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Collection of in vivo Mature Oocytes and Fertilized Oocyte   

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摘要: 猪是多胎动物,自然发情一次可排10-15个卵母细胞,为了获得更多卵母细胞或胚胎用于胚胎学研究,我们通常对供体母猪进行超数排卵。经过超排卵,一头供体母猪可排除20-30个卵母细胞或者受精卵,用于后续体外受精或者受精卵注射。

关键词: 超数排卵, 体内成熟, 卵母细胞

材料与试剂

  1. 75% 酒精(安徽安特生物化学有限公司) 
  2. DPBS (Gibco,catalog number: D8537) 
  3. 0.9% 生理盐水(衡阳制药有限公司)
  4. 石蜡油(Sigma,catalog number: M5310)
  5. NaCl(Sigma,catalog number: S5886)
  6. KCl(Sigma,catalog number: P4504)
  7. NaHCO3(Sigma,catalog number: S8875)
  8. NaH2PO4(Sigma,catalog number: S5011)
  9. MgCl2·6H2O(Sigma,catalog number: M0250)
  10. Hepes(Sigma,catalog number: H3784)
  11. Penicillin(Sigma,catalog number: P3032)
  12. phenol Red(Sigma,catalog number: 5530)
  13. CaCl2·2H2O(Sigma,catalog number: C7902)
  14. Polyvinyl alcohol(PVA,catalog number: Sigma P8136)
  15. Sorbitol(Sigma,catalog number: S1876)
  16. Gentamicin(Sigma,catalog number: G1264)
  17. Sodium pyruvate(Sigma,catalog number: P4562)
  18. H2O(Sigma,catalog number: W-1503)
  19. Na Lactate(Sigma,catalog number: L7900) 
  20. TCM-199(Gibco,catalog number: 11150-059)
  21. D-glucose(Sigma,catalog number: G7021)
  22. Penicillin(Sigma,catalog number: P3032)
  23. Streptomycin(Sigma,catalog number: S1277)
  24. Cysteine(Sigma,catalog number: C8152) 
  25. Hyaluronidase(Sigma,catalog number: H3506) 
  26. Mannitol(Sigma,catalog number: M9647)
  27. BSA(Sigma,catalog number: A8022)
  28. KH2PO4(Sigma,catalog number: P-5655)
  29. MgSO4·7H2O(Sigma,catalog number: M1880)
  30. Na-Pyruvate(Sigma,catalog number: 4562)
  31. Ca.(Lactate)2·5H2O(Fisher,catalog number: C110-500)
  32. L-Glutamine(Sigma,catalog number: G-7029)
  33. Hypotaurine(Sigma,catalog number: H-1384)
  34. Regu-mate(Intervet RM480150)
  35. Estrumate (Bayer)
  36. hCG(Intervet,catalog number: CