Method for temperature-induced triploidy in haliotis discus hannai inkedo

Abstract

The present application relates to the field of genetic breeding of shellfish, and particularly relates to a method for artificially inducing triploid of Haliotis discus hannai. The method comprises the following steps: 1) promoting maturity and selection of seed abalones: selecting healthy seed abalones to promote maturity synchronously in a seed abalone culture pond, and selecting diploid abalones with good gonad development; 2) artificial spawning: performing spawning on the selected seed abalones until the female and male seed abalones spawn and ejaculate; 3) selection and mixed fertilization of gametes: filtering impurities in sperm and eggs respectively, mixing the sperm and the eggs to perform artificial fertilization, and obtaining zygotes; 4) heat shock induction: after 15 minutes of fertilization, placing the zygotes in seawater with a preheated temperature of 26-31 DEG C for 10-15 minutes to inhibit the discharge of the second polar body of the zygotes; and 5) incubation: immediately collecting the zygotes into an incubation tank after the heat shock to perform incubation. The triploid of Haliotis discus hannai obtained by the method has heat resistance, and can reduce economic losses of fishermen caused by difficulty of abalones in summer. By improving the spawning mode of seed abalones and optimizing the induction conditions, the triploid of Haliotis discus hannai can be obtained efficiently and stably.

Description

[0001] manual Technical Field

[0002] The present invention relates to the field of shellfish genetic breeding in marine agriculture, and in particular to a method for artificially inducing triploids of Haliotis discus hannai. Background Art

[0003] The wrinkled abalone (Haliotis discus hannai) is an important economic shellfish for marine aquaculture in my country. In recent years, it has gradually become the main species of abalone aquaculture in the southern sea areas of my country.

[0004] Because triploid abalone's gametes are aneuploid, all metabolic energy used for gonadal development is used for growth. Consequently, triploid abalone boast advantages such as large size, rapid growth, and superior meat quality. Furthermore, their sterility and heterozygosity contribute to improved growth and stress resistance. Therefore, triploid abalone has enormous market potential and commercial value, significantly accelerating time to market and producing higher-quality individuals.

[0005] Common methods for inducing triploid abalone are primarily physical and chemical, including cold shock, hydrostatic pressure, and drug induction. These methods all block meiosis to produce the most commercially valuable homologous and heterologous triploid abalone. Alternatively, triploid individuals can be produced by mating tetraploid and diploid abalone, but the survival rate of tetraploid adults is currently low, making widespread use difficult. Chemical induction methods are associated with issues of drug residue and toxicity, and also have a high rate of larval malformations.

[0006] To address these technical challenges, this application proposes artificially inducing triploid abalone (Halium discus hannai) using a temperature shock method. Compared to existing technologies, the temperature shock method requires simple equipment, is inexpensive, and is more suitable for large-scale production. The triploid abalone bred using this method is heat-resistant, potentially reducing the economic losses to fishermen caused by abalone's difficulty surviving the summer heat. Therefore, research on triploid induction technology for abalone discus hannai and the production of triploid seedlings is of great significance for promoting technological advancement in my country's abalone discus hannai industry. Summary of the Invention

[0007] In view of this, the present invention provides a method for artificially inducing triploids of Haliotis discus hannai, by which triploids of Haliotis discus hannai can be efficiently obtained.

[0008] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0009] The present invention provides a method for artificially inducing triploids of Haliotis discus hannai, comprising the following steps:

[0010] The sperm and eggs are mixed and artificially inseminated. 15 minutes after fertilization, the fertilized eggs are placed in preheated seawater for heat shock treatment to inhibit the discharge of the second polar body of the fertilized eggs. The heat shock treatment temperature is 26 to 31 degrees Celsius and the time is 10 to 15 minutes.

[0011] Preferably, the volume ratio of sperm to egg is (9-11):1.

[0012] In a specific embodiment provided by the present invention, the volume ratio of sperm to egg is 10:1.

[0013] Preferably, the fertilized egg is subjected to heat shock treatment 15 minutes after fertilization.

[0014] Preferably, the temperature of the heat shock treatment is 28-30° C., and the time is 10-15 minutes.

[0015] More preferably, the temperature of the heat shock treatment is 28-30° C., and the time is 12-15 minutes.

[0016] The present invention provides a method for artificially inducing triploids in Haliotis discus hannai. The method comprises the following steps:

[0017] 1) Ripening and Selection of Seedling Abalone: ​​Abalone with a shell length of 65 mm to 80 mm, good vitality, and no external damage was selected as seedling abalone. Males and females were placed in a breeding abalone cultivation pond for ripening, and diploid abalone with well-developed and plump gonads were selected. The seedling abalone were 3-year-old Abalone discus hannai. Females with well-developed gonads had plump, dark green gonads. Ploidy was determined by clipping the upper legs of the abalone for flow cytometry. The abalone in the breeding pond must have a single sex.

[0018] 2) Artificial induced spawning: After drying the abalone in the shade for 2 to 2.5 hours, separate the male and female individuals into separate tanks and inject them with ultraviolet-treated seawater at an intensity of 800 to 1200 mWh / L. The seawater is replaced every hour until gametes are released. During this period, care is taken to prevent accidental fertilization and the abalone's condition is checked promptly.

[0019] 3) Gamete selection and mixed fertilization: Use a 300-mesh sieve to filter out impurities from the gametes in step 2) and select high-quality eggs and sperm with high motility under a microscope. Mix the sperm with high motility and high-quality eggs at a mass ratio of (9-11):1 and divide them into treatment and control groups. The fertilization temperature can be 18-22°C and the salinity can be 30-35.

[0020] 4) Heat shock induction: 15 minutes after gamete fertilization, when the proportion of first polar bodies in zygotes is observed to be 60% to 80%, the zygotes are collected and placed in seawater at a temperature of 26 to 31°C for 10 to 15 minutes;

[0021] 5) Incubation: Immediately after the heat shock, the fertilized eggs were collected and placed in an incubation tank at 18-22°C. They were incubated according to conventional incubation and post-cultivation methods. The triploid rate of the trochophore larvae was subsequently determined using flow cytometry.

[0022] The technical effects of the present invention are:

[0023] The present invention discloses a method for artificially inducing triploid abalone (Halion discus hannai) by heat shock induction, achieving the cultivation of artificial triploid abalone seedlings and improving the efficiency of polyploid seedling production. Compared with existing methods for preparing triploid abalone, the present invention improves the method for inducing spawning of parent abalone and optimizes induction conditions (heat shock treatment temperature and duration), thereby achieving efficient and stable production of triploid abalone (Halion discus hannai). This technology is easy to operate, highly effective and practical, and is suitable for large-scale promotion. The triploid abalone bred by this method is heat-resistant, which can reduce the economic losses caused by the difficulty of abalone surviving the summer. The method has significant application value and broad application prospects in the production of Halion discus hannai and the selection of superior polyploid varieties.

[0024] 1. Highly efficient induction method: using shade drying combined with ultraviolet irradiation and seawater stimulation to ensure that the breeding abalone can smoothly discharge high-quality gametes, facilitating subsequent fertilization and induction.

[0025] 2. Optimize induction conditions: 5 minutes after the sperm and egg mixture, continuously observe the fertilized eggs using a standard optical microscope. Approximately 15 minutes after fertilization, when 60% to 80% of the first polar body appears, initiate induction treatment to ensure experimental stability and increase the triploid rate.

[0026] 3. Simple and harmless induction method: The heat shock method of inducing triploidy of Abalone discus hannai has simple equipment, low price and is more suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Images of triploid trochophore larvae of Haliotis discus hannai; A is the heat shock method; B is the chemical method;

[0028] Figure 2 The ploidy results of diploid and triploid detected by flow cytometry, 2N is diploid, 3N is triploid; DETAILED DESCRIPTION

[0029] The present invention discloses a method for artificially inducing triploids in Haliotis discus hannai. Those skilled in the art can refer to the contents of this document and appropriately modify the process parameters to achieve the desired effect. It should be noted that all similar substitutions and modifications will be apparent to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and it is apparent that those skilled in the art can modify or appropriately alter and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0030] The reagents and instruments used in the present invention can be purchased from the market.

