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 PDF

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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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cryopreservation
liquid nitrogen
sea cucumber
sperm
cryotube
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PCT/CN2020/090969
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French (fr)
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常亚青
湛垚垚
赵谭军
宋坚
张伟杰
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大连海洋大学
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Priority to KR1020207032341A priority Critical patent/KR102199675B1/ko
Priority to JP2021507063A priority patent/JP6983452B2/ja
Publication of WO2021012763A1 publication Critical patent/WO2021012763A1/zh

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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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  • 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)
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Abstract

本发明公开一种海参精子超低温冷冻保存方法，依次按照如下步骤进行：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；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，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；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。

Description

海参精子超低温冷冻保存方法 技术领域
本发明属于海参低温生物学及种质超低温冷冻保存技术领域，尤其涉及一种海参精子超低温冷冻保存方法。
背景技术
海参属于棘皮动物门(Echinodermata)，游走亚门(Eleutherozea)，海参纲(Holothuroidea)，广泛分布于全球温带区及热带区海洋，是最常见的海洋高等无脊椎动物。海参“其性温补，足敌人参”富含多种营养元素和生物活性物质，是药食同源的滋补佳品。随着海参市场需求以及产业规模逐年扩大，野生海参的过度采捕日趋严重，使得野生海参的资源量和种质质量急剧下降，养殖海参亦出现单产降低和种质退化等问题。为此，目前研究人员多采用地理隔离较大的南北海参杂交方法以提高海参种质质量。但是，由于南北雌雄海参并不同步成熟，以至于在海参杂交过程中均需采用催熟、促排等手段，不仅操作复杂、成本高，而且还会出现因催熟、促排失败而导致杂交无法实现等问题。
2006年，Shao等人发表的论文(Shao M Y,Zhang Z F,Yu L,et al.Cryopreservation of sea cucumber Apostichopus japonicus(Selenka)sperm[J].Aquaculture Research,2006,37(14):1450-1457.)中使用保存液对海参精子稀释后进行冷冻保存。该方法操作时间达1小时，精子活力损失较大；保存液配方为423.00mM NaCl、9.00mM KCl、9.27mM CaCl 2、22.94mM MgCl 2、22.50mM MgSO 4和10mM Hepes缓冲液+15％二甲基亚砜。Hepes缓冲液不仅价格较高且暴露于可见光下会产生具一定生物毒性的过氧化氢；精子保存时间短(1小时)，解冻后，精子寿命仅为1200s，难以应用于生产。
发明内容
本发明是为了解决现有技术所存在的上述技术问题，提供一种海参精子超低温冷冻保存方法。
本发明的技术解决方案是：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。
本发明操作时间短(15～20min)，避免精子活力损失较大的问题；所用保存液成本低且无毒；保存时间长(对精子冷冻保存17天)，精子最高成活率达到19.00±4.00％，寿命达2817.33s；解冻复苏的精子的受精率可达83.72～83.74％，卵裂率(统计2～4细胞期以上)达到77.67～87.05％，受精20小时后，测定囊胚孵化率为56.47～65.00％，适用于海参人工繁殖、杂交育种以及种质资源保存等方面。
具体实施方式
a.利用大连海洋大学农业农村部北方海水增养殖重点实验室培育的雄性海参，体长20～25cm，体重450～650g。在2019年5月份繁殖季节将海参从水中取出并将海参身体表面海水擦干(避免有海水激活精子)，用灭菌消毒的剪刀腹部解剖海参，避免将性腺剪破，用镊子挑出完整性腺，用无菌滤纸将性腺表面体腔液吸干，移至到无菌培养皿中，将性腺剪碎，用5ml注射器吸取，并用300目的筛绢(高温消毒并60℃烘干)包住注射口进行过滤，过滤过程中避免体腔液与海水混入，将过滤好的精子收集到50ml离心管，暂置在有冰块的保温盒中；
b.利用移液枪吸取过滤后精液和冷冻保存液各300μl，加入2ml无菌冷冻保存管中混合均匀，所述冷冻保存液是在每升蒸馏水中加入12g葡萄糖、7g氯化钠、0.7g氯化钾、5g海藻糖、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(-196℃)保存。
实验：
精子活力检测：蘸取少量a步骤过滤后的精液涂在载玻片上，滴加一滴海水激活，在显微镜下观察精子活力，统计分析显示精子活力为85.55±5.00％。
冷冻精子的解冻及活力检测：将c步骤的冷冻管从液氮中取出，在液氮表面平衡30s后，立即放入30℃恒温水浴中解冻，解冻过程中不断摇动冷冻管，直至完全融解。解冻冷冻保存17天的精子，进行精子活力观察和统计：取少量解冻后的精液涂抹在载玻片上，滴加一滴海水进行激活，在显微镜下观察精子活力，统计分析显示，解冻后其精子活力最高可达到19.00±4.00％，寿命2817.33s。
冷冻精子解冻激活后的受精检测：将解冻的冷冻保存17天的精子注入含有卵子的海 水中(卵子密度13～26颗/ml)，按照常规方法，每隔1小时上下搅动海水，促进精卵结合完成受精。受精4小时后，用300目筛绢洗卵，洗去多余精子，将受精卵放入21～22℃海水中进行孵化。统计分析显示，解冻复苏的精子的受精率可达83.72～83.74％，卵裂率(统计2～4细胞期以上)达到77.67～87.05％；受精20小时后，测定囊胚孵化率为56.47～65.00％。
与现有的鱼类、海胆和贝类的有关冷冻保存液对比：本发明实施例的冷冻保存液替换成鱼类精子冷冻保存液，其它步骤不变冷冻保存海参精子48小时，解冻后海参精子活力低(最高：11±2.33％)且解冻后精子寿命较短(539s)；本发明实施例的冷冻保存液分别替换成海胆和贝类精子冷冻保存液，其它步骤不变冷冻保存海参精子48小时，解冻后海参精子活力几乎为零。说明鱼类、海胆和贝类的冷冻保存液并不适用于海参精子冷冻保存。

Claims (1)

  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。
PCT/CN2020/090969 2019-07-19 2020-05-19 海参精子超低温冷冻保存方法 WO2021012763A1 (zh)

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