CN113951194B - Method for producing triploid oysters by interspecific hybridization of Fujian oysters and sikauri oysters - Google Patents
Method for producing triploid oysters by interspecific hybridization of Fujian oysters and sikauri oysters Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/50—Culture of aquatic animals of shellfish
- A01K61/54—Culture of aquatic animals of shellfish of bivalves, e.g. oysters or mussels
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
- A01K61/17—Hatching, e.g. incubators
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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Abstract
The invention relates to a method for producing triploid oysters by interspecific hybridization of Fujian oysters and panda oysters, which belongs to the technical field of aquaculture variety genetic breeding, and comprises the steps of obtaining excellent tetraploid Fujian oysters, strongly optimizing parents, breeding parents, artificial insemination, hatching and breeding larvae; the female parent of the sida ursinum and the male parent of the tetraploid Fujian oyster are hybridized to obtain the triploid sida ursinum, on one hand, the problems of small size and slow growth speed are solved on the basis of keeping the fine and delicious taste of the sida ursinum, and on the other hand, the sida ursinum hybrid triploid oyster which can be put on the market all year round is obtained through the characteristic of high sterility of the triploid.
Description
Technical Field
The invention belongs to the technical field of aquaculture variety genetic breeding, and particularly relates to a method for producing triploid oysters by interspecific hybridization of Fujian oysters and sida oysters.
Background
Oyster (Crassostrea angusta), also called portuguese oyster, belongs to the family of oysters (Ostridae), and oyster (Crassostrea) is mainly distributed in high-temperature and high-salt sea areas in the south of the Yangtze river of China, mainly in the province of Fujian, has the advantages of fast growth, high yield and the like, and is an important oyster cultivation variety in China.
The Crassostrea sikamea belongs to the family of oyster (Ostridae) and the genus Crassostrea (Crassostrea), is mainly distributed in the Japanese famous sea, the Korean peninsula and the central and south China sea area, is suitable for high-temperature and medium-high-salt sea areas, and is mainly in Zhejiang province. Has become an important oyster breeding variety abroad. Compared with the crassostrea gigas and the Fujian oysters, the crassostrea gigas has a small size, fine and delicious meat quality and high economic value.
The triploid shellfish has three sets of chromosomes, so the gonad high sterility has excellent breeding characteristics of fast growth and high sterility, and the triploid shellfish has high glycogen content and better mouthfeel. Has high economic value in shellfish culture.
The tetraploid Fujian oyster has four sets of chromosomes, and can be obtained by fertilization of an ovum and a normal diploid sperm of the triploid oyster and polar body release inhibition (CN 100415085C, CN107494358A, CN109730008A and CN 113016673A).
Different scholars preliminarily study the interspecific hybridization of the diploid crassostrea sikamea and the diploid Fujian oyster, and at present, reports on the production of the Xifu triploid oyster by the hybridization of the crassostrea sikamea (male parent) and the tetraploid Fujian oyster (female parent) do not exist in China. There is a need for a stable and efficient sikawa oyster tetraploid production and sikafu interspecific hybridization method.
Disclosure of Invention
The invention aims to provide a method for producing triploid oysters by crossing tetraploid Fujian oysters and Xiufu, which provides excellent Fujian oyster tetraploid and Xiufu hybrid triploid for oyster cultivation.
The invention is realized by the following technical scheme:
a method for producing triploid oyster by interspecific hybridization of Fujian oyster and bear oyster comprises obtaining excellent tetraploid Fujian oyster, strong optimization of parent, parent cultivation, artificial insemination and incubation and larva cultivation;
obtaining the excellent tetraploid Fujian oyster: stimulating triploid Fujian oyster at 26 deg.c for 5 days, maturing the triploid Fujian oyster in 23 deg.c water for 30-40 days, and fertilizing with the largest diameter 1/3 of the ovum of the triploid female shellfish and the sperm of diploid Fujian oyster; treating fertilized eggs with 0.5-0.8mg/L Cytochalasin B (CB) or 30-80 mg/L6-dimethylaminopurine (6 DMAP) for 15 minutes from 5 minutes after fertilization to inhibit first polar body discharge; the temperature of the indoor cultivation stage is 23 ℃, the salinity is 30 per mill, and after larvae are attached and metamorphosed, adult shellfish are cultivated on the sea;
the parent is strongly preferred: identifying adult tetraploid Fujian oyster by flow cytometry, selecting maximum 10% of individuals as seed shellfish to promote ripening, and selecting maximum 10% of individuals of the oyster of bear as seed shellfish to promote ripening;
the parent breeding: when parent gonad ripening is carried out, in order to synchronize gonad development of the sika oyster and the Fujian oyster, the sika oyster is ripened 30-40 days earlier than the Fujian oyster, gonad ripening work of the tetraploid Fujian oyster is started according to gonad development conditions of the sika oyster, when ripening is carried out, the tetraploid Fujian oyster is firstly stimulated for 7 days at high temperature of 27 ℃, then the water temperature is reduced to 1 ℃ every day and is gradually reduced to 24 ℃ for cultivation for 30-40 days until gonad development is ripened, and the salinity is 30 per thousand; the initial temperature of diploid parent strain of the oyster is 16 ℃, and then the temperature is gradually increased to 27 ℃; adjusting the initial salinity to 24-26 per mill, adjusting the salinity to 30-32 per mill of normal seawater after 15 days, and culturing until the gonad is mature;
the artificial insemination and hatching: selecting female oyster of the first 10% of maximum individual crassostrea sikamea, collecting ovum, soaking the collected ovum in seawater for 40-60 min, and aging; selecting the first 10% tetraploid Fujian oyster male shellfish with the largest individual to obtain sperms; inseminating each ovum according to the proportion of 200 active sperms, wherein the whole inseminating and hatching process adopts filtered and ultraviolet-sterilized seawater with the temperature of 27 ℃, the salinity of 24 per mill and the hatching density of 50 ova/ml;
and (3) larva cultivation: the larva culture density at the early stage is 10-15/ml, the temperature is 27 ℃, the salinity is 24 per mill, the golden algae is initial bait, the larva culture density is adjusted along with the growth of the larva, and the bait feeding amount is gradually increased; when the length of the larva shells is more than 150 mu m, the temperature and the salinity of the culture pond are adjusted, the temperature is reduced by 0.5 ℃ every day to 23-24 ℃, and the salinity is adjusted by 0.5 every day to be less than or equal to 2 different from the salinity of the culture sea area.
Furthermore, in the parent cultivation, when the gonad interstitial fluid of the oyster of the giant salamander is observed to be reduced, the egg cells are clear or the sperm activity is high, the gonad ripening work of the tetraploid Fujian oyster is started.
Compared with the prior art, the invention has the beneficial effects that:
1) The invention adopts a unique triploid Fujian oyster parent ripening method, firstly uses seawater at 26 ℃ to stimulate gonad development, so that the gonad development is faster, and more and better eggs are obtained; the prior art method adopts naturally mature triploid, and can not obtain a large amount of high-quality ova; 2) The eggs produced by the first 1/3 triploid Fujian oyster female shellfish with large eggs are selected, the large eggs have high maturity, and the tetraploid Fujian oyster is more beneficial to survival; the prior art does not screen the size of the ovum. The most critical restriction point of inducing tetraploid Fujian oyster with triploid Fujian oyster ovum is that a large amount of ovum can not be obtained. The two innovations enable the method to obtain and utilize a large amount of high-quality triploid Fujian oyster eggs to obtain a large amount of pure tetraploid Fujian oysters, and the pure tetraploid Fujian oysters can be normally cultured to adults, so that a foundation is laid for selecting excellent tetraploid Fujian oysters to serve as hybrid oysters.
The bear-Fu hybrid triploid can be generated by inhibiting the induction of a second polar body by using normal diploid hybrid sperm eggs, but the artificially induced bear-Fu hybrid triploid has low doubling rate and survival rate and cannot be applied to large-scale production. The invention provides a novel method for producing excellent hybrid sika-traploid oyster, which improves the fertility rate of the ova of the sika oyster by hybridizing a strong and preferred tetraploid sika oyster and a preferred diploid sika oyster and using high-concentration sperms of the tetraploid sika oyster.
The female parent of the crassostrea sikamea and the male parent of the tetraploid Fujian oyster are hybridized to obtain the triploid Fufu oyster, on one hand, the problems of small size and slow growth speed are solved on the basis of keeping the delicate and delicious taste of the crassostrea sikamea, and on the other hand, the triploid hybrid triploid oyster which can be put into the market all year round is obtained through the highly sterile characteristic of the triploid.
Detailed description of the preferred embodiment
The following examples are illustrative and not limiting of the present invention.
Example 1
A method for producing Fujian oyster and bear oyster interspecific hybridization triploid oyster includes obtaining excellent tetraploid Fujian oyster, optimizing parent, culturing parent, artificial insemination, hatching and culturing larva.
1) Obtaining tetraploid Fujian oyster: in 2019 and 2 months, respectively putting 120 identified 2-age artificially induced triploid Fujian oysters and 50 2-age diploid Fujian oysters into parent oyster cultivation pools with salinity of 32 per thousand and the temperature of 16 ℃, cultivating the diploid Fujian oysters according to a conventional method, wherein the cultivation mode of the triploid Fujian oysters is as follows: standing for one day, heating to 5 deg.C from day 2 to 26 deg.C for 3 days, maintaining at 26 deg.C for 5 days, cooling to 23 deg.C from day 8, feeding 8 times per day, and changing water in the pond for 100%. Triploid and diploid sikaea oysters reached sexual maturity on day 40.
Opening 106 triploid Fujian oysters to obtain 60 female oysters, respectively putting the female oysters into 60 beakers after dearraying and ovum washing, taking an average value by measuring the diameter of 10 ova from each sample of each beaker, mixing the ova in the first 20 beakers with the largest ovum diameter to obtain 3000 thousands of ova in total, and diluting the ova to 10000 ova/ml. Opening 42 diploid Fujian oysters to obtain 24 male oysters, taking 15 largest diploid Fujian oysters for deplanation, filtering and mixing in equal amount. After 5 minutes from fertilization, fertilized eggs were treated with CB (0.5 mg/L) or 6DMAP (50 mg/L) to inhibit the first polar body, washed after 15 minutes, and then placed in 350L of water for incubation, and after incubation, the eggs were placed in 10D-shaped plating baths per ml for normal culture. All treatment operations were carried out at 23-24 deg.C with seawater, sand filtered and UV disinfected, to a salinity of 32% o. When the larva grows to the stage of creeping larva, putting scallop shells to string and cover the seedlings, and after seven days, going to the sea to hang and culture, and culturing according to the existing culturing method of the Fujian oysters. The statistical breeding results are as follows: the hatchability of fertilized eggs is 72 percent, the tetraploid doubling rate of D-type larvae is 81 percent, the survival rate of a workshop culture stage is 73 percent, the survival rate of first-instar sea area culture is 86 percent, and the doubling rate of first-instar tetraploids is 76 percent.
2) The parent is preferably: in 3 months of 2020, cultivated one-year-old tetraploid Fujian oysters are taken, 600 cultured tetraploid Fujian oysters are identified by using a flow cytometer, the largest 60 (10%) cultured tetraploid Fujian oysters are selected as breeding shells, and meanwhile, 600 wild panda oysters are taken, and the largest 60 (10%) cultured tetraploid Fujian oysters are selected as breeding shells.