[0031] The present invention will be further described below in conjunction with the embodiments:

[0032] Example 1

[0033] The method for obtaining artificially induced triploid Haliotis discus hannai of the present invention comprises the following steps

[0034] 1) Ripening and selection of abalone

[0035] Healthy and vigorous three-year-old Haliotis discus henna, free of external damage, were selected as breeding abalone. Their shell length was between 72.3±6.5mm, shell width was 46.5±4.3mm, and wet weight was 43.14±9.89g. Male and female abalone were placed in a breeding abalone culture tank at 20-22℃ to accelerate maturation. Diploid abalone with well-developed and plump gonads were selected from them.

[0036] 2) Artificial induction of labor

[0037] After drying the abalone in the shade for 2 to 2.5 hours, separate the male and female individuals and place them in separate water tanks. Inject ultraviolet-treated seawater with an irradiation intensity of 800 to 1200 mWh / L. Replace the seawater every hour until gametes are released. During this period, take care to prevent accidental fertilization and check the abalone's condition in a timely manner.

[0038] 3) Gamete selection and mixed fertilization

[0039] Fresh sperm was diluted with seawater filtered through a 0.22 μm membrane to a concentration of approximately 5 × 10 6 The eggs were filtered and washed with a 300-mesh sieve and set aside. Under microscopic examination, high-quality eggs and sperm with strong motility were selected and mixed at a mass ratio of 1: (9-11) and divided into treatment and control groups. The fertilization temperature was 18-22°C and the salinity was 30-35.

[0040] 4) Heat shock induction

[0041] After 5 minutes of mixing, the fertilized eggs were observed continuously under a conventional optical microscope. 8 minutes after mixing, the first polar body began to appear. When 60% to 80% of the first polar body appeared, the fertilized eggs in the treatment group were quickly placed in preheated seawater at 26 to 31°C for 10 to 15 minutes. After that, they were placed in seawater at 20 to 22°C filtered through a 25μm silk sieve to terminate the induction.

[0042] 5) Incubation

[0043] Immediately after the heat shock, the fertilized eggs were collected and incubated in an incubation tank at 18-22°C. Triploid seedlings of Abalone discus hannai were obtained using conventional incubation and post-cultivation methods. The trochophore larvae hatch rate was measured 14 hours after fertilization, and the doubling rate of trochophore larvae (10-14 hours) was determined by flow cytometry.

[0044] The present invention optimizes the method and induction conditions for abalone induction, and can obtain triploid seedlings safely, harmlessly and efficiently. The operation is simple and can be widely promoted. Compared with chemical induction using 6-DMAP at a concentration of 30 mg / L for 15 minutes, this method has a low teratogenicity rate and does not have drug residues and toxicity problems. The teratogenicity of trochophore larvae by the two methods is as follows: Figure 1 shown.

[0045] Figure 1 (A) The triploid trochophore larvae of Abalone discus hannai induced by heat shock method have intact morphology and strong ciliary activity. Figure 1 (B) The triploid trochophore larvae of Haliotis discus hannai were chemically induced using 6-DMAP. Due to the high toxicity of the reagent, many fertilized eggs did not develop into trochophore larvae, and the remaining trochophore larvae were deformed and difficult to survive.

[0046] Example 2

[0047] To initially explore induction conditions, two heat shock temperatures (27°C and 30°C) were set, with heat shock durations of 10 and 15 minutes, respectively, for each temperature, with three replicates. Fifteen minutes after fertilization, the fertilization rates of the heat shock treatment groups (27°C and 30°C, respectively) for 10 minutes were higher than those for the 15-minute treatment group (82.71±2.42% and 79.86±1.27%). Prolonged treatment time resulted in fertilization egg death, which in turn decreased the fertilization rate, but the differences were not significant.

[0048] At 27°C and 30°C, the hatching rate and triploid rate of the group treated for 15 minutes at 27°C were 69.50±3.78% and 76.23±1.65% at 27°C, and 63.34±0.78% and 79.10±2.29% at 30°C. The triploid rate of the group treated for 15 minutes was significantly higher than that of the group treated for 10 minutes (59.70±1.27% and 66.30±2.73%), while the hatching rate was significantly lower than that of the group treated for 10 minutes (75.92±1.94% and 69.44±1.43%).

