JP6983452B2 - ナマコの精子の超低温凍結保存法 - Google Patents
ナマコの精子の超低温凍結保存法 Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0236—Mechanical aspects
- A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
- A01N1/0252—Temperature controlling refrigerating apparatus, i.e. devices used to actively control the temperature of a designated internal volume, e.g. refrigerators, freeze-drying apparatus or liquid nitrogen baths
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0601—Invertebrate cells or tissues, e.g. insect cells; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0608—Germ cells
- C12N5/061—Sperm cells, spermatogonia
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Description
繁殖期のナマコを取り、ナマコの体を乾かし、腹部から解剖した後、完全な性腺を摘出し、完全な性腺の表面の体液を吸収した後、滅菌ペトリ皿内に移して細かくせん断し、精液を吸い取り、300メッシュの篩絹でろ過するステップaと、
ろ過した精液と凍結保存液を1:1〜2の体積比で凍結チューブに注入して均一に混合し、前記凍結保存液は、1リットルあたりの蒸留水に8〜15gのグルコース、7〜9gの塩化ナトリウム、0.5〜0.7gの塩化カリウム、3〜5gのトレハロース、0.05〜0.1gの無水塩化カルシウム、及び純度≧99.7%の80〜120mLのジメチルスルホキシドを加えることによって得られるステップbと、
凍結チューブを液体窒素の表面から10〜15cmの距離で10min吊り下げ、次に、凍結チューブを液体窒素の表面に5min吊り下げ、最後に、凍結チューブを液体窒素に浸して凍結保存するステップcと、
を順次に行うことを特徴とする。
b.ピペットを利用して、ろ過した精液と凍結保存液をそれぞれ300μl吸い取り、2mlの滅菌凍結保存チューブに加えて均一に混合し、前記凍結保存液は、1リットルあたりの蒸留水に12gのグルコース、7gの塩化ナトリウム、0.7gの塩化カリウム、5gのトレハロース、0.1gの無水塩化カルシウム、及び純度≧99.7%の80〜120mLのジメチルスルホキシドを加えることによって得られ、
c.凍結チューブを液体窒素の表面から15cmの距離で10min吊り下げ、次に、凍結チューブを液体窒素の表面に5min吊り下げ、最後に、凍結チューブを液体窒素に浸して凍結(−196℃)保存する。
精子の運動性試験:ステップaでろ過した少量の精液を取り、スライドガラスに塗布し、海水を1滴滴下して活性化させ、顕微鏡で精子の運動性を観察し、統計分析によると、精子の運動性が85.55±5.00%であることを示した。
Claims (1)
- 繁殖期のナマコを取り、ナマコの体を乾かし、腹部から解剖した後、完全な性腺を摘出し、完全な性腺の表面の体液を吸収した後、滅菌ペトリ皿に移して細かくせん断し、精液を吸い取り、300メッシュの篩絹でろ過するステップaと、
0.3mlのろ過した精液と0.3mlの凍結保存液を、2mlの凍結チューブに注入して均一に混合し、前記凍結保存液は、1リットルあたりの蒸留水に8〜15gのグルコース、7〜9gの塩化ナトリウム、0.5〜0.7gの塩化カリウム、3〜5gのトレハロース、0.05〜0.1gの無水塩化カルシウム、及び純度≧99.7%の80〜120mLのジメチルスルホキシドを加えることによって得られるステップbと、
前記凍結チューブの全体を液体窒素の表面から10〜15cmの距離で10min吊り下げ、次に、前記凍結チューブを前記液体窒素の表面に5min吊り下げ、最後に、前記凍結チューブの全体を前記液体窒素に浸して凍結保存するステップcと、
を順次に行うことを特徴とするナマコの精子の超低温凍結保存法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910653260.1 | 2019-07-19 | ||
CN201910653260.1A CN110326610B (zh) | 2019-07-19 | 2019-07-19 | 海参精子超低温冷冻保存方法 |
PCT/CN2020/090969 WO2021012763A1 (zh) | 2019-07-19 | 2020-05-19 | 海参精子超低温冷冻保存方法 |
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JP2021525790A JP2021525790A (ja) | 2021-09-27 |
JP6983452B2 true JP6983452B2 (ja) | 2021-12-17 |
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KR (1) | KR102199675B1 (ja) |
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CN116034992B (zh) * | 2023-03-08 | 2023-06-27 | 中国科学院海洋研究所 | 刺参精子低温保存液及应用和刺参精子的保存方法 |
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- 2020-05-19 KR KR1020207032341A patent/KR102199675B1/ko active IP Right Grant
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