Classifications

• • 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
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
  • A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
  • A01K67/0334—Genetically modified Molluscs
• • 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
• • 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
  • A01K2217/00—Genetically modified animals
  • A01K2217/03—Animals modified by random mutagenesis, e.g. using ENU, chemicals
• • 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
  • A01K2227/00—Animals characterised by species
  • A01K2227/70—Invertebrates
• • 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
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/02—Animal zootechnically ameliorated
• • 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
  • Y02A40/81—Aquaculture, e.g. of fish

Definitions

• One embodiment of the present invention is directed to a method for efficiently producing viable tetraploid oysters.
• Meiosis is the process of reducing the number of chromosomes by half to prevent doubling the number of chromosomes in each generation. This is a two step process, whereby one diploid cell becomes four haploid cells, each with one set of chromosomes. One or all four of these haploid cells can mature into functional eggs or sperm known as spouses.
• Tetraploid oysters are important for a variety of purposes, including triploid, hybridization and other breeding programs.
• Triploid oysters have been known to have certain commercial advantages, such as better taste and improved growth rate, compared to normal diploid oysters during the normal reproduction of diploids.
• triplet oysters are produced from normal diploid oysters using a specific chromosome set manipulation technique, whereby meiosis in oocytes causes the oocytes to release the second polar body during the second meiosis. Engineered to retain in oocytes.
• PB1 first polar body
• One embodiment of the present invention provides a method for efficiently producing a viable tetraploid oyster.
• One embodiment of the present invention comprises the steps of fertilizing the eggs of the diploid female oyster with sperm of the diploid male oyster; Retarding the release of the first polar body from the fertilized egg; And culturing the fertilized eggs to produce viable tetraploid oysters; wherein the release of the first polar is inhibited in cytogalasin B (CB) and 6-dimethylaminopurine (DMAP).
• CBD cytogalasin B
• DMAP 6-dimethylaminopurine
• the method includes the step of fertilizing the eggs of triploid female oysters with the sperm of diploid male oysters.
• Triploid oysters are commercially available (Allen, Jr., S.K. (1988); Oceanus 31, 58-63).
• Triploid oysters use chromosomal set manipulation techniques to allow meiosis to be manipulated in oocytes to retain the second polar body in oocytes instead of releasing the second polar body during second meiosis. (diploid oyster) can be produced.
• Triploid oysters can be examined by flow cytometry prior to spawning to confirm their ploidy. Eggs of triploid oysters can be collected by strip spawning.
• the eggs can be washed with filtered seawater and held on a suitable screen such as a 25 ⁇ m screen.
• the triploid oyster used in one embodiment of the present invention may be conditioned by placing in an environment with high temperature and abundant food. The conditioning preferably begins at an early stage of gametogenesis immediately after winter dormancy.
• the eggs are then fertilized with sperm obtained from normal diploid males.
• the amount of sperm used for fertilization may be about 10 or more sperm cells per egg.
• the modification can be made by known methods. For example, it can be made by contacting the eggs and sperm in seawater.
• the method also includes retarding the release of the first polar body from the fertilized egg.
• the inhibition of release of the first polar body may comprise contacting the modified egg with cytogalacin B (CB) and 6-dimethylaminopurine (DMAP).
• the contact may be in the time range of 30% to 90% of the time the fertilized egg releases the first polar body.
• the contact may be, for example, made in 5 to 15 minutes after the correction.
• the contact may be the simultaneous contact of the fertilized egg with cytogalasin B (CB) and 6-dimethylaminopurine (DMAP).
• the contact may be a sequential contact of the fertilized egg and cytogalacin B (CB) and 6-dimethylaminopurine (DMAP).
• the contact may be contacting cytogalasin B (CB) with the fertilized egg followed by contacting 6-dimethylaminopurine (DMAP) with the fertilized egg.
• the contact may be, for example, contacting cytogalasin B (CB) with the fertilized egg in a time range of 30% to 90% of the time at which the fertilized egg releases the first polar body, followed by 6-dimethylaminopurine (DMAP) and the fertilized egg may be contacted in a time range of 50% to 90% of the time that the fertilized egg releases the first polar body.
• One embodiment of the contact is a contact between the cytogalsin B (CB) and the fertilized egg from 5 minutes to 15 minutes after fertilization, and then 6-dimethylaminopurine (DMAP) and the fertilized egg after fertilization 8.3 May be contacting in minutes to 15 minutes.
• the contact was modified in 6-dimethylaminopurine (DMAP) at 0.25-1.0 ppm concentration in cytogallasin B (CB) and 0.25-1.0 ppm concentration in dimethyl sulfoxide (DMSO) in dimethyl sulfoxide (DMSO). It may be to add the eggs.
• DMAP 6-dimethylaminopurine
• the contact is carried out to 6-dimethylaminopurine (DMAP) at a concentration of 0.25 to 0.75 ppm cytotolacin B (CB) and 0.25 to 0.75 ppm in dimethyl sulfoxide (DMSO) in dimethyl sulfoxide (DMSO). It may be to add the modified egg.
• the method also includes culturing the fertilized egg to produce a viable tetraploid oyster.