[0049] Example 3

[0050] Heat shock treatment has a significant impact on the survival of fertilized eggs, and the hatching rate decreases with the increase of treatment temperature. To further optimize the conditions, two experimental groups were set at 26℃, 28℃, and 31℃, and the treatment methods were as follows: (1) 15 minutes after fertilization, heat shock treatment continued for 10 minutes; (2) 15 minutes after fertilization, heat shock treatment continued for 15 minutes;

[0051] Under conditions (2), the highest hatching rate was in the 26°C group (71.35±2.99%), and the lowest was in the 31°C group (59.07±4.22%). The hatching rate in the 28°C group was about 65±1.79%, significantly lower than that in the control group (88.29±0.68%), indicating that 31°C is the limit temperature for induction. The hatching rate is an indicator calculated under various temperature conditions. Under conditions (1), the hatching rates at the three temperatures were 74.28±2.86%, 72.34±1.39%, and 60.16±2.96%, respectively. In order to ensure the hatching rate and facilitate the promotion of abalone seedlings, it is preferred that the temperature of heat shock treatment be 28-30°C and the time be 10-15 minutes.

[0052] Example 4

[0053] Three experimental groups were set up at 28℃ and 30℃, and the treatment methods were as follows: (1) 15 minutes after fertilization, heat shock treatment continued for 10 minutes; (2) 15 minutes after fertilization, heat shock treatment continued for 12 minutes; (3) 15 minutes after fertilization, heat shock treatment continued for 15 minutes.

[0054] The triploid rates of the cells treated at different induction temperatures and the same treatment time were: (1) 63.33±2.17%, (2) 71.65±3.67%, (3) 77.10±2.73%. To ensure the triploid rate, the heat shock treatment temperature was preferably 28-30°C and the treatment time was 12-15 min.

[0055] Example 5

[0056] Rapid identification method of triploid abalone seedlings

[0057] 1) Sampling: Collect trochophore larvae 10 to 14 hours after fertilization into a 1.5 mL centrifuge tube, briefly discard the supernatant, add 1 mL of 1× PBS (Phosphate buffered saline), and gently pipette several times.

[0058] 2) Fixation: Mix the sample solution in step 1) with 3 mL of anhydrous ethanol using a 1 mL pipette to prepare a cell suspension, aspirate and filter (300 mesh) into a 5 mL centrifuge tube and store for more than 24 hours.

[0059] 3) Staining: After centrifugation (300×g, 7 min), discard the supernatant and add 500 μL of 1× PBS. Filter the sample through a 50 μm filter membrane. Add 33.33 μL of PI (1 mg / mL) and stain for 10 min. Analyze cellular DNA content using flow cytometry. This process takes only 2–3 min. Using the normal control group as a diploid control, randomly test samples in the experimental group and calculate the diploid and triploid rates.

[0060] The fertilization rates of both the normal H. discus henna control group and the triploid experimental group were consistently above 80%, but hatching rates varied slightly due to differences in egg quality at different stages. Although triploids have an extra set of chromosomes, the fertilization rate was not significantly different from the control group, and the hatching deformity rate was lower than that of the chemical induction method, resulting in a relatively stable overall hatching rate.

[0061] Figure 2 Figure 3 is the ploidy result of diploid and triploid by flow cytometry. The DNA content of the heat shock treatment group was 1.5 times that of the diploid control group.

[0062] Example 6

[0063] Identification of triploid abalone seedlings by chromosome analysis

[0064] 1) Pretreatment: The trochophore larvae were collected by filtration using a sieve, added to a colchicine solution to a final colchicine concentration of 0.05%, and allowed to stand for 1 hour.

[0065] 2) Hypotonic: Add a small amount of 0.075M KCl hypotonic solution, aspirate the waste liquid after 15 minutes, then add hypotonic solution again and let it stand for 1 hour.

[0066] 3) Fixation: Aspirate the waste solution and add pre-chilled Carnoy's fixative (methanol:acetic acid = 3:1). Change the fixative every hour for three times. Store the fixed sample in a -20°C freezer.

