CN111587876B - 中间球海胆胚胎的快速玻璃化冷冻保存与复苏方法 - Google Patents

中间球海胆胚胎的快速玻璃化冷冻保存与复苏方法 Download PDF

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CN111587876B
CN111587876B CN202010446846.3A CN202010446846A CN111587876B CN 111587876 B CN111587876 B CN 111587876B CN 202010446846 A CN202010446846 A CN 202010446846A CN 111587876 B CN111587876 B CN 111587876B
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常亚青
湛垚垚
赵谭军
张伟杰
宋坚
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Abstract

本发明公开一种中间球海胆胚胎的快速玻璃化冷冻保存方法,所用玻璃化是将二甲基亚砜(DMSO)、甲醇(MeOH)及1,2丙二醇(1,2‑PG)与过滤消毒海水混合,置于2℃预冷15min,二甲基亚砜、甲醇及丙二醇的体积浓度分别为1.0~2.0%、1.5~2.0%和1.5~2.0%;复苏方法则是采用25℃水浴解冻等处理,解冻后的中间球海胆胚胎原肠完整率可达到47.81%,具有解冻后胚胎原肠完整率高、易操作及成本低等优点。

Description

中间球海胆胚胎的快速玻璃化冷冻保存与复苏方法
技术领域
本发明属于海胆种质保存领域,尤其涉及一种中间球海胆胚胎的快速玻璃化冷冻保存与复苏方法。
背景技术
中间球海胆,又称虾夷马粪海胆,属于棘皮动物门(Echinodermata),游走亚门(Eleutherozea),海胆纲(Echinoidea)。原产于日本北海道和俄罗斯远东等地沿海,1989年自日本引入,现已在大连、山东等地形成一定的人工养殖规模,是我国北方沿海最重要的经济海胆种类。近年来,海胆的市场需求以及产业规模逐年扩大,但是海胆的资源量和种质质量却急剧下降。为了维持海胆优良品种的种质质量以及保护海胆资源多样性,目前已有在海胆繁殖季节对海胆进行常规人工催产和授精后得到的受精卵或囊胚期胚胎进行冷冻保存进而复苏孵化的研究,如慢速冷冻保存或程序降温冷冻保存。慢速冷冻保存,是将海胆受精卵或囊胚期胚胎置于二甲基亚砜中,在-76℃中平衡10 min后转入液氮保存(Asahina E, Takahashi T . CRYOPRESERVATION OF SEA URCHIN EMBRYOS AND SPERM[J].Development Growth and Differentiation, 1979, 21(5):423-430.)。程序降温冷冻保存,是将海胆受精卵或囊胚期胚胎置于1.5M二甲基亚砜和0.04M海藻糖(TRE)中,4℃下初始平衡2min,随后在-1℃min至-12℃冷却平衡2min,后以-1℃/min降至-80℃后平衡2 min,最后转移到液氮中进行储存(Bellas J , Estefanía Paredes. Advances in thecryopreservation of sea-urchin embryos: Potential application in marine waterquality assessment[J]. Cryobiology, 2011, 62(3):0-180.)。但是,以上两种技术所需要的仪器较多且操作比较繁琐,不利于大规模应用。
目前,已有应用于鱼类和甲壳类水产动物的胚胎快速玻璃化冷冻保存技术,是将包裹有目标细胞的高浓度保护剂(玻璃化液)在超低温环境下凝固,形成不规则的玻璃化样固体。该技术可使细胞本身及玻璃化液冷冻时呈现黏稠而不产生结晶的玻璃化状态,可大幅度提高细胞存活率。现有用于鱼类胚胎快速玻璃化冷冻保存技术的玻璃化液是以蒸馏水100毫升、2.472%氯化钠、0.086%氯化钾、0.146%二水氯化钙、0.0486%六水合氯化镁和0.019%碳酸氢钠混合而成,而用于虾类胚胎快速玻璃化冷冻保存技术的玻璃化液是由30%甘油、6%乙二醇、3 mg/mL牛血清蛋白、10 mg/mL聚蔗糖和0.5 mol/L蔗糖混合而成。但是使用这两种玻璃化液对海胆胚胎进行玻璃化冷冻保存,解冻后的海胆胚胎几乎全部破裂,难以找到完整胚胎,即现有玻璃化液并不适合中间球海胆胚胎的快速玻璃化冷冻保存。
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,提供一种中间球海胆胚胎的快速玻璃化冷冻保存与复苏方法。
本发明的技术解决方案是:一种中间球海胆胚胎的快速玻璃化冷冻保存方法,依次按照如下步骤进行:
a. 胚胎的收集:对中间球海胆进行人工催产和授精,收集受精卵发育至原肠期的胚胎;
b. 玻璃化液配制:将二甲基亚砜、甲醇及1,2丙二醇与过滤消毒海水混合,置于0~4℃预冷15min,所述二甲基亚砜、甲醇及丙二醇的体积浓度分别为1.0~2.0%、1.5~2.0%和1.5~2.0 %;
c. 胚胎冷冻前的平衡处理:将收集的胚胎依次在1/5、1/4、1/3、1/2和1浓度的玻璃化液中各浸泡5~6min;
d. 胚胎超低温冷冻保存:将经过平衡处理的胚胎连同250μl最后一次平衡处理用玻璃化液吸入麦管中并热压封口,立即置入液氮中冷冻保存。
一种以上述方法冷冻后的中间球海胆胚胎复苏方法,将冷冻的麦管从液氮中取出,立即放入25℃水浴中,待麦管中玻璃化液乳白色即将消失时,将麦管取出并剪去封口,用1~2mL的0.075M的蔗糖溶液洗脱5~10min,之后每5 min加入同体积过滤海水进行清洗,共清洗3次,用新鲜无菌过滤海水培养。
本发明是在消毒过滤海水中添加二甲基亚砜、甲醇和1,2丙二醇作为玻璃化液实现对中间球海胆胚胎的快速玻璃化冷冻保存,并经过25℃水浴解冻等专用复苏方法,使得解冻后的中间球海胆胚胎原肠完整率可达到47.81%,具有解冻后胚胎原肠完整率高、易操作及成本低等优点。
具体实施方式
本发明的一种中间球海胆胚胎的快速玻璃化冷冻保存方法,依次按照如下步骤进行:
a. 胚胎的收集:在2019年10月份繁殖季节将中间球海胆(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中间球海胆)从水中取出并将海胆身体表面海水擦干,利用氯化钾对中间球海胆进行人工催产,倒置于培养皿中,待产精(白色)或产卵(橙黄色)后,随机混合3只以上海胆的精子与卵子均匀混合于5L烧杯中,持续观察至原肠期,收集原肠期的胚胎;
b. 玻璃化液配制:将二甲基亚砜(DMSO)、甲醇(MeOH)及1,2丙二醇(1,2-PG)与过滤消毒海水混合,置于2℃预冷15min,所述二甲基亚砜、甲醇及丙二醇的体积浓度分别为1%、2%和2 %;
c. 胚胎冷冻前的平衡处理:将收集的胚胎依次在1/5、1/4、1/3、1/2和1浓度的玻璃化液中在烧杯中各浸泡5min;每一次浸泡时尽量清除上次玻璃化液并用500目筛绢置于烧杯顶部,逐滴加入玻璃化液,利用玻璃化液自然渗透筛绢后落入烧杯,不断滴加玻璃化液使胚胎一直处于玻璃化液中,直至达到平衡时间(5min),五步共计时25min;
d. 胚胎超低温冷冻保存:将经过平衡处理的胚胎连同250μl最后一次浸泡处理用玻璃化液吸入直径3 mm、长度约为10 cm的麦管中,酒精灯灼烧管口,镊子热压封口后,立即丢入液氮(-196℃)中冷冻保存;
需要复苏时,将冷冻的麦管从液氮中取出,立即放入25℃水浴使其快速解冻,待麦管中玻璃化液的乳白色即将消失时,将麦管取出剪去封口,将中间球海胆胚胎置于培养皿中,用1.5 mL的0.075M的蔗糖溶液洗脱5 min,立即放置于显微镜下进行观察,原肠期胚胎的完整率达47.81%;之后每5min加入5mL过滤海水进行清洗,共清洗3次,最后用新鲜无菌过滤海水培养。

