Method for obtaining and preserving tetraploid male oyster species

Abstract

The application discloses a method for obtaining and preserving tetraploid male Crassostrea gigas, and belongs to the technical field of genetic breeding of aquatic breeding varieties. The method comprises the following steps: accelerating the maturity of tetraploid Crassostrea gigas by regulating water temperature and bait, identifying the male and female of the tetraploid Crassostrea gigas by nuclear magnetic resonance technology, further accelerating the maturity of the tetraploid male Crassostrea gigas by regulating water temperature and bait, and preserving the tetraploid male Crassostrea gigas by well storage seawater. The method has the advantages of simple operation, no influence of external air temperature and seawater temperature, no damage to the tetraploid Crassostrea gigas, and can provide the tetraploid male Crassostrea gigas stably throughout the year, thereby laying a solid foundation for the stable cultivation of triploid Crassostrea gigas throughout the year.

Description

TECHNICAL FIELD

[0001] The application relates to a method for obtaining and preserving male oyster seed, in particular a method for obtaining and preserving tetraploid Crassostrea gigas male seed, and belongs to the technical field of genetic breeding of aquatic breeding varieties. BACKGROUND

[0002] The triploid Crassostrea gigas is extremely valuable in the shellfish breeding industry due to its rapid growth and natural sterility. The tetraploid Crassostrea gigas is the core germplasm for breeding full triploid Crassostrea gigas. The tetraploid Crassostrea gigas carries four sets of chromosomes, and can be crossed with the diploid Crassostrea gigas to obtain 100% full triploid Crassostrea gigas. However, the gonad discharge of the tetraploid Crassostrea gigas varies with the seasons, which limits the production of triploid Crassostrea gigas seedlings. In order to ensure the year-round supply of triploid Crassostrea gigas seedlings, it is necessary to ensure the long-term and stable supply of tetraploid Crassostrea gigas male seed. SUMMARY

[0003] The purpose of the present application is to provide a method for providing tetraploid Crassostrea gigas male seed stably throughout the year.

[0004] In order to achieve the above-mentioned target, the present application adopts the following technical solution:

[0005] A method for obtaining and preserving tetraploid Crassostrea gigas male seed, comprising the following steps:

[0006] (1) Artificially maturing tetraploid Crassostrea gigas seed: placing the tetraploid Crassostrea gigas seed in a breeding pond, the seawater in the breeding pond has a base temperature of 10 DEG C, the water is changed once a day, the water temperature is increased by 0.5 DEG C to 1.0 DEG C per day, and the effective accumulated temperature is increased to 300 DEG C·day;

[0007] (2) Identifying the male and female tetraploid Crassostrea gigas seed in vivo by nuclear magnetic resonance technology: using a nuclear magnetic resonance scanning imaging system to scan the tetraploid Crassostrea gigas seed with an effective accumulated temperature of 300 DEG C·day, identifying the male and female according to the gray value of the gonad, and identifying the tetraploid Crassostrea gigas male seed according to the fact that the gray value of the gonad of the tetraploid Crassostrea gigas male seed is significantly lower than that of the female;

[0008] (3) Further maturing the tetraploid Crassostrea gigas male seed: placing the tetraploid Crassostrea gigas male seed back into the original breeding pond for further breeding, changing the water once a day, keeping the water temperature unchanged, and starting to reduce the temperature when the effective accumulated temperature reaches 380 DEG C·day, and the gonad of the tetraploid Crassostrea gigas male seed is fully matured when the effective accumulated temperature reaches 400 DEG C·day;

[0009] (4) Preserving the tetraploid Crassostrea gigas male seed by storing seawater in a well: placing the tetraploid Crassostrea gigas male seed with fully matured gonad in a well storing seawater, and preserving the tetraploid Crassostrea gigas male seed by storing seawater in the well with a temperature ranging from 10 DEG C to 17 DEG C.

[0010] Preferably, in step (1) and step (3), the water temperature for promoting the maturation of the seed oysters is not higher than 25℃.

