WO2021012763A1 - 海参精子超低温冷冻保存方法 - Google Patents
海参精子超低温冷冻保存方法 Download PDFInfo
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- WO2021012763A1 WO2021012763A1 PCT/CN2020/090969 CN2020090969W WO2021012763A1 WO 2021012763 A1 WO2021012763 A1 WO 2021012763A1 CN 2020090969 W CN2020090969 W CN 2020090969W WO 2021012763 A1 WO2021012763 A1 WO 2021012763A1
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- WO
- WIPO (PCT)
- Prior art keywords
- cryopreservation
- liquid nitrogen
- sea cucumber
- sperm
- cryotube
- Prior art date
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Classifications
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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
Definitions
- the invention belongs to the technical field of sea cucumber cryobiology and germplasm ultra-low temperature cryopreservation, and in particular relates to a method for ultra-low temperature cryopreservation of sea cucumber sperm.
- Sea cucumbers belong to the phylum Echinodermata (Echinodermata), Eleutherozea (Eleutherozea), and Holothuroidea (Holothuroidea). They are widely distributed in the temperate and tropical oceans of the world and are the most common marine higher invertebrates. Sea cucumber is rich in various nutrients and biologically active substances. It is a good nourishing product with the same medicine and food. With the market demand for sea cucumbers and the expansion of the industry scale year by year, the over-harvesting of wild sea cucumbers has become more and more serious, causing the resource quantity and germplasm quality of wild sea cucumbers to decline sharply, and the problems of lower yield and germplasm degradation of farmed sea cucumbers have also occurred.
- the operation time of this method is up to 1 hour, and the sperm motility loss is large; the preservation solution formula is 423.00mM NaCl, 9.00mM KCl, 9.27mM CaCl 2 , 22.94mM MgCl 2 , 22.50mM MgSO 4 and 10mM Hepes buffer + 15% dimethyl Base sulfoxide. Hepes buffer is not only expensive but also produces certain biologically toxic hydrogen peroxide when exposed to visible light. The sperm storage time is short (1 hour). After thawing, the sperm life span is only 1200s, which is difficult to use in production.
- the present invention aims to solve the above-mentioned technical problems in the prior art, and provides a method for cryopreservation of sea cucumber spermatozoa at ultra-low temperature.
- the technical solution of the present invention is: a method for cryopreservation of sea cucumber spermatozoa at ultra-low temperature, which is characterized by sequentially following the steps:
- a Take the sea cucumbers in the breeding period and dry the sea cucumbers. After dissecting from the abdomen, pick out the integrity glands, suck up the body fluids on the integrity glands, then transfer them to a sterile petri dish and cut them into pieces, and then filter the semen with a 300-mesh sieve. ;
- the cryopreservation solution is 8-15g glucose and 7-9g sodium chloride per liter of distilled water. , 0.5 ⁇ 0.7g potassium chloride, 3 ⁇ 5g trehalose, 0.05 ⁇ 0.1g anhydrous calcium chloride and 80 ⁇ 120mL dimethyl sulfoxide with purity ⁇ 99.7%;
- the present invention has short operation time (15-20min), avoids the problem of large sperm motility loss; low cost and non-toxic storage solution used; long storage time (sperm cryopreservation for 17 days), and the highest sperm survival rate reaches 19.00 ⁇ 4.00% ,
- the life span is 2817.33s; the fertilization rate of thawed and recovered sperm can reach 83.72 ⁇ 83.74%, the cleavage rate (statistically above 2 ⁇ 4 cell stage) reaches 77.67 ⁇ 87.05%, 20 hours after fertilization, the blastocyst hatching rate is determined to be 56.47 ⁇ 65.00%, suitable for artificial propagation, hybrid breeding and germplasm resource preservation of sea cucumbers.
