A method for culturing tetraploid Haliotis discus hannai

Abstract

The present invention relates to a method for cultivating tetraploid Haliotis discus hannai, belonging to the technical field of genetic breeding of aquaculture varieties. The method includes the selection of broodstock, the ripening promotion of broodstock, the spawning induction of broodstock, artificial insemination, and the inhibition of polar body release of fertilized eggs; the selected male and female broodstock are separately ripened; the female broodstock is induced to spawn by means of air-drying, running water stimulation, and soaking in hydrogen peroxide seawater; the eggs are preferably selected and fertilized by adding high-concentration sperm; cytochalasin B is used to inhibit the release of the first polar body of the fertilized eggs. The unique three-stage gonad ripening promotion method and spawning induction method for triploid female abalones of the present invention can obtain more high-quality eggs; the present invention uses cytochalasin B to inhibit the release of the second polar body of fertilized eggs to obtain tetraploids, with a stable induction rate of more than 96%, and a large number of tetraploid Haliotis discus hannai can be obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic breeding of aquaculture varieties, and particularly relates to a method for cultivating tetraploid Haliotis discus hannai. Background Art

[0002] Haliotis discus hannai is the main cultivated variety of abalone in China and is known as the top of the eight treasures of seafood products. China is the largest country for cultivating Haliotis discus hannai, and it is cultivated along the coast from Liaoning to Fujian. Under natural conditions, abalones have two gonad discharge periods in spring and autumn. After the gonad discharge, the physical condition of abalones is weak, and they are prone to death due to environmental stress during summer and winter.

[0003] In recent years, due to the fast growth rate and strong high-temperature adaptability of green abalone (American green abalone × Haliotis discus hannai), the scale of its cultivation has increased rapidly year by year, resulting in a large-scale reduction in the cultivation space of the native variety Haliotis discus hannai in China. Therefore, there is an urgent need for a new variety of Haliotis discus hannai with a fast growth rate, high yield, and strong high-temperature adaptability to change the current situation of the sharp reduction in the cultivation scale of the native variety.

[0004] Triploid shellfish have three sets of chromosomes and thus have excellent cultivation traits such as fast growth and high sterility. Triploid Pacific oysters, triploid Fujian oysters, and triploid hybrid oysters have become the main oyster cultivation varieties in the north and south of China. The triploidization of Haliotis discus hannai may be an important direction to solve the current problems in the Haliotis discus hannai industry in China.

[0005] Hybridization of tetraploid (♂) and diploid (♀) is currently the common method for producing triploid oyster seedlings internationally. Therefore, obtaining viable tetraploid Haliotis discus hannai is the key to producing triploids. In 1994, Guo Ximing and Allen successfully induced viable tetraploid Pacific oysters and invented the technology of hybridizing tetraploid oysters with diploids to produce 100% triploid oysters. According to existing reports, tetraploid Haliotis discus hannai can be obtained by inhibiting the release of polar bodies in normal fertilized eggs, or by inhibiting the release of the first polar body (PB1) of fertilized eggs after fertilizing eggs of triploid Haliotis discus hannai with diploid sperm (CN 114600807 A).

[0006] Currently, in the process of producing tetraploid Haliotis discus hannai, there are problems such as difficulties in promoting the maturation of the female gonads of triploid Haliotis discus hannai, it is difficult to obtain a large number of mature oocytes, low hatching rate after induction, low larval survival rate, unstable ploidy rate of juvenile tetraploids, and it is difficult to obtain viable adult tetraploid Haliotis discus hannai. As a result, there is currently no report on obtaining adult tetraploid Haliotis discus hannai. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for culturing tetraploid Haliotis discus hannai, which can greatly increase the quantity and quality of ovulation of triploid female parents, stably obtain tetraploid Haliotis discus hannai, and successfully cultivate it to adulthood for the first time, laying a foundation for the triploidization of the Haliotis discus hannai industry.

[0008] The technical solutions and steps of the present invention include the selection of broodstock, the ripening of broodstock, the induced spawning of broodstock, artificial insemination, and the inhibition of polar body release of fertilized eggs.

[0009] The selection of the broodstock: Both male and female broodstock are required to be 2 - 3 years old, with a shell length ≥ 10 cm and no external injuries.