[0011] Preferably, in step (1), step (3) and step (4), during the artificial maturation promotion of the seed oysters and the low-temperature storage of the seed oysters, live unicellular algal feed and yeast selenium are fed, wherein the live unicellular algal feed is fed at a feeding amount of 6-15×10 15 cells / 100kg seed oysters each time, and the yeast selenium is fed at a feeding amount of 10g / 100kg seed oysters each time, and the feeding is performed 3 times a week.

[0012] Preferably, in step (2), the nuclear magnetic resonance scanning imaging system is a BioSpec 70 / 20USR magnetic resonance spectrometer, and the parameter settings of the BioSpec 70 / 20USR magnetic resonance spectrometer are as follows: the scanning orientation is set to a transverse position, the scanning thickness is set to 2mm, the scanning layer number is set to 20 layers, the field of view is adjusted to 35mm×35mm, the layer resolution is set to 256×256, the echo time is set to 20ms under the condition of longitudinal relaxation time, and the echo time is set to 30ms under the condition of transverse relaxation time.

[0013] Preferably, in step (3), the water is changed once a day during the temperature reduction, and the temperature is reduced by 0.5℃-1.0℃ per day.

[0014] Preferably, in step (4), Bacillus is added to the well-stored seawater every day, and the concentration of the Bacillus in the seawater is 0.5g / m³.

[0015] The method for obtaining and storing the tetraploid Crassostrea gigas male seed oysters provided by the application is simple in operation, is not affected by external air temperature and seawater temperature, does not damage the tetraploid Crassostrea gigas seed oysters, and can stably provide the tetraploid Crassostrea gigas male seed oysters throughout the year, thereby laying a solid foundation for the stable cultivation of triploid Crassostrea gigas throughout the year. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a nuclear magnetic resonance scanning imaging result diagram of the internal structure of the diploid Crassostrea gigas;

[0017] Figure 2 is a manual annotation diagram of the internal structure of the diploid Crassostrea gigas;

[0018] Figure 3 is an MRI image of the internal structure of the diploid Crassostrea gigas obtained under different TE values under T1 weighted imaging conditions;

[0019] Figure 4 is an MRI image of the internal structure of the diploid Crassostrea gigas obtained under different TE values under T2 weighted imaging conditions;

[0020] Figure 5 Figure is the comparison chart of fat content (g / 100g) and gray value (gonad / drop of water) of male and female oysters. DETAILED DESCRIPTION

[0021] The application will be specifically introduced below in combination with the drawings and specific examples.

[0022] I. Obtaining and preserving tetraploid C. gigas male seed oysters

[0023] 1. Artificially accelerating the maturation of tetraploid C. gigas seed oysters

[0024] Select tetraploid C. gigas seed oysters with regular shape, healthy gonads and vigorous vitality, remove the attachments on the shell of the seed oysters, and place the seed oysters in a breeding pond. The seawater in the breeding pond has a base temperature of 10℃, and the water is changed once a day. The temperature is raised by 0.5-1.0℃ per day, and the water temperature for accelerating the maturation of the seed oysters is not higher than 25℃. The seed oysters are bred until the effective accumulated temperature reaches 300℃·day (at this time, the gonads of the tetraploid C. gigas seed oysters have developed to a certain stage).

[0025] During the period of artificially accelerating the maturation of the seed oysters, only live monosomatous algae feed is fed, or live monosomatous algae feed and yeast selenium are simultaneously fed. The live monosomatous algae feed is fed to fatten the tetraploid C. gigas seed oysters, and the feeding amount is 6-15×10 15 cells / 100kg of seed oysters each time, and the feeding is performed 6-8 times a day. The yeast selenium is fed to improve the sperm motility of the tetraploid C. gigas male seed oysters, thereby improving the fertilization rate, and the feeding amount is 10g / 100kg of seed oysters each time, and the feeding is performed 3 times a week.

[0026] 2. Identifying the male and female of tetraploid C. gigas seed oysters in vivo by nuclear magnetic resonance technology

[0027] Before the application uses nuclear magnetic resonance technology to identify the male and female of tetraploid C. gigas seed oysters, the relationship between the gray value of the gonad of C. gigas in the nuclear magnetic resonance imaging (MRI) scan and the gender is first studied.