[0067] 4) Slide preparation: After centrifuging the sample, replace the Carnoy's fixative and pipette several times. Let it sit for several minutes. Pipette the suspension from a high place and drop it onto a pre-chilled glass slide. Immediately heat the slide with an alcohol burner and stain with Giemsa. After staining, store in a refrigerator at 4°C.

[0068] 5) Observation: Observe the chromosome karyotype image, find the mitotic field of view under a 10x microscope, find the appropriate mitotic phase under a 40x microscope, and take pictures under a 100x oil immersion lens.

[0069] The conventional chromosome preparation method was used to analyze the chromosomes of the trochophore larvae of Haliotis discus hannai. The number of chromosomes in the trochophore larvae treated with heat shock was 54, which was a chromosome number ratio of 1.5:1 to that of the ordinary Haliotis discus hannai, proving that these juvenile abalone were triploid.

[0070] It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered as the scope of protection of the present invention.

Claims

1. A method for artificially inducing triploids of Haliotis discus hannai, characterized in that: Sperm and eggs are mixed and artificially inseminated. 15 minutes after fertilization, when the proportion of the first polar body in the fertilized egg is observed to be 60% to 80%, the fertilized egg is placed in preheated seawater for heat shock treatment to inhibit the expulsion of the second polar body of the fertilized egg. The heat shock treatment temperature is 26 to 31 degrees Celsius and the time is 10 to 15 minutes. The specific steps include: 1) Ripening and selection of abalone; 2) artificial induction of labor; 3) selection of gametes and mixed fertilization; 4) heat shock induction; 5) incubation; The specific method of artificial induction of labor in step 2) is: inducing labor in individuals with mature gonads by using a method of drying in the shade combined with ultraviolet irradiation of seawater stimulation until male and female abalone lay eggs and ejaculate; the method of drying in the shade combined with ultraviolet irradiation of seawater stimulation is: after the abalone is dried in the shade for 2 hours to 2.5 hours, the male and female individuals are strictly separated and placed in different water tanks, and ultraviolet-treated seawater with an irradiation intensity of 800 to 1200 mWh / L is injected, and the seawater is replaced every 1 hour until gametes are discharged. During this period, care is taken to prevent accidental fertilization and the abalone condition is checked in time.

2. The method for artificially inducing a triploid Haliotis discus hannai according to claim 1, wherein In step 1), the specific method for promoting the ripening and selecting the seed abalone is: taking abalone with a shell length of 65 mm to 80 mm, good individual vitality and no external damage as the seed abalone, placing them in a breeding abalone cultivation pond according to gender to promote ripening, and selecting diploid abalone with well-developed and plump gonads; the seed abalone is a 3-year-old abalone.

3. The method for artificially inducing a triploid Haliotis discus hannai according to claim 1, wherein the triploid In step 3), the specific method for selecting the gametes is: using a 300-mesh sieve to filter out impurities in the gametes of step 2), and selecting high-quality eggs and highly motile sperm under a microscope; the high-quality eggs refer to eggs in fresh egg fluid with intact egg membranes, regular shapes, and no accidental fertilization; the highly motile sperm refer to fresh sperm with good mobility and dispersion.

4. The method for artificially inducing a triploid Haliotis discus hannai according to claim 1, wherein the triploid In step 3), the specific method of mixed fertilization is: the sperm and eggs with good vitality and high quality are mixed and divided into a treatment group and a control group, the mass ratio of the sperm to the egg is (9-11):1; the fertilization temperature is 18-22°C, and the salinity is 30-35.

5. The method for artificially inducing a triploid Haliotis discus hannai according to claim 1, wherein In step 4), the stability of the seawater temperature is continuously monitored during the heat shock induction process, and the development of the fertilized eggs is continuously observed using a microscope.

6. The method for artificially inducing a triploid Haliotis discus hannai according to claim 1, wherein In step 5), the specific method of incubation is: immediately after the heat shock ends, the fertilized eggs are collected into an incubation tank at 18-22° C.; conventional incubation and post-cultivation methods are followed, and the triploid rate of the trochophore larvae is subsequently determined using a flow cytometer.

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