[0010] The ripening of the broodstock: The male and female broodstock are ripened separately. To synchronize the development of female parents, female broodstock are ripened 30 - 40 days earlier than male broodstock; the initial water temperature for ripening triploid female broodstock is 10 °C, and it is heated by 1 - 2 °C per day to 15 °C, and this temperature is maintained for 15 - 20 days; thereafter, it is heated by 4 - 5 °C per day to 27 °C, and this high - temperature stimulation is carried out for 8 days; then it is cooled by 1 °C per day to 22 °C, and it is cultured at this temperature for 100 - 110 days until the gonads are developed and mature; diploid male broodstock are ripened according to the conventional method; during the ripening process, the seawater salinity is maintained at 30‰.

[0011] The induced spawning of the broodstock: Triploid female broodstock are air - dried at room temperature of 20 °C for 2 - 3 hours; immediately after air - drying, they are put into saturated oxygen - filled seawater at 20 °C and stimulated by running water for 1 - 1.5 h; then they are soaked in pre - prepared seawater containing hydrogen peroxide for 1 h; the soaked female broodstock are put into seawater irradiated by ultraviolet light to await spawning; diploid male broodstock are induced to spawn according to the conventional method.

[0012] The artificial insemination: The female broodstock are sorted according to the diameter of the eggs they produce from large to small, and the top 50% of the female broodstock are selected, and their eggs are collected with a 300 - mesh silk screen; fertilization is carried out according to the ratio of the number of eggs to sperm of 1:400, and the fertilization temperature is 23 °C and the salinity is 30‰.

[0013] The inhibition of polar body release of fertilized eggs: When the fertilized eggs begin to release the first polar body, the fertilized eggs are put into seawater containing 0.3 - 1.2 mg / L cytochalasin B for treatment for 15 - 20 min; the fertilized eggs are washed to remove the drug and excess sperm; they are hatched and cultured according to the conventional method; the tetraploid ploidy rate is measured at the veliger larva stage.

[0014] Furthermore, the female broodstock is triploid Haliotis discus hannai, and the male broodstock is diploid Haliotis discus hannai.

[0015] Furthermore, the method for ripening male broodstock: After male broodstock enter the room, they are first temporarily cultured at the natural water temperature for 2 - 3 days, and then the temperature is continuously increased at a gradient of 1 °C per day. When the water temperature rises to 20 °C, they are cultured at a constant temperature until the gonads are mature.

[0016] Further, the prepared seawater containing hydrogen peroxide has been irradiated with ultraviolet light at 1500 mW·h / L and contains 3×10 -3 M.

[0017] Advantages of the present invention compared with the prior art:

[0018] 1. Selecting triploid female abalones preferably: Select triploid female abalones whose gonads have developed under natural conditions. The selected female abalones are required to have a long and narrow gonad area and a large coverage area, and then their ploidy is determined by a flow cytometer. This method is beneficial to obtaining triploid female abalones with easily developed gonads and a large amount of mature oocytes, and can save production costs.

[0019] 2. Due to the difficulty in gonad development and spawning of triploid female abalones, the prior art cannot obtain a large number of high-quality eggs. The present invention uses a unique three-stage method for promoting gonad maturation and inducing spawning of triploid female abalones. Three different temperatures are used in the maturation process: first, culture at 15°C for 15 - 20 days. At this time, the food intake of abalones is normal, the gonads hardly develop, and the triploid female abalones are more plump, accumulating energy for subsequent gonad development; then stimulate the triploid female abalones at a high temperature of 27°C for 8 days to stimulate gonad development and make the gonads develop faster; finally, develop in seawater at 22°C until the gonads are mature. The three steps of inducing spawning are also different from the prior art: the drying time is increased, the flowing water stimulation is increased, and hydrogen peroxide is added to the seawater with high-dose ultraviolet irradiation for soaking female abalones. With this three-stage method for promoting maturation and inducing spawning, more high-quality eggs can be obtained from triploid female abalones.

[0020] 3. The prior art does not screen the size of eggs, and conventional sperm concentration is used for fertilization. The present invention ranks the female parents according to the diameter of the eggs they produce from large to small, and selects the eggs of the top 50% of the female parents. The selected eggs are large and have a high maturity, with more cytoplasm to support the development of tetraploids, which is more conducive to the survival of tetraploid Haliotis discus hannai. The present invention uses a high concentration of diploid male abalone sperm during fertilization, effectively improving the fertilization rate and the fertilization synchronization rate.