[0028] (1) Materials

[0029] The diploid C. gigas samples cultured in the sea area of Kongdong Island in Yantai City, Shandong Province were collected. Individuals with a weight of 100g-120g were selected to ensure that the samples were representative in terms of size, weight and health status.

[0030] (2) Instruments and equipment

[0031] The instruments and equipment used include an electron microscope, an analytical balance, a freeze dryer, a forced air drying oven, a BioSpec 70 / 20USR magnetic resonance spectrometer (BRUKER), and a fat tester.

[0032] (3) Preparation of samples

[0033] Before MRI scanning, the collected diploid long oyster samples were left to stand at room temperature for 1 h (to reduce physiological stress caused by temperature changes). After that, the diploid long oysters were gently fixed on the coil of the BioSpec 70 / 20USR magnetic resonance spectrometer (to ensure that the diploid long oysters remained stationary during scanning). Then, a drop of water was added below the diploid long oysters as a control (the drop of water served as a stable reference signal that could be used to correct the instability of the magnetic resonance spectrometer and inconsistencies during imaging; in addition, by comparing the gray value of the gonad region of the diploid long oyster with that of the drop of water, the signal intensity of the gonad could be more accurately evaluated).

[0034] (4) Optimization of MRI acquisition sequence and parameters

[0035] The specific parameter settings of the BioSpec 70 / 20USR magnetic resonance spectrometer were as follows: the scanning orientation was transverse; the scanning thickness was set to 2 mm, and the number of scanning layers was 20 to obtain a complete image of the oyster gonad; the field of view (FOV) was adjusted to 35 mm x 35 mm, and the layer resolution was set to 256 x 256 to obtain a high-resolution image of the oyster gonad.

[0036] The oyster nuclear magnetic resonance scanning imaging parameters were optimized through experiments, i.e., the optimal echo time (TE) value was designed and obtained, and the repetition time (TR) value was not set but changed accordingly with the TE value.

[0037] (5) Determination of fat content

[0038] After the MRI scan was completed, the shell of the diploid oyster was carefully opened using anatomical tools, and the gonad was extracted. Under a microscope, the characteristics of the cells and tissue structures were observed to further confirm the gender of the diploid oyster.

[0039] After the gender of the diploid oyster was determined, the sample was dried using the freeze-drying method for 24 h, and then ground into fine powder. 0.2900-0.3000 g of the sample was placed in a filter paper cylinder, which was then placed in an extraction tube containing petroleum ether and placed on a round-bottom flask. The extraction tube and the round-bottom flask were connected, and heated in a water bath at 60°C. The heating caused the solvent in the flask to evaporate, and the vapor was then condensed through a condenser tube and dropped back into the flask. As the solvent gradually dissolved the fat in the extraction tube, it would automatically return to the round-bottom flask through the siphon effect when it accumulated to a certain height. This process was repeated until all the soluble fat in the sample was extracted. After the extraction was completed, the filter paper cylinder was placed in an oven at 100°C to dry to a constant weight, which was recorded. This process was repeated three times for each sample.

[0040] (6) Analysis of MRI images

[0041] The MRI images were analyzed for gray value using the DICOM Viewer 3.6.1 software. The software was opened and the MRI image data of the diploid L. longamus were imported into the software using the import function. The background noise in the images was reduced and the signal of the gonad region was enhanced using the denoising function. The gonad region was manually circled in the software. The area of the gonad was selected to be consistent among all samples for effective comparison. Water droplets were also selected as controls for subsequent comparative analysis. The gray value of the selected regions was measured using the analysis tool provided with the software. The average gray value, minimum and maximum gray value, and other statistical information of the gonad and water droplet control regions were recorded.

[0042] (7) Data processing

[0043] The data obtained from the MRI image analysis and fat content determination were statistically processed using statistical software (SPSS 20.0 software). Descriptive statistical analysis was used to describe the basic situation of the samples, and independent sample t-test was used to compare the differences in gray value and fat content between the gonads of male and female L. longamus. For significance analysis, the significance level was set at 0.05.

