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Cryopreservation and Resuscitation of Sperm   

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摘要:精子冷冻与复苏,是指利用冷冻保护剂和逐步降温程序,将精子冻存于液氮中进长期保存。在需要使用的时候,将冻存的精子在适当温度下进行复苏使其重新获得游动活力。复苏后挑选活力良好的精子可用于后续的辅助生殖,最终得到存活动物个体。本文详细描述了猴精子冷冻与复苏的方法及流程。

关键词: 冷冻, 复苏, 辅助生殖

材料与试剂

  1. 冷冻麦管(IMV,catalog number: 005565)
  2. 移液枪及枪头 (Eppendorf)
  3. 1.5 ml 离心管(AXYGEN,catalog number:MCT-150-C-S)
  4. 15 ml 离心管(Corning,catalog number:430791)
  5. 3.5 cm 培养皿(CORNING,catalog number: 430165)
  6. 一次性使用无菌注射器带针(KDL 1ml)
  7. 镊子(吉尔泰)
  8. 液氮
  9. 矿物油(Sigma,catalog number:M8410)
  10. 精子冷冻保护液(Irvine Scientific,catalog number:90128)
  11. TH3液(见溶液配方)
  12. T3液(见溶液配方)

仪器设备

  1. 恒温试管架(Cook)
  2. 培养箱(Thermo Scientific, model:Forma 3111、Forma 371)
  3. 冰箱(海信)
  4. 水浴锅(bluepard)
  5. 离心机 (Eppendorfcentrifuge,model:5702)

实验步骤

一、精子冷冻

  1. 解冻精子冷冻保护液,放培养箱37 °C加热 30 min,摇匀精子冷冻保护液;
  2. 将TH3放于37 °C恒温试管架上预热 30 min;
  3. 提前在精子冷冻麦管上标注好具体信息;
  4. 将采集的精子放于37 °C恒温试管架上液化15-20 min;
  5. 将液化好的精子用移液枪吸入1.5 ml 的离心管中,根据精子的液化浓度加入适量的TH3液,即精子液化量与TH3液的总量是三的倍数;
  6. 按照冷冻保护液:精液等于1:3的比例,用移液枪在离心管内加入冷冻保护液,加冷冻液时应一滴一滴慢慢加,加入一滴就轻摇一下,冷冻液加完后,用移液枪轻轻吹打;
  7. 用注射器连接上提前标记好的冷冻麦管,先吸一段40 μl的TH3液,再吸一段空气,然后吸80 μl的精子,再吸一段空气,然后再吸40 μl的TH3,最后分别将麦管两头在酒精灯上烧软化后捏紧封口;(见图1)


    图1. 精液吸入冷冻麦管后将两头封口

  8. 先放入4 °C的冰箱内1 h,然后再将麦管放于距液氮面2 cm的液氮蒸汽上30 min,最后放入精子冷冻液氮罐内;
  9. 做好详细记录。

二、精子复苏

  1. 提前半小时打开37 °C水浴锅让水温升至37 °C;
  2. 提前半小时将TH3和T3放于37 °C恒温试管架上预热;
  3. 用镊子从液氮中取出冷冻麦管,在空气中停留10 s,然后放37 °C的水浴锅中 1 min;
  4. 加入1 ml TH3液体在 15 ml的离心管内,将精子冷冻麦管两头剪开,用注射器将精子推至TH3液体中,静置 10 min;
  5. 再加入4 ml TH3 液体在精子离心管内,静置10 min;
  6. 将精子1,800 rpm,离心5 min,去上清,加入5 ml的T3液体;
  7. 再将精子1,800 rpm,离心5 min,去上清,加入1 ml的T3液体;
  8. 再次1,800 rpm,离心5 min,离心后等待精子上游;
  9. 上游后取中层的精子,在培养皿内制备精子滴,盖上矿物油,放入培养箱待用。

溶液配方

  1. Tyrode's Albumin Lactate Pyruvate medium with HEPES (TH3)(表1)

    表1. Tyrode's Albumin Lactate Pyruvate medium with HEPES (TH3) 配制表
    成分/100ml/50ml备注
    NaCl3.329 ml1.665 ml200 mg/ml stock
    KCl239 μl119.5 μl100 mg/ml stock
    CaCl2·2H2O294 μl147 μl100 mg/ml stock
    MgCl2·6H2O102 μl51 μl100 mg/ml stock
    NaH2PO4 48 μl24 μl100 mg/ml stock
    NaHCO3 210.6 mg105.3 mg
    HEPES240 mg120 mg
    D-Glucose90 mg45 mg
    Sodium Pyruvate12 mg6 mg
    Sodium lactate (60% syrup)0.17 ml85 μl
    BSA300 mg150 mg
    penicillin-streptomycin (100X)1 ml500 μl
    Phenol Red (0.01%)10 μl5 μl
    pH7.4
  2. Tyrode's Albumin Lactate Pyruvate medium without HEPES (T3)(表2)

    表2. Tyrode's Albumin Lactate Pyruvate medium without HEPES (T3) 配制表
    成分/100ml/10ml备注
    NaCl3.329 ml333 μl200 mg/ml stock
    KCl239 μl23.9 μl100 mg/ml stock
    CaCl2·2H2O294 μl29.4 μl100 mg/ml stock
    MgCl2·6H2O102 μl10.2 μl100 mg/ml stock
    NaH2PO4 48 μl4.8 μl100 mg/ml stock
    NaHCO3 210.6 mg21.06 mg
    Glcose90 mg9 mg
    Sodium Pyruvate12 mg1.2 mg
    Sodium lactate (60% syrup)0.17 ml17 μl
    BSA300 mg30 mg
    penicillin-streptomycin (100X)1 ml100 μl
    Phenol Red (0.01%)10 μl1 μl
    pH7.4

参考文献

  1. 胡京美,许成岩,李媛,耿玲,马刚,王丽,陈子江. 冷冻保护剂与精液比例对冷冻复苏后精子活力的影响. 中华男科学杂志,2009年5期
  2. 朱文兵,范立青,卢光琇. 不同配比的保护剂对人类精子冷冻复苏后活动能力的影响. 中国现代医学杂志,2005年6月第15卷第11期
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Copyright: © 2023 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:王燕, 许玉婷, 李春杨, 孙强. (2023). 精子冷冻与复苏. // 实验动物胚胎操作实验手册. Bio-101: e1010984. DOI: 10.21769/BioProtoc.1010984.
How to cite: Wang, Y., Xu, Y. T., Li, C. Y., and Sun, Q. (2023). Cryopreservation and Resuscitation of Sperm. // Embryo Manipulation Manual of Laboratory Animals. Bio-101: e1010984. DOI: 10.21769/BioProtoc.1010984.
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