Cross breeding method for anti-ichthyophthirius mongholicus schizothorax prenanti

Abstract

The invention discloses a cross breeding method for anti-ichthyophthirius mongholicus schizothorax prenanti. The method comprises the following steps: 1) selecting parents; (2) carrying out intensive cultivation on the parents; 3) synchronous artificial spawning induction; 4) artificial insemination; 5) hatching management; (6) breeding the schizothorax prenanti (female) and percocypris pingi (sex) hybrid fry; and (7) measuring the ichthyophthirius multifiliis resistance and high-temperature tolerance of the schizothorax prenanti (female) * percocypris pingi (sex) hybrid fry. The ichthyophthirius ichthyophthirius infection rate of the bred schizothorax prenanti (female) and percocypris pingi (sex) hybrid offspring is reduced by 31.95% compared with that of the schizothorax prenanti (female) and percocypris pingi (sex) selfing offspring; the death rate is only 0.33% and is reduced by 73% compared with that of the selfing filial generation of the schizothorax prenanti; the high-temperature tolerance range of the schizothorax prenanti selfing offspring is improved by 4-5 DEG C compared with that of the schizothorax Therefore, the method disclosed by the invention can be used for successfully culturing a hybrid variety which is high in ichthyophthirius multifiliis resistance, relatively high in economic value and relatively high in high-temperature tolerance.

Description

Technical Field

[0001] This invention relates to the field of animal breeding technology, specifically to a method for hybrid breeding of schizothorax fish resistant to Ichthyophthirius multifiliis. Background Technology

[0002] *Schizothorax prenanti*, belonging to the Cyprinidae family and the *Schizothorax* genus, is a rare and endemic fish species found in the upper reaches of the Yangtze River. With its delicious and nutritious flesh, *Schizothorax prenanti* is a valuable economic fish species, particularly important in Sichuan, Chongqing, and Guizhou provinces. However, juvenile *Schizothorax prenanti* are highly susceptible to Ichthyophthirius multifiliis (white spot disease), and currently, there is no effective treatment for this infection in juveniles, resulting in extremely low survival rates for fry. Furthermore, *Schizothorax prenanti* is intolerant of high temperatures, with an optimal culture temperature of 10–25°C. In many parts of Sichuan, summer water temperatures consistently exceed 25°C, severely limiting the aquaculture range. Therefore, developing hybrid varieties with strong resistance to Ichthyophthirius multifiliis and greater heat tolerance is an urgent problem to be solved. Distant hybridization between different species is an important method for creating new fish varieties. Distant hybridization, by combining the genomic information of two species, produces hybrids that possess the genetic characteristics of both parents and may even exhibit traits that surpass those of both parents. However, due to nucleocytoplasmic incompatibility and other reasons, most species cannot engage in distant hybridization. Even after artificial insemination, the hybrids often fail to survive or exhibit extremely poor viability. Even if some species do survive, they may not necessarily demonstrate significant hybrid vigor. Extensive research is needed to determine whether distant hybridization is possible between fish species, and the performance of the hybrids must be evaluated to decide whether they are suitable for artificial aquaculture. Summary of the Invention

[0003] The purpose of this invention is to solve the above-mentioned problems existing in the prior art and to provide a method for hybrid breeding of schizothorax fish resistant to Ichthyophthirius multifiliis.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A method for hybrid breeding of ichthyophthirius multifiliis-resistant schizothorax fish, comprising the following steps:

[0005] S1, Parent Selection

[0006] In the first year, from June to August, select 4-6 year old sexually mature male Golden Sand Bass and Carp as the father; select 4-6 year old sexually mature female Qi-Mouth Split-Bellied Fish as the mother.

[0007] S2, Parental Enhancement Breeding

[0008] From the selection of parent fish until the second year of breeding, maintain the culture water temperature of female Schizothorax 'Qikou' and male Carp 'Jinsha' at 14-21℃; dissolved oxygen in the water ≥6mg / L;

[0009] S3, Artificial labor induction

[0010] When the female *Schizothorax schistosomiasis* reaches sexual maturity, an oxytocin agent is injected. Approximately 10 days before the female reaches sexual maturity, the male *Carassius amurensis* is injected with an oxytocin agent at a dose 10% of the female's dose. The oxytocin agent is administered via a single injection at the base of the pectoral fin, with a needle depth ≤0.5cm. After injection, the female *Schizothorax schistosomiasis* and the male *Carassius amurensis* are transferred to two separate oxytocin tanks, and the water flow is increased to stimulate estrus. The estrus response behavior of the female *Schizothorax schistosomiasis* is observed. When a small number of eggs are released from the abdomen of the female *Schizothorax schistosomiasis* by gentle squeezing, artificial insemination is immediately performed.