• the culture may be cultured by a conventional method for culturing fertilized eggs known in the art.
• the culture may be made at 18 °C to 30 °C, for example, 23 °C to 28 °C.
• the culture may be at a salinity of 17 ppt to 33 ppt, for example, about 20 ppt to 22 ppt.
• the oysters may be of the genus Crassostrea .
• Crassostrea genus may be selected from the group consisting of Crassostrea gigas , Crassostrea sikamai , Crassostrea rivularis and Crassostrea virginica .
• the liquor may also be a genus of Pinctada .
• the oyster may be Pinctada magaratifera .
• a tetraploid oyster can be produced that can grow and mature in natural conditions in which a conventional diploid oyster can naturally grow, and the tetraploid oyster is crossed with the diploid oyster to produce a triploid oyster. Can be generated.
• tetraploid oysters can be efficiently produced from triplex oysters and diploid oysters.
• triploid Pacific oyster used in this example was 2 years old and was prepared by retarding the release of the second polar body (PB2).
• Triploid oysters were individually identified by flow cytometry before spawning. Spouses were collected by strip spawning.
• the eggs were passed through an 85 ⁇ m screen to remove large tissue debris and washed on a 25 ⁇ m screen. All modifications and treatments were performed in 25 ° C. to 28 ° C. seawater filtered on a 2 ⁇ m screen.
• the salinity of the seawater used in this example was about 20 ppt to 22 ppt.
• Eggs derived from triploids were fertilized with haploid sperm derived from diploids. After fertilization, fertilized eggs were divided into the following groups: control group, control group, and experimental group.
• CB cytogalasine B
• DMSO dimethylsulfoxide
• Comparative Group 1 and 2 fertilized eggs containing 0.25% 0.5% DMSO
• Comparative Group 2 0.5mg / l containing 0.5% DMSO
• Comparative Group 3 the fertilized eggs were treated with 6-dimethylaminopurine (hereinafter abbreviated as DMAP) to arrest the release of the first polar body.
• DMAP 6-dimethylaminopurine
• CB and DMAP were prepared by dissolving in DMSO, and fertilized eggs containing 0.25 mg CB / l and 0.25 mg DMAP / l (Experiment 1) containing 0.5% DMSO and 0.5 mg CB / l and 0.5 mg DMAP containing 0.5% DMSO. / l (experimental group 2) at the final concentration.
• each treatment started 5 minutes after fertilization and continued for 15 minutes. After each treatment, fertilized eggs were washed with 1% DMSO-sea water and incubated at a density of 65 eggs / ml.
• the fertilized eggs were treated with CB and DMAP to arrest the release of the first polar body.
• CB and DMAP were prepared by dissolving in DMSO.
• the fertilized eggs were treated with a final concentration of 0.25 mg CB / l containing 0.5% DMSO at 5 minutes after fertilization and a final concentration of 0.25 mg DMAP / l at 10 minutes after fertilization. It was added to and continued until 15 minutes after fertilization (Experimental group 3).
• the fertilized eggs were treated with CB and DMAP to arrest the release of the first polar body.
• CB and DMAP were prepared by dissolving in DMSO.
• the fertilized eggs were treated with a final concentration of 0.5 mg CB / l containing 0.5% DMSO at 5 minutes after fertilization and a final concentration of 0.5 mg DMAP / l at 10 minutes after fertilization. It was added until the lasting 15 minutes after fertilization (Experimental group 4).
• fertilized eggs were washed with 1% DMSO-sea water and incubated at a density of 65 eggs / ml.
• the ploidy composition of surviving larvae on these sampling days was determined using flow cytometry.
• live oysters were harvested to count body weight and chromosome number.
• chromosome analysis oysters were treated with colchicine (0.005%) for 12 hours with intensive feeding. Oysters were separated from the shells and weighed. Next, the whole body was cut and fixed in an acetic acid / methanol mixture (1: 3). Appropriate amount of fixed sample was poured onto the slides and air dried. Slides were Leishman's stain. At least 10 metaphase cells that did not show signs of chromosomal loss were counted for each oyster. Only individuals whose chromosome number was clearly determined were used for the analysis.
• Oysters with 20, 30 and 40 chromosomes were named diploid, triploid and tetraploid, respectively.
• Table 2 shows the results of measuring the weight and the number of stains of 50 oysters derived from the experimental group at 3 months after fertilization. Obviously only chromosome counts were included in the data, and oysters without measurable mid-term cells were excluded from the analysis.
• the produced tetraploid oysters were able to grow under the conditions of natural diploid oysters.
• the tetraploid oysters were able to produce triploid oysters by crossing with diploid oysters.
• the tetraploid oysters could produce other tetraploid oysters by crossing between the tetraploid oysters.

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• Life Sciences & Earth Sciences (AREA)
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• Engineering & Computer Science (AREA)
• Biotechnology (AREA)
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• Animal Husbandry (AREA)
• Biodiversity & Conservation Biology (AREA)
• Animal Behavior & Ethology (AREA)
• Marine Sciences & Fisheries (AREA)
• Farming Of Fish And Shellfish (AREA)
• Meat, Egg Or Seafood Products (AREA)

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• 2010
  • 2010-08-06 CN CN201080046057.5A patent/CN102573453B/zh active Active
  • 2010-08-06 WO PCT/KR2010/005169 patent/WO2011019166A2/ko active Application Filing
  • 2010-08-06 JP JP2012524635A patent/JP5613240B2/ja active Active
• 2012
  • 2012-03-09 CO CO12041581A patent/CO6511223A2/es unknown

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