- Perform filtration avoid mixing of body cavity fluid with seawater during the filtration process, collect the filtered sperm into a 50ml centrifuge tube, and temporarily place it in a heat preservation box with ice;
- cryopreservation solution is to add 12g glucose, 7g sodium chloride, 0.7g per liter of distilled water Potassium chloride, 5g trehalose, 0.1g anhydrous calcium chloride and 80 ⁇ 120mL dimethyl sulfoxide with a purity of ⁇ 99.7%;
- Sperm motility test dip a small amount of semen filtered in step a and apply it on a glass slide, add a drop of sea water to activate it, observe sperm motility under a microscope, and statistical analysis shows that sperm motility is 85.55 ⁇ 5.00%.
- Frozen sperm thawing and vitality test take the cryotube of step c out of liquid nitrogen, and after 30 seconds of equilibrium on the surface of liquid nitrogen, immediately put it in a 30°C constant temperature water bath to thaw, and shake the cryotube until it is completely thawed.
- Thaw and store the frozen sperm for 17 days to observe and count sperm motility take a small amount of thawed semen and smear it on a glass slide, add a drop of sea water to activate it, observe sperm motility under a microscope, and statistical analysis shows that the sperm motility after thawing The highest can reach 19.00 ⁇ 4.00%, and the life span is 2817.33s.
- Fertilization test after activation of frozen sperm thawing Inject thawed and cryopreserved sperm into seawater containing eggs (egg density 13 ⁇ 26/ml), according to the conventional method, stir the seawater up and down every 1 hour to promote sperm and eggs The combination completes fertilization. 4 hours after fertilization, the eggs were washed with a 300-mesh sieve to remove excess sperm, and the fertilized eggs were incubated in 21-22°C seawater.
- cryopreservation solution of the embodiment of the present invention is replaced with the cryopreservation solution of fish sperm, and the other steps remain unchanged.
- the sea cucumber sperm is frozen for 48 hours, and the sea cucumber sperm is thawed.
- the cryopreservation solution of the embodiment of the present invention is replaced with sea urchin and shellfish sperm cryopreservation solution, and other steps remain unchanged.
- the cryopreservation solution of fish, sea urchin and shellfish is not suitable for cryopreservation of sea cucumber sperm.
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
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- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims (1)
- 一种海参精子超低温冷冻保存方法,其特征在于依次按照如下步骤进行:a.取处于繁殖期的海参并擦干参体海水,由腹部解剖后挑出完整性腺,将完整性腺表面体液吸干后移至无菌培养皿中剪碎,吸取精液用300目的筛绢过滤;b.取过滤后的精液与冷冻保存液按照体积比为1:1~2注入冷冻管中混合均匀,所述冷冻保存液是在每升蒸馏水中加入8~15g葡萄糖、7~9g氯化钠、0.5~0.7g氯化钾、3~5g海藻糖、0.05~0.1g无水氯化钙和80~120mL纯度≥99.7%的二甲基亚砜;c.将冷冻管悬置在距离液氮表面10~15cm处10min,然后再将冷冻管悬置在液氮表面处5min,最后将冷冻管浸入液氮中冷冻保存。
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KR1020207032341A KR102199675B1 (ko) | 2019-07-19 | 2020-05-19 | 해삼정자 초저온 냉동 보존방법 |
JP2021507063A JP6983452B2 (ja) | 2019-07-19 | 2020-05-19 | ナマコの精子の超低温凍結保存法 |
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CN116034992A (zh) * | 2023-03-08 | 2023-05-02 | 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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CN110326610B (zh) * | 2019-07-19 | 2021-09-24 | 大连海洋大学 | 海参精子超低温冷冻保存方法 |
CN112075415B (zh) | 2020-09-25 | 2021-08-31 | 中国科学院海洋研究所 | 一种冷冻保护液及刺参精子超低温冷冻保存方法 |
CN112352777A (zh) * | 2020-11-19 | 2021-02-12 | 中山大学 | 一种精子冻存液及其在中华乌塘鳢精子冷冻保存中的应用 |
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