[0021] 4. The present invention uses cytochalasin B to inhibit the release of the first polar body of the fertilized egg. This method efficiently and stably induces tetraploid Haliotis discus hannai, and the induction rate reaches more than 96%, and a large number of tetraploid Haliotis discus hannai can be obtained. Specific embodiments

[0022] The technical solution of the present invention will be further explained below through examples, but the protection scope of the present invention is not limited by any form of the examples.

[0023] Example 1

[0024] A method for culturing tetraploid Haliotis discus hannai, the method comprising the selection of broodstock, the ripening of broodstock, the spawning induction of broodstock, artificial insemination, and the inhibition of polar body release of fertilized eggs, and the specific steps are as follows:

[0025] 1) Selection of broodstock:

[0026] A) Selection of female broodstock: Select triploid Haliotis discus hannai individuals with a shell length of ≥10 cm, no external injuries, a narrow and long gonad area, a large coverage area, and a small number of oocytes from a population of 2-year-old triploid Haliotis discus hannai, and determine their ploidy using a flow cytometer.

[0027] B) Selection of male broodstock: Select male individuals of diploid Haliotis discus hannai aged 2 - 3 years with a shell length of ≥10 cm and no external injuries.

[0028] 2) Ripening of broodstock: The identified triploid female abalones and diploid male abalones are cultured separately. The initial breeding water temperature is 10°C, and the temperature is increased by 1°C per day to 15°C, and they are cultured at this temperature for 15 days. In order to synchronize the development of triploid female abalones and diploid male abalones, the triploid female abalones are ripened by advancing the temperature increase: The water temperature in the culture pond of triploid female abalones is increased to 19°C on the first day, 23°C on the second day, 27°C on the third day and maintained for 8 days, and then the water temperature is decreased to 22°C (1°C decrease per day). After 30 days of cultivation, it is observed under a microscope that the gonad tissue fluid of triploid female abalones is less and the oocytes are clear. At this time, the diploid male abalones start to be gonadally ripened and induced to spawn by the conventional method: After the male broodstock enters the room, it is first temporarily cultured in natural water temperature for 2 - 3 days, and then the temperature is continuously increased at a gradient of 1°C per day. When the water temperature rises to 20°C, it is cultured at a constant temperature until the gonads are mature. The daily management methods of triploid female abalones and diploid male abalones are the same, and the water is changed 100% every day, and the seawater salinity is 30‰.

[0029] 3) Spawning induction of broodstock: After the triploid female abalones and diploid male abalones develop synchronously to sexual maturity, 100 triploid female abalones with well-developed gonads are taken, air-dried in a seeding room at 20°C for 2.5 h, and then placed in a saturated oxygenated water tank at 20°C and stimulated with running water at 20°C. After 1 h of running water, the triploid female abalones have strong adsorption ability and are active. Then the female abalones are placed in the prepared seawater at 23°C (hydrogen peroxide 3X10 -3 M, ultraviolet irradiation 1500 mW.h / L), and after 1 h of stimulation, the triploid female abalones are respectively transferred into 100 beakers containing seawater irradiated with ultraviolet light (1500 mW.h / L) until the eggs are discharged. In order to make the triploid female abalones and diploid male abalones discharge synchronously, after the triploid female abalones are air-dried, 20 diploid male abalones are induced to spawn by the conventional method (the male parent is air-dried for 50 - 60 min and placed in seawater irradiated with ultraviolet light at 800 - 900 mW.h / L until the gonads discharge).

[0030] 4) Inhibition of polar body release from fertilized eggs: All 100 selected triploid female abalones released eggs. The diameters of 10 eggs were measured from each beaker sample, and the average value was taken. The eggs in the first 50 beakers with the largest egg diameters were mixed, and a total of 3 million eggs were obtained. The eggs were diluted to 600 eggs / ml. The sperm of 20 diploid male abalones was filtered through a 300-mesh silk screen and mixed equally, and fertilization was carried out with highly concentrated active sperm (the egg-sperm ratio was 1:400). Starting from 12 minutes after fertilization (when a small amount of the first polar body was released), cytochalasin B (0.8 mg / L) was used to treat the fertilized eggs to inhibit the first polar body. After treatment for 15 minutes, they were rinsed and incubated and cultured by conventional methods. Throughout the fertilization and hatching process, seawater filtered and disinfected by ultraviolet light was used, with a salinity of 30‰, a fertilization temperature of 23°C, and a hatching temperature of 21°C. Sufficient oxygen was maintained during hatching.

