL.

[0068] Twelve hours later, the development of the female fish was checked. Artificial insemination was performed on the females who successfully ovulated. This involved squeezing out the female eggs, applying male sperm onto the eggs, adding water and mixing thoroughly, letting it stand for two minutes, washing away mucus and impurities, and obtaining fertilized eggs. Females that failed to ovulate were returned to the water, and checked every hour. Ultimately, four females were successfully induced to spawn, a spawning rate of 40%, with a total of approximately 1200 eggs laid.

[0069] Fertilized eggs were placed in an incubation tank at a rate of 100 eggs / L. The flow rate was controlled to keep the fertilized eggs slowly tumbling in the water. Oxygen was continuously introduced during incubation. Every other day, the eggs were soaked in a 2% sodium chloride solution for 30 minutes. The water temperature was maintained at 18-20℃. Fry hatching was observed 118 hours after fertilization. Since the egg membrane of the Tibetan blackhead fish is relatively thick, after hatching was observed, the oxygen and water intake of the incubation tank were turned off, and half of the water was drained to increase the concentration of membrane-dissolving enzymes in the incubation tank, thus aiding hatching. Embryonic development was observed, with approximately 300 neurites developing, a fertilization rate of 25%. Ultimately, 260 Tibetan blackhead fish fry hatched, achieving a hatching rate of 86.67%.

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

[0071] From 0 to 3 days old, the eggs are not yet swimming horizontally and receive all nutrition from the yolk sac; no feeding is required.

[0072] For fish aged 4-39 days, feed them three times evenly over 24 hours a day, each time overfeeding the entire pond (10% of the total weight of the fry) with crushed tilapia feed. Turn off the oxygen before feeding and suck out the uneaten feed and feces 30 minutes after feeding.

[0073] After 40 days of age, the fish were fed twice daily, at 8:00 AM and 8:00 PM, with each feeding consisting of 3% of their total body weight in crushed tilapia feed. The feeding amount was adjusted every two weeks by weighing the fish. Significant size differences existed among the fry, and some were deficient in certain nutrients. Ultimately, 120 Tibetan tilapia fry were obtained, representing a survival rate of 46.15%.

[0074] Comparative Example 3

[0075] On March 15, 2024, ten male broodstock were selected, exhibiting prominent head tubercles and a milky white semen that dissipated upon contact with water when the lower abdomen was gently pressed. Ten female broodstock were also selected, exhibiting no prominent head tubercles, a soft lower abdomen, and swollen, red genital pores. The females were injected with luteinizing hormone-releasing hormone A2 (LARHA2) at 3 μg / kg. Ten hours later, they were injected again with LARHA2 at 10 μg / kg, dioxin maleate (DOM) at 10 mg / kg, and human chorionic gonadotropin (HCG) at 500 IU / kg. The males were injected with LARHA2 at 5 μg / kg, DOM at 5 mg / kg, and HCG at 400 IU / kg during the second injection of the females. All solvents were 0.9% physiological saline, and the single-tail injection dose was 0.5 mL.

[0076] Twelve hours later, the development of the female fish was checked. Artificial insemination was performed on the females who successfully ovulated. This involved squeezing out the female eggs, applying male sperm onto the eggs, adding water and mixing thoroughly, letting it stand for 2 minutes, washing away mucus and impurities, and obtaining fertilized eggs. Females that failed to ovulate were returned to the water, and checked every hour. Ultimately, nine females were successfully induced to spawn, achieving a spawning rate of 90%, with a total of approximately 2500 eggs laid.

[0077] Fertilized eggs were placed in an incubation tank at a rate of 100 eggs / L. The flow rate was controlled to keep the fertilized eggs slowly tumbling in the water. Oxygen was continuously introduced during incubation. Every other day, the eggs were soaked in a 2% sodium chloride solution for 30 minutes. The water temperature was maintained at 18-20℃. Fry hatching was observed 118 hours after fertilization. Since the egg membrane of the Tibetan blackhead fish is relatively thick, after hatching was observed, the oxygen and water intake of the incubation tank were turned off, and half of the water was drained to increase the concentration of membrane-dissolving enzymes in the incubation tank, thus aiding hatching. Embryonic development was observed, with approximately 2000 neurites developing, a fertilization rate of 80%. Ultimately, 1700 Tibetan blackhead fish fry hatched, a hatching rate of 85%.