[0011] S4, Artificial Insemination

[0012] Wipe the water off the female Schizothorax 'Qikou' and gently press her abdomen to squeeze the mature eggs into a dry basin. Then squeeze in the sperm from the male Carp and Perch. Stir the eggs and sperm evenly with a feather, then add water. The volume ratio of eggs to water should be 1:3 to 1:5. After letting it stand for 2 minutes, place it in an incubation box for running water incubation.

[0013] S5, Incubation

[0014] Fertilized eggs are evenly spread in the hatching frame at a density of 10,000 eggs / plate and hatched in flowing water at a temperature of 14-17℃. After 6-8 days of incubation, the hybrid fish fry hatch.

[0015] S6, Hybrid Fish Breeding

[0016] After the hybrid fry swim horizontally, they are transferred to the fry rearing pond for their first feeding, which consists of Artemia nauplii. When they are hybrid juveniles, they are fed fresh bloodworms and artificial microparticle feed.

[0017] S7. Hybrid Fish Performance Testing

[0018] At 90 days of age, the hybrid fish's resistance to Ichthyophthirius multifiliis and its tolerance to high temperatures were determined.

[0019] Preferably, the female *Schizothorax chevron* weighs over 1.8 kg, is robust, disease-free, injury-free, and deformed, and has a noticeably enlarged abdomen that is soft and elastic to the touch, with a red genital pore.

[0020] Preferably, the male carp of the Golden Sand Bass weighs over 1.5 kg, is robust, disease-free, injury-free, and deformed, and has a slightly protruding genital opening, with a milky white substance flowing out when the abdomen is gently squeezed.

[0021] Preferably, in S2, from February of the second year to the breeding season, the pond is kept flushed; the breeding season is April of the second year; male golden sand perch are fed small miscellaneous fish, and female fish with open mouths and split bellies are fed artificial compound feed.

[0022] Further preferably, the oxytocin is a mixture of human chorionic gonadotropin (HCG), luteinizing hormone-releasing hormone analog LHRH-A2, and dioxone maleate (DOM), with an injection dosage of: HCG 1000U / kg, LHRH-A2 10ug / kg, and DOM 3mg / kg.

[0023] In a further preferred embodiment, in step S3, the development of the parent fish is checked every 6 hours 24 hours after the injection of the oxytocin.

[0024] The beneficial effects of this invention are as follows:

[0025] 1. The hybrid fish of *Schizothorax chevron* bred by the method of this invention have strong resistance to Ichthyophthirius multifiliis. When Ichthyophthirius multifiliis infection test was conducted at 90 days of age, it was found that the infection rate of Ichthyophthirius multifiliis in the hybrid fish was reduced by 31.95% compared with the self-crossed offspring of *Schizothorax chevron*; the mortality rate was only 0.33%, which was 73% lower than that of the self-crossed offspring of *Schizothorax chevron*.

[0026] 2. The hybrid fish of *Schizothorax davidii* bred by the method of this invention are resistant to high temperatures, with a high temperature tolerance range that is 4-5°C higher than that of *Schizothorax davidii*. They maintain a 100% survival rate at a continuous 24-hour water temperature of 31°C, while the survival rate of self-crossed offspring of *Schizothorax davidii* is only 80% at a continuous 24-hour water temperature of 29°C, and only 0% at a continuous 24-hour water temperature of 31°C. Attached Figure Description

[0027] Figure 1 This is the natural state of the hybrid fish, *Schizothorax micranthum*, bred in Example 1 of this invention, in water.

[0028] Figure 2 This refers to the hybrid fish of the Qikou Schizothorax fish bred in Example 1 of this invention. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0030] Example 1

[0031] 1. Parent selection

[0032] In the first year, from June to August, select sexually mature female schizothorax bream aged 4-6 years from the Yalong River basin. The females should be over 1.8 kg in size, healthy, disease-free, injury-free, and without deformities. Their abdomens should be noticeably swollen, soft and elastic to the touch, and their genital openings should be red.