[0031] 5) Table 1 compares Example 1 with the prior art. Table 1 shows that Example 1 is significantly higher than the prior art method in terms of average egg production, hatching rate, the tetraploid doubling rate at the veliger larva stage and the eyed larva stage (measured using a flow cytometer), and the survival rate from larvae to the eyed larva stage.

[0032] Table 1

[0033]

[0034] The method of the prior art described in Table 1 is specifically as follows: After the parent abalones were placed in the room, they were first temporarily cultured at the natural water temperature for 2 - 3 days, and then the temperature was continuously increased at a gradient of 1°C per day. When the water temperature reached 20°C, they were fed at a constant temperature until sexual maturity. After the parent abalones were air-dried for 50 - 60 minutes, they were placed in a spawning tank with seawater irradiated by ultraviolet light (800 - 900 mW·h / L) until gonadal discharge. The eggs were not screened before fertilization.

[0035] 6) At 8 months old, tetraploid juvenile Haliotis discus hannai were detected and cultured to the adult stage.

Claims

1. A method for cultivating tetraploid Haliotis discus hannai, characterized in that, The method includes the selection of broodstock, the ripening acceleration of broodstock, the induced spawning of broodstock, artificial insemination, and the inhibition of polar body release of fertilized eggs; The selection of broodstock: Both male and female broodstock are required to be 2 - 3 years old, with a shell length ≥ 10 cm and no external injuries; The ripening acceleration of broodstock: Male and female broodstock are ripened separately. To synchronize the development of female parents, female broodstock are ripened 30 - 40 days earlier than male broodstock. The initial water temperature for ripening triploid female broodstock is 10 °C, and it is heated by 1 - 2 °C per day to 15 °C, and this temperature is maintained for 15 - 20 days; then it is heated by 4 - 5 °C per day to 27 °C, and stimulated at this temperature for 8 days; then it is cooled by 1 °C per day to 22 °C, and cultivated at this temperature for 100 - 110 days until the gonads are developed and mature. Diploid male broodstock are ripened by the conventional method. During the ripening process, the seawater salinity is maintained at 30‰; The induced spawning of broodstock: Triploid female broodstock are air - dried at room temperature of 20 °C for 2 - 3 hours; immediately after air - drying, they are put into 20 °C seawater saturated with oxygen and stimulated with flowing water for 1 - 1.5 h; then soaked in prepared seawater containing hydrogen peroxide for 1 h; the soaked female broodstock are put into seawater irradiated by ultraviolet light to await spawning. Diploid male broodstock are induced to spawn by the conventional method; Artificial insemination: Female broodstock are sorted according to the diameter of the eggs they produce from large to small, and the top 50% of female broodstock are selected, and their eggs are collected with a 300 - mesh silk screen; Fertilization is carried out according to the ratio of the number of eggs to sperm of 1:400, and the fertilization temperature is 23 °C and the salinity is 30‰; The inhibition of polar body release of fertilized eggs: When the fertilized eggs begin to release the first polar body, the fertilized eggs are put into seawater containing 0.3 - 1.2 mg / L cytochalasin B for treatment for 15 - 20 min; the fertilized eggs are washed and hatched and cultivated by the conventional method. The tetraploid doubling rate is measured at the veliger larva stage.

2. The method for cultivating tetraploid Haliotis discus hannai according to claim 1, characterized in that, The female broodstock is triploid Haliotis discus hannai, and the male broodstock is diploid Haliotis discus hannai.

3. The method for cultivating tetraploid Haliotis discus hannai according to claim 1, characterized in that, The method for ripening male broodstock: After male broodstock enter the room, they are first temporarily cultured at the natural water temperature for 2 - 3 days, and then the temperature is continuously increased at a gradient of 1 °C per day. When the water temperature rises to 20 °C, it is cultured at a constant temperature until the gonads are mature.

4. The method for cultivating tetraploid Haliotis discus hannai according to claim 1, characterized in that, The prepared seawater containing hydrogen peroxide has been irradiated with ultraviolet light at 1500 mW·h / L and contains 3×10 -3 M.

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