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

[0079] From 0 to 3 days old, the eggs are not yet able to swim horizontally and are provided with all their nutrition by the yolk sac; no feeding is required.

[0080] Starting from 4 days old, algae were fed at 5% of the fry's body weight. The fry's feeding was poor, and the survival rate was 32.15%.

[0081] Comparative Example 4

[0082] On March 20, 2024, ten male parent fish with prominent head tubercles and milky white semen flowing out when the lower abdomen was gently pressed, which dispersed upon contact with water, were selected. Ten female parent fish without prominent head tubercles, with soft lower abdomens and swollen genital pores were also selected. The females were injected with luteinizing hormone-releasing hormone A2 (LARHA2) at 3 μg / kg. Ten hours later, they were injected again with LARHA2 at 10 μg / kg, dioxin maleate (DOM) at 10 mg / kg, and human chorionic gonadotropin (HCG) at 500 IU / kg. The males were injected with LARHA2 at 5 μg / kg, DOM at 5 mg / kg, and HCG at 400 IU / kg during the second injection of the females. All solvents were 0.9% physiological saline, and the single-fish injection dose was 0.5 mL.

[0083] Twelve hours later, the development of the female fish was checked. Artificial insemination was performed on the females who successfully ovulated. This involved squeezing out the female eggs, applying male sperm onto the eggs, adding water, mixing thoroughly, letting it stand for 2 minutes, washing away mucus and impurities, and obtaining fertilized eggs. Females that failed to ovulate were returned to the water, and checked every hour. Ultimately, 8 females were successfully induced to spawn, achieving an spawning rate of 80%, with a total of approximately 2400 eggs laid.

[0084] Fertilized eggs were placed in an incubation tank at a rate of 100 eggs / L. The flow rate was controlled to keep the fertilized eggs slowly tumbling in the water. Oxygen was continuously introduced during incubation. Every other day, the eggs were soaked in a 2% sodium chloride solution for 30 minutes. The water temperature was maintained at 18-20℃. Fry hatching was observed 118 hours after fertilization. Since the egg membrane of the Tibetan blackhead fish is relatively thick, after hatching was observed, the oxygen and water intake of the incubation tank were turned off, and half of the water was drained to increase the concentration of membrane-dissolving enzymes in the incubation tank, thus aiding hatching. Embryonic development was observed, with approximately 1700 neurulas developed, representing a fertilization rate of 70.8%. Ultimately, 1500 Tibetan blackhead fish fry hatched, achieving a hatching rate of 88.24%.

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

[0086] From 0 to 3 days old, the eggs are not yet able to swim horizontally and are provided with all their nutrition by the yolk sac; no feeding is required.

[0087] At 4-6 days old, feed the fish 12 times evenly over 24 hours a day. Each time, feed the fish with brine shrimp equal to 8% of their total body weight. Turn off the oxygen before feeding and remove any uneaten food and feces 30 minutes after feeding.

[0088] For fish aged 7-20 days, feed them 8 times a day at a fixed point each time, with each feeding consisting of brine shrimp and crushed tilapia feed mixed in a 1:1 mass ratio. Turn off the oxygen before feeding and remove any uneaten food and feces 30 minutes after feeding.

[0089] From 21 to 40 days old, the fish were fed four times a day at a fixed location, each time with 5% of their total body weight in feed, consisting of crushed tilapia feed. Aeration was turned off before feeding, and uneaten feed and feces were removed 30 minutes after feeding. After 40 days old, the fish were fed twice a day, at 8:00 AM and 8:00 PM, each time with 3% of their total body weight in feed, consisting of crushed tilapia feed. The feeding amount was adjusted every two weeks by weighing the fish. Significant size differences existed among the fry, and some were deficient in certain nutrients. Ultimately, 1000 Tibetan blackhead fry were obtained, with a survival rate of 66.67%.

[0090] Comparative Example 5

[0091] On March 20, 2024, ten male parent fish with prominent head tubercles and milky white semen flowing out when the lower abdomen was gently pressed, which dispersed upon contact with water, were selected. Ten female parent fish without prominent head tubercles, with soft lower abdomens and swollen genital pores were also selected. The females were injected with luteinizing hormone-releasing hormone A2 (LARHA2) at 3 μg / kg. Ten hours later, they were injected again with LARHA2 at 10 μg / kg, dioxin maleate (DOM) at 10 mg / kg, and human chorionic gonadotropin (HCG) at 500 IU / kg. The males were injected with LARHA2 at 5 μg / kg, DOM at 5 mg / kg, and HCG at 400 IU / kg during the second injection of the females. All solvents were 0.9% physiological saline, and the single-fish injection dose was 0.5 mL.

[0092] Twelve hours later, the development of the female fish was checked. Artificial insemination was performed on the females who successfully ovulated. This involved squeezing out the female eggs, applying male sperm onto the eggs, adding water and mixing thoroughly, letting it stand for 2 minutes, washing away mucus and impurities, and obtaining fertilized eggs. Females that failed to ovulate were returned to the water, and checked every hour. Ultimately, nine females were successfully induced to spawn, achieving a spawning rate of 90%, with a total of approximately 2500 eggs laid.

[0093] Fertilized eggs were placed in an incubation tank at a rate of 100 eggs / L. The flow rate was controlled to keep the fertilized eggs slowly tumbling in the water. Oxygen was continuously introduced during incubation. Every other day, the eggs were soaked in a 2% sodium chloride solution for 30 minutes. The water temperature was maintained at 18-20℃. Fry hatching was observed 118 hours after fertilization. Since the egg membrane of the Tibetan blackfish is relatively thick, after observing hatching, the oxygen and water intake of the incubation tank were turned off, and half of the water was drained to increase the concentration of membrane-dissolving enzymes in the incubation tank, thus aiding hatching. Embryonic development was observed, with approximately 2100 eggs developing into neurites, representing a fertilization rate of 84.0%. Ultimately, 1800 Tibetan blackfish fry hatched, achieving a hatching rate of 85.71%.

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

[0095] Do not feed infants aged 0-3 days;

[0096] At 4-6 days old, feed the fish 12 times evenly over 24 hours a day. Each time, feed the fish with brine shrimp equal to 8% of their total body weight. Turn off the oxygen before feeding and remove any uneaten food and feces 30 minutes after feeding.

[0097] For fish aged 7-20 days, feed them 8 times a day at a fixed point each time, with each feeding consisting of 5% of the total body weight of the fish. The feed is a mixture of algae powder and crushed tilapia feed in a mass ratio of 4:1. Turn off the oxygen before feeding and remove any uneaten feed and feces 30 minutes after feeding.

[0098] For fish aged 21-40 days, feed them 4 times a day at a fixed point each time, with each feeding consisting of 5% of the total body weight of the fish. The feed is a mixture of algae powder and crushed tilapia feed in a mass ratio of 4:1. Turn off the oxygen before feeding and remove any uneaten feed and feces 30 minutes after feeding.

[0099] After 40 days of age, the fish were fed twice daily, at 8:00 AM and 8:00 PM, with each feeding consisting of 3% of their total body weight. Algae powder and crushed tilapia feed were mixed at a 4:1 mass ratio. The feeding amount was adjusted every two weeks by weighing the fish. Significant size differences existed among the fry, and they suffered from nutritional deficiencies. Ultimately, 800 Tibetan blackhead fry were obtained, with a survival rate of 44.44%.