3) Parent breeding: in order to synchronize gonad development of tetraploid Fujian oyster and diploid bear oyster, heating the diploid bear oyster in advance to promote maturity, keeping the temperature of the bear oyster in a pool at 16 ℃ and 24-26 per thousand, standing for 2 days, heating to 1 ℃ every day, heating to 27 ℃ for keeping, culturing for 15 days, and adjusting the salinity to 30-32 per thousand of normal seawater until the gonad is mature and ready to produce; the tetraploid Fujian oyster is kept in a pool with the water temperature of 16 ℃ and kept standing, and the maturation promotion method is that when the gonad interstitial fluid of the oyster is little, the egg cells are clear or the sperm activity is high through the observation of a microscope, the tetraploid Fujian oyster begins to mature: heating to 22 deg.C in the first day, heating to 27 deg.C in the second day, maintaining for 7 days, cooling to 24 deg.C (1 deg.C per day), and culturing until gonad is mature. The daily management method of the crassostrea sikamea and the tetraploid Fujian oyster is the same, 8 times of feeding are carried out every day, and 100 percent of water is changed after the pond is inverted.
4) After the oysters containing the sida ursinum and the tetraploid Fujian oysters synchronously develop to sexual maturity, 51 oysters containing the sida ursinum are opened, 26 female individuals are identified under a microscope to obtain 3 hundred million eggs, and the collected eggs need to be soaked in seawater for 40-60 minutes for curing; opening strong and preferable 45 tetraploid Fujian oysters, identifying under a microscope to obtain 22 male individuals, obtaining sperms, and filtering impurities by using a 300-mesh donation net. Sperm was inseminated with a high concentration of sperm, i.e., 200 motile sperm per egg. The whole insemination and hatching process adopts filtered and ultraviolet sterilized seawater with the temperature of 27 ℃ and the salinity of 24 per mill, and the oxygen is kept sufficient during hatching. Hatching fertilized eggs at a density of 50 eggs/ml, developing to D type after 24 hours, culturing larvae of the D type at the early stage at a density of 10 eggs/ml at a culturing temperature of 27 ℃, with salinity of 24 per mill, feeding golden algae as initial feed at a daily feeding density of 5 multiplied by 10 4 2X 10 units/mL 4 Feeding every mL for 4-6 times, wherein the bait feeding amount is gradually increased along with the growth of the larvae; when the shell of larva grows more than 150 μm, adjusting larva density to 5/ml, adding appropriate amount of Platymonas mellea and Chlorella, etc., and feeding bait daily to reach 2 × 10 4 one/mL-8X 10 4 Feeding for 6-8 times per mL, adjusting the temperature and salinity of the culture pond, reducing the temperature by 0.5 ℃ every day to 24 ℃, adjusting the salinity by 0.5% every day to 32 per mill, growing to a larva creeping stage, putting scallop shells into the culture pond for stringing seedlings, and taking the scallop shells out of the sea for hanging culture after 10 days. The statistical breeding results of the sidford hybrid triploid oysters are as follows: the hatchability of the fertilized eggs is 62 percent, the triploid doubling rate of the D-type larvae is 100 percent, the survival rate of the workshop culture stage is 67 percent, and the survival rate of the first-instar sea area culture is 82 percent. The sikok hybrid triploid oyster keeps the delicate and delicious taste of the sikok oyster, and simultaneously, compared with the common sikok oyster under the same culture condition, the sikok height of the sikok hybrid triploid oyster is improved by 22 to 28 percent, the fresh weight is improved by 48 to 62 percent, and the survival rate is improved by 12 to 20 percent. Meanwhile, due to the characteristic of high sterility of the triploid, the sikok hybrid triploid oyster which can be put into the market all the year round is obtained.