[0044] (8) Results

[0045] (i) Results of nuclear magnetic resonance scanning imaging of internal tissues of diploid L. longamus

[0046] The results of nuclear magnetic resonance scanning imaging of internal tissues of diploid L. longamus are shown in Figure 1 , and the manual labeling of internal structures is shown in Figure 2 .

[0047] As can be seen, by scanning and imaging L. longamus using MRI technology, a series of images can be obtained that reveal the internal structure of L. longamus in detail. These images clearly show the main organs such as the gonad, hepatopancreas, adductor muscle and gill.

[0048] (ii) Optimal echo time

[0049] The MRI images of the internal tissues of diploid L. longamus obtained under T1-weighted imaging conditions at different TE values (10 ms, 20 ms and 30 ms) are shown in Figure 3 ; and the MRI images obtained under T2-weighted imaging conditions at different TE values (30 ms, 40 ms and 50 ms) are shown in Figure 4 .

[0050] Considering the specific parameters, performance and time limit of the BioSpec 70 / 20USR magnetic resonance spectrometer, the optimal TE value is set to 20 ms under the condition of T1 weighted imaging, and the optimal TE value is set to 30 ms under the condition of T2 weighted imaging, at which the signal contrast of the obtained MRI image is the highest, and different tissue structures can be clearly distinguished.

[0051] (iii) Fat content and gray value of male and female diploid C. gigas

[0052] It is detected that the fat content of female diploid C. gigas is 33.46±0.32 g / 100 g, and the gray value (gonad / water droplet) is 25.72±3.32; the fat content of male diploid C. gigas is 16.52±0.21 g / 100 g, and the gray value (gonad / water droplet) is 16.80±0.77. The graph is shown in Figure 5 .

[0053] It can be seen that the fat content and the gray value of the gonad of female diploid C. gigas are significantly higher than those of male. This gender difference shows that after MRI scanning, the male and female C. gigas can be distinguished according to the gray value of the gonad tissue.

[0054] The gray value difference reveals the potential of MRI technology in distinguishing the gender and physiological state of C. gigas. In the MRI image, the fat tissue usually shows high signal intensity. The higher gray value of female C. gigas may be due to its higher fat content, and the significant manifestation of this gender difference in the MRI gray value provides a basis for using MRI technology to identify the gender and evaluate the physiological state of C. gigas and other aquatic organisms.

[0055] After determining the relationship between the gray value of C. gigas in MRI scanning and gender, the nuclear magnetic resonance technology can be used to distinguish the gender of tetraploid C. gigas seed, and the specific method is as follows:

[0056] (1) Fix the tetraploid C. gigas seed with effective accumulated temperature of 300℃·day on the nuclear magnetic resonance scanning imaging system (for example: BioSpec 70 / 20USR magnetic resonance spectrometer), and wait for nuclear magnetic resonance scanning;

[0057] (2) Set the parameters of the nuclear magnetic resonance scanning imaging system (for example: set the scanning orientation of the BioSpec 70 / 20USR magnetic resonance spectrometer to transverse position, set the scanning thickness to 2 mm, set the scanning layer number to 20, adjust the field of view to 35 mm×35 mm, set the layer resolution to 256×256, set the echo time to 20 ms under the condition of longitudinal relaxation time, and set the echo time to 30 ms under the condition of transverse relaxation time), so as to obtain complete, high-resolution and high-signal-intensity images of the gonad of tetraploid C. gigas;

[0058] (3) MRI images were obtained by performing nuclear magnetic resonance scanning on tetraploid oysters using a nuclear magnetic resonance imaging system;

[0059] (4) Gray value analysis was performed on the MRI images, and the sex of the tetraploid long oyster was identified based on the gray value of the gonads. The gray value of the gonads of the male tetraploid long oyster was significantly lower than that of the gonads of the female tetraploid long oyster, thus identifying the male tetraploid long oyster.