[0100] The statistical results of induced spawning 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] As shown in Table 1, compared with the comparative example, the spawning, fertilization and hatching methods of the present invention have high spawning rate, fertilization rate and hatching rate and stable effect. The spawning injection method of the present invention can significantly improve the spawning rate and hatching rate; assisted hatching can significantly increase the hatching rate of Tibetan blackfish; in the seedling cultivation process of the embodiment, by optimizing the feeding method, feeding the seedlings in batches in stage IV, and adding algae powder to the feed, the seedling survival rate can be further significantly increased to over 90%.

[0104] This invention details the selection of parent fish, formulation of spawning stimulants, duration of effect, fertilization method, hatching method, and fry rearing method in the artificial breeding of Tibetan blackfish. The method achieves high spawning rate, fertilization rate, hatching rate, and fry survival rate. Following this method, artificial breeding and fry rearing of Tibetan blackfish can be completed, enabling the artificial breeding, large-scale production, and stock enhancement of Tibetan blackfish. This is of great significance for the protection and development of Tibetan blackfish resources.

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

Claims

1. A method for artificially breeding Tibetan cuttlefish, characterized in that, Includes the following steps: Sexually mature male and female parent fish were selected. The female fish were injected with oxytocin twice. The first injection was luteinizing hormone-releasing hormone A2 at a dose of 3 μg / kg. The second injection was luteinizing hormone-releasing hormone A2, dioxonone maleate, and human chorionic gonadotropin (hCG) at a dose of 10 μg / kg, dioxonone maleate at a dose of 10 mg / kg, and hCG at a dose of 500 IU / kg. The interval between the two injections was 10 hours. The male fish was injected with luteinizing hormone-releasing hormone A2, dioxonone maleate, and human chorionic gonadotropin in a single injection during the second injection of the female fish; the injection dose of luteinizing hormone-releasing hormone A2 was 5 μg / kg, the injection dose of dioxonone maleate was 5 mg / kg, and the injection dose of human chorionic gonadotropin was 400 IU / kg. In vitro artificial insemination was performed on the female fish according to her developmental status to obtain fertilized eggs; The fertilized eggs were incubated. During the incubation process, after the fry broke through the membrane, the concentration of membrane-dissolving enzymes in the incubation system was increased to artificially assist in the membrane breaking, thus obtaining Tibetan blackhead fish fry. The Tibetan cuttlefish fry were fed in stages; The phased feeding is as follows: Phase I: 0-3 days old, do not feed; Phase II: 4-6 days old, feed brine shrimp every 2 hours, each time feeding 8% of the fish's total body weight; Stage III: 7-20 days old, feed a mixture of brine shrimp, crushed tilapia feed and algae powder, once every 3 hours; Stage IV: 21-40 days old. The fry are divided into two batches based on their average body length: those greater than the average body length and those less than or equal to the average body length. They are fed separately, once every 6 hours. The feed consists of a mixture of crushed tilapia feed and algae powder. Phase V: After 40 days, transfer to an outdoor pond for rearing. Feed once a day at 8:00 and 20:

00. The feed is a mixture of crushed tilapia feed and algae powder. Weigh the fish every two weeks to adjust the feeding amount. In stage III, the mass ratio of the brine shrimp, the crushed tilapia feed, and the algae powder is 1:1:1, and the feeding amount each time is 5% of the total fish weight. In stage IV, the mass ratio of the crushed tilapia feed to the algae powder is 2:1, and the feeding amount is 5% of the total fish weight each time. In stage V, the mass ratio of the crushed tilapia feed to the algae powder is 4:1, and the amount fed each time is 3% of the total fish weight.

2. The method for artificial breeding of Tibetan cuttlefish according to claim 1, characterized in that, The in vitro artificial insemination process involves squeezing out the female fish eggs, then squeezing the male fish sperm onto the eggs, adding water and stirring to mix evenly, letting it stand for 2 minutes, washing away the mucus and impurities, and obtaining fertilized eggs.

3. The method for artificial breeding of Tibetan cuttlefish 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 for 30 minutes every other day at a temperature of 18-20°C.

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