Claims (2)
1. A method for producing triploid oysters by interspecific hybridization of Fujian oysters and Ursus oysters is characterized in that the method comprises the steps of obtaining excellent tetraploid Fujian oysters, strongly optimizing parents, cultivating parents, artificial insemination, hatching and cultivating larvae;
obtaining the excellent tetraploid Fujian oyster: stimulating the triploid Fujian oyster at 26 deg.c for 5 days, maturing the triploid Fujian oyster in 23 deg.c water for 30-40 days, arranging the eggs of the triploid female oyster in the size from large to small, and fertilizing the eggs in the first 1/3 of the size and the sperms of the diploid Fujian oyster; treating fertilized eggs with 0.5-0.8mg/L cytochalasin B or 30-80 mg/L6-dimethylaminopurine for 15 minutes from 5 minutes after fertilization to inhibit the first polar body discharge; the temperature of the indoor cultivation stage is 23 ℃, the salinity is 30 per mill, and after larvae are attached and metamorphosed, adult shellfish are cultivated on the sea;
the parent is strongly preferred: identifying and confirming adult tetraploid Fujian oysters by using a flow cytometer, arranging the tetraploid Fujian oysters from large to small, selecting the first 10 percent of individuals as the seed shells to promote ripening, simultaneously selecting the oysters of the bear from large to small, and selecting the first 10 percent of individuals as the seed shells to promote ripening;
the parent breeding: when parent gonad ripening is carried out, in order to synchronize gonad development of the sika oyster and the Fujian oyster, the sika oyster is ripened 30-40 days earlier than the Fujian oyster, gonad ripening work of the tetraploid Fujian oyster is started according to gonad development conditions of the sika oyster, when ripening is carried out, the tetraploid Fujian oyster is firstly stimulated for 7 days at high temperature of 27 ℃, then the water temperature is reduced to 1 ℃ every day and is gradually reduced to 24 ℃ for cultivation for 30-40 days until gonad development is ripened, and the salinity is 30 per thousand; the initial temperature of diploid parent strain of the oyster is 16 ℃, and then the temperature is gradually increased to 27 ℃; adjusting the initial salinity to 24-26 per mill, adjusting the salinity to 30-32 per mill after 15 days, and culturing until the gonad is mature;
the artificial insemination and hatching: selecting female oysters of the first 10 percent of the oysters from big to small, collecting eggs, soaking the collected eggs in seawater for 40-60 minutes, and curing; selecting the first 10% tetraploid Fujian oyster male shells with the individuals arranged from big to small to obtain sperms; insemination is carried out according to the proportion of 200 active sperms of each ovum, the whole insemination and hatching process adopts filtered and ultraviolet sterilized seawater, the temperature is 27 ℃, the salinity is 24 per mill, and the hatching density of fertilized ova is 50 ova/ml;
and (3) larva cultivation: the larva culture density at the early stage is 10-15/ml, the temperature is 27 ℃, the salinity is 24 per mill, the chrysophytes are initial baits, the larva culture density is adjusted along with the growth of the larva, and the bait feeding amount is gradually increased; when the length of the larva shell is more than 150 mu m, the temperature and the salinity of the culture pond are adjusted, the temperature is reduced by 0.5 ℃ every day to 23-24 ℃, and the salinity is adjusted by 0.5 every day until the salinity is different from the salinity of the culture sea area by less than or equal to 2.
2. The method as claimed in claim 1, wherein the parent culture is performed by observing that the gonad maturation promoting work of tetraploid Fujian oyster is started when the gonad tissue fluid of the oyster is low, the egg cell is clear or the sperm activity is high.
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| DE945503T1 (en) * | 1994-01-21 | 2000-02-17 | Rutgers, The State University Of New Jersey | Tetraploid shellfish |
| JP5613240B2 (en) * | 2009-08-14 | 2014-10-22 | シーエバー カンパニーリミテッド | How to produce viable tetraploid oysters |
| CN102100197A (en) * | 2010-12-20 | 2011-06-22 | 山东海益宝水产股份有限公司 | Method for breading triploid monomer oysters |
| CN102077800A (en) * | 2010-12-20 | 2011-06-01 | 山东海益宝水产股份有限公司 | Method for breeding oyster |
| CN102763617B (en) * | 2012-08-20 | 2013-10-30 | 中国海洋大学 | Artificial breeding method of sikamea oysters |
| CN107637551B (en) * | 2017-11-15 | 2020-09-29 | 中国海洋大学 | Method for breeding new strain of northern low-temperature-resistant crassostrea sikamea |
| CN109730008B (en) * | 2019-03-11 | 2021-01-01 | 中国海洋大学 | Method for cultivating grapevine oyster tetraploid |
| CN113016673A (en) * | 2021-03-03 | 2021-06-25 | 世倍(厦门)海洋科技有限公司 | Method for producing oyster of Fujian oyster of yellow shell/black shell triploid in large scale |
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