[0060] Nuclear magnetic resonance (NMR) technology is a technique that uses strong magnetic fields and radio frequency pulses to acquire detailed images of the internal structure of objects without any form of radiation exposure. It enables non-destructive testing and is currently used in brain structure imaging in mice and in moisture content detection in aquatic products. This invention uses NMR technology to distinguish the sex of tetraploid Pacific oyster larvae, achieving radiation-free, high-resolution imaging. It solves the problems of traditional Pacific oyster sex identification requiring dissection (which not only destroys the sample but is also inefficient and cannot meet the needs of large-scale aquaculture) and the inability to detect broodstock live. This improves the efficiency and accuracy of Pacific oyster sex identification, enabling effective control of tetraploid Pacific oyster larvae.

[0061] 3. Further promote maturation of male tetraploid oyster larvae.

[0062] The male tetraploid oysters were returned to their original culture ponds for continued cultivation. The feeding method remained unchanged, the water was changed once a day, and the water temperature remained constant. When the effective accumulated temperature reached 380℃·day, the temperature was lowered, the water was changed once a day, and the temperature was lowered by 0.5℃-1.0℃ per day. When the effective accumulated temperature reached 400℃·day, the gonads of the male tetraploid oysters were fully mature.

[0063] 4. Preservation of tetraploid male oyster larvae using well-stored seawater

[0064] Seawater is stored in wells (15-30 meters deep). Because the seawater in the wells is below the ground and is not exposed to direct sunlight all year round, the seawater temperature in the wells is maintained at 10-17℃. This temperature can inhibit the further development of the gonads of male tetraploid oysters and prevent the release of male gametes.

[0065] Put the gonad of all mature tetraploid Crassostrea gigas male seed into the well storing seawater, and store the tetraploid Crassostrea gigas male seed in the well-stored seawater with the temperature in the range of 10-17 DEG C, and the feeding method remains unchanged during the storage period. Alternatively, Bacillus is added to the well-stored seawater every day (the addition of Bacillus can reduce the ammonia nitrogen content in the water body, and effectively prevent the tetraploid Crassostrea gigas male seed from discharging sperm), and the concentration of Bacillus in the seawater is 0.5 g / m3, and the tetraploid Crassostrea gigas male seed is stored in the well-stored seawater with the temperature in the range of 10-17 DEG C and the addition of Bacillus, and the feeding method remains unchanged during the storage period.

[0066] The well-stored seawater is used to store the tetraploid Crassostrea gigas male seed, which not only realizes the purpose of low-cost storage of male seed, but also realizes energy saving and environmental protection.

[0067] II. Detecting the survival rate, sperm-discharging seed proportion, sperm activity and fertilization rate of the stored tetraploid Crassostrea gigas male seed

[0068] 1. Only feeding live monochrysalis bait, and storing the male seed in the well-stored seawater without adding Bacillus

[0069] In the case of only feeding live monochrysalis bait, after the tetraploid Crassostrea gigas male seed is stored in the well-stored seawater without adding Bacillus at 15 DEG C for 4 months, it is detected that the ammonia nitrogen content in the water body is 0.70±0.05%, the survival rate of the seed is 82±3%, the proportion of sperm-discharging seed is 11%, the sperm is active, and the fertilization rate is 85%.

[0070] 2. Simultaneously feeding live monochrysalis bait and yeast selenium, and storing the male seed in the well-stored seawater with the addition of Bacillus

[0071] In the case of simultaneously feeding live monochrysalis bait and yeast selenium, after the tetraploid Crassostrea gigas male seed is stored in the well-stored seawater with the addition of Bacillus (the concentration is 0.5 g / m3) at 15 DEG C for 4 months, it is detected that the ammonia nitrogen content in the water body is 0.03±0.01%, the survival rate of the seed is 95±2%, the proportion of sperm-discharging seed is 3%, the sperm is very active, and the fertilization rate is 90%.