Claims (1)

1.一种中间球海胆胚胎的快速玻璃化冷冻保存方法,其特征在于依次按照如下步骤进行:
a. 胚胎的收集:从水中取出并将海胆身体表面海水擦干,利用氯化钾对中间球海胆进行人工催产,倒置于培养皿中,待产精或产卵后,随机混合3只以上海胆的精子与卵子均匀混合于5L烧杯中,持续观察至原肠期,收集原肠期的胚胎;
b. 玻璃化液配制:将二甲基亚砜、甲醇及1,2丙二醇与过滤消毒海水混合,置于2℃预冷15min,所述二甲基亚砜、甲醇及丙二醇的体积浓度分别为1.0%、2.0%和2.0 %;
c. 胚胎冷冻前的平衡处理:将收集的胚胎依次在1/5、1/4、1/3、1/2和1浓度的玻璃化液中各浸泡5min,每一次浸泡时尽量清除上次玻璃化液并用500目筛绢置于烧杯顶部,逐滴加入玻璃化液,利用玻璃化液自然渗透筛绢后落入烧杯,不断滴加玻璃化液使胚胎一直处于玻璃化液中,直至达到平衡时间,五步共计时25min;
d. 胚胎超低温冷冻保存:将经过平衡处理的胚胎连同250μl最后一次平衡处理用玻璃化液吸入麦管中并热压封口,立即置入液氮中冷冻保存;
e. 将冷冻的麦管从液氮中取出,立即放入25℃水浴中,待麦管中玻璃化液乳白色即将消失时,将麦管取出并剪去封口,将中间球海胆胚胎置于培养皿中,用1.5 mL 的0.075M的蔗糖溶液洗脱5min,之后每5 min加入同体积过滤海水进行清洗,共清洗3次,用新鲜无菌过滤海水培养。
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