[0033] Select sexually mature golden perch sires aged 4-6 years from the Yalong River basin. The male fish should be over 1.5 kg in size, healthy, disease-free, injury-free, and without deformities. The genital opening should be slightly protruding, and a milky white substance should flow out when the abdomen is gently squeezed.

[0034] 2. Enhanced parent breeding;

[0035] From the selection of parent fish until breeding in the second year (generally mid-April), maintain the water temperature at 14-24℃ and dissolved oxygen ≥6mg / L. From February of the second year until breeding time, keep the pond flushed. Feed the golden sand perch and common carp with small miscellaneous fish, and feed the Qi-mouthed schizothorax with formulated feed.

[0036] 3. Artificial induction of labor;

[0037] Approximately 10 days before the female *Schizothorax bream* reaches sexual maturity, male *Perelope bream* are injected with an oxytocin-inducing agent at a dose 10% of the dose used on the female, ensuring synchronized maturation of their gonads. The oxytocin-inducing agent is injected again when the female reaches sexual maturity. A single injection is used, with the injection site at the base of the pectoral fin and a needle depth ≤0.5 cm. The oxytocin-inducing agents consist of a mixture of human chorionic gonadotropin (HCG), luteinizing hormone-releasing hormone analogue (LHRH-A2), and dioxin maleate (DOM), with the following dosages: HCG 1000 U / kg, LHRH-A2 10 ug / kg, and DOM 3 mg / kg. After injection, the broodstock are transferred to a spawning tank, and the water flow is increased to stimulate estrus. The estrus response behavior of the broodstock is observed. Twenty-four hours after injection, the development of the broodstock is checked every 6 hours to prevent over-maturation and degeneration of the eggs. When a small number of eggs are released from the abdomen of a female golden sand perch by gently squeezing it, artificial insemination should be performed immediately.

[0038] 4. Artificial insemination (hybridization: female Schizothorax 'Qikou' × male Carp 'Jinsha');

[0039] Wipe the water off the female Schizothorax bream's body, gently press its abdomen to squeeze the mature eggs into a dry basin, and squeeze in the sperm from the male Carp. Stir the eggs and sperm evenly with a feather, then add an appropriate amount of water. After letting it stand for 2 minutes, it can be placed in an incubation frame for flowing water incubation.

[0040] 5. Hatching;

[0041] Fertilized eggs are evenly spread in hatching frames at a density of 10,000 eggs per frame and incubated in flowing water at a temperature maintained at 14-17℃. After 6-8 days of incubation, the fry hatch.

[0042] 6. Hybrid fish breeding;

[0043] After the fry swim horizontally, they are transferred to a fry rearing pond for their first feeding, which consists of nauplius larvae of Artemia. During the juvenile stage, they are fed fresh bloodworms and artificial microparticle feed.

[0044] Example 2

[0045] Performance tests were conducted on the hybrid offspring of *Schizothorax girdorax* bred in Example 1, the self-crossed offspring of *Carassius amurensis* collected from the Yalong River Basin, and the self-crossed offspring of *Schizothorax girdorax*.

[0046] 1. Ichthyophthirius multifiliis resistance test

[0047] Three types of fish fry aged 90 days were placed in the same rearing tank of goldfish infected with Ichthyophthirius multifiliis for a 7-day experiment. The infection rate and mortality rate of Ichthyophthirius multifiliis in the three types of fish fry were observed and statistically analyzed (Table 1 and Table 2). The results showed that the infection rate of the offspring of *Schizothorax micranthum* (♀) × *Carassius amurensis* (♂) was 69.05% on the seventh day, which was 31.95% lower than that of the self-crossed offspring of *Schizothorax micranthum*; the mortality rate was only 0.33%, which was 73% lower than that of the self-crossed offspring of *Schizothorax micranthum*.

[0048] Table 1. Infection rate of Ichthyophthirius multifiliis in two types of fish fry

[0049]

[0050] Table 2. Mortality rate of Ichthyophthirius multifiliis in two types of fish fry

[0051]

[0052] 2. High temperature resistance test

[0053] Schizothorax 'Qikou' (♀) × Golden Sand Bass The offspring maintained a 100% survival rate at a continuous 24-hour water temperature of 31℃, while the survival rate of self-crossed offspring of *Schizothorax cherubim* was only 80% at a continuous 24-hour water temperature of 29℃, and only 0% at a continuous 24-hour water temperature of 31℃. The data are shown in Table 3.