[0072] In summary, the acquisition and storage method of the tetraploid Crassostrea gigas male seed provided by the application can efficiently store the tetraploid Crassostrea gigas male seed (high survival rate, low proportion of sperm-discharging seed), and the sperm activity is good, the fertilization rate is high, and the tetraploid Crassostrea gigas male seed can be stably provided throughout the year, which lays a solid foundation for the stable cultivation of triploid Crassostrea gigas throughout the year.

[0073] It should be noted that the above examples are only used to clearly illustrate the present application, and are not intended to limit the embodiments of the present application. For those skilled in the art, on the basis of the above description, other different forms of changes or variations can also be made. Here, all the embodiments cannot be exhausted. Any obvious changes or variations derived from the technical scheme of the present application are still within the scope of protection of the present application.

Claims

1. A method for obtaining and preserving tetraploid male oyster Crassostrea gigas, characterized in that, The method comprises the following steps: (1) artificially promoting maturity of tetraploid Crassostrea gigas seed oysters: the tetraploid Crassostrea gigas seed oysters are placed in a culture pond, the seawater in the culture pond has a base temperature of 10℃, the seawater is replaced once a day, the water temperature is increased by 0.5-1.0℃ per day, and the seed oysters are cultured until the effective accumulated temperature reaches 300℃·day; (2) identifying the male and female tetraploid Crassostrea gigas seed oysters in vivo by using a nuclear magnetic resonance technology: the nuclear magnetic resonance scanning imaging system is used to scan the tetraploid Crassostrea gigas seed oysters with an effective accumulated temperature of 300℃·day, the male and female seed oysters are identified according to the gray value of the gonad of the seed oysters, the gray value of the gonad of the male tetraploid Crassostrea gigas is significantly lower than that of the female, and thus the male tetraploid Crassostrea gigas seed oysters are identified; (3) further promoting maturity of the male tetraploid Crassostrea gigas seed oysters: the male tetraploid Crassostrea gigas seed oysters are placed back into the original culture pond for further culture, the seawater is replaced once a day, the water temperature is kept unchanged, the temperature is decreased when the effective accumulated temperature reaches 380℃·day, and the gonad of the male tetraploid Crassostrea gigas seed oysters is fully matured when the effective accumulated temperature reaches 400℃·day. (4) Preserving tetraploid Crassostrea gigas male broodstock by well storage of seawater: placing tetraploid Crassostrea gigas male broodstock with fully mature gonads in a well with a depth of 15-30 meters storing seawater, preserving tetraploid Crassostrea gigas male broodstock by well storage of seawater with a temperature in the range of 10-17℃, adding Bacillus spores to the well storage of seawater every day, and the concentration of Bacillus spores in seawater is 0.5g / m 3 .

2. The method of claim 1, wherein, In steps (1) and (3), the water temperature for promoting maturity of the seed oysters is not higher than 25℃.

3. The method of claim 1, wherein, In step (1), step (3) and step (4), the live unicellular algal feed and yeast selenium are fed during the artificial maturation of the seed shellfish and during the low-temperature preservation of the seed shellfish, wherein the live unicellular algal feed is fed at a feeding amount of 6-15×10 15 cells / 100kg seed shellfish each time, and 6-8 times a day; and the yeast selenium is fed at a feeding amount of 10g / 100kg seed shellfish each time, and 3 times a week.

4. The method of claim 1, wherein, In step (2), the nuclear magnetic resonance scanning imaging system is a BioSpec 70 / 20USR magnetic resonance spectrometer.

5. The method of claim 4, wherein, The parameter settings of the BioSpec 70 / 20USR magnetic resonance spectrometer are as follows: the scanning orientation is set to the transverse position, the scanning thickness is set to 2mm, the scanning layers are set to 20 layers, the field of view is adjusted to 35mm×35mm, the layer resolution is set to 256×256, the echo time is set to 20ms under the condition of longitudinal relaxation time, and the echo time is set to 30ms under the condition of transverse relaxation time.

6. The method of claim 1, wherein, In step (3), the seawater is replaced once a day when the temperature is decreased, and the water temperature is decreased by 0.5-1.0℃ per day.

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