[0054] Table 3. Behavior and survival rate of two types of fish fry under different water conditions

[0055]

[0056]

[0057] As shown in Table 1-3, at 90 days of age, the resistance to Ichthyophthirius multifiliis and the high temperature tolerance range of the hybrid fish were measured. The resistance to Ichthyophthirius multifiliis of the hybrid fish was significantly higher than that of the self-crossed offspring of Schizothorax giardinis. The high temperature tolerance range of the hybrid fish was 4-5℃ higher than that of the self-crossed offspring of Schizothorax giardinis.

[0058] The specification and drawings of this invention are intended to be illustrative rather than restrictive. Based on this invention, those skilled in the art can make substitutions and modifications to some of the technical features without creative effort, and all such modifications are within the scope of protection of this invention.

Claims

1. A method for hybrid breeding of *Schizothorax micranthum* resistant to *Ichthyophthirius multifiliis*, characterized in that, Includes the following steps: S1, Parent Selection In the first year, from June to August, select 4-6 year old sexually mature male Golden Sand Bass and Carp as the father; select 4-6 year old sexually mature female Qi-Mouth Split-Bellied Fish as the mother. S2, Parental Enhancement Breeding From the selection of parent fish until the second year of breeding, maintain the culture water temperature of female Schizothorax 'Qikou' and male Carp 'Jinsha' at 14-21℃; dissolved oxygen in the water ≥6mg / L; S3, Artificial labor induction When the female *Schizothorax schistosomiasis* reaches sexual maturity, an oxytocin agent is injected. Approximately 10 days before the female reaches sexual maturity, the male *Carassius amurensis* is injected with an oxytocin agent at a dose 10% of the female's dose. The oxytocin agent is administered via a single injection at the base of the pectoral fin, with a needle depth ≤0.5cm. After injection, the female *Schizothorax schistosomiasis* and the male *Carassius amurensis* are transferred to two separate oxytocin tanks, and the water flow is increased to stimulate estrus. The estrus response behavior of the female *Schizothorax schistosomiasis* is observed. When a small number of eggs are released from the abdomen of the female *Schizothorax schistosomiasis* by gentle squeezing, artificial insemination is immediately performed. S4, Artificial Insemination Wipe the water off the female Schizothorax 'Qikou' and gently press her abdomen to squeeze the mature eggs into a dry basin. Then squeeze in the sperm from the male Carp and Perch. Stir the eggs and sperm evenly with a feather, then add water. The volume ratio of eggs to water should be 1:3 to 1:

5. After letting it stand for 2 minutes, place it in an incubation box for running water incubation. S5, Incubation Fertilized eggs are evenly spread in the hatching frame at a density of 10,000 eggs / plate and hatched in flowing water. The water temperature is maintained at 14-17℃. After 6-8 days of incubation, the hybrid fish fry hatch. S6, Hybrid Fish Breeding After the hybrid fry swim horizontally, they are transferred to the fry rearing pond for their first feeding, which consists of Artemia nauplii. When they are hybrid juveniles, they are fed fresh bloodworms and artificial microparticle feed. S7. Hybrid Fish Performance Testing At 90 days of age, the hybrid fish's resistance to Ichthyophthirius multifiliis and its tolerance to high temperatures were determined.

2. The method according to claim 1, characterized in that, The female *Schizothorax schizophrenia* should weigh over 1.8 kg, be robust, disease-free, injury-free, and without deformities, with a noticeably enlarged abdomen that is soft and elastic to the touch, and a red genital pore.

3. The method according to claim 1, characterized in that, The male carp of the Golden Sand Bass must weigh over 1.5kg, be robust, free from disease, injury, and deformity, and have a slightly protruding genital opening. A milky white substance will flow out when the abdomen is gently squeezed.

4. The method according to claim 1, characterized in that, In S2, from February of the second year to the breeding season, the pond is kept flushed; the breeding season is April of the second year; male golden sand perch are fed small miscellaneous fish, and female fish with open mouths and split bellies are fed artificial compound feed.

5. The method according to claim 1, characterized in that, The oxytocin is a mixture of human chorionic gonadotropin (HCG), luteinizing hormone-releasing hormone analog LHRH-A2, and dioxone maleate (DOM), with an injection dosage of: HCG 1000U / kg, LHRH-A2 10ug / kg, and DOM 3mg / kg.

6. The method according to claim 1, characterized in that, In S3, 24 hours after the injection of the oxytocin, the development of the parent fish is checked every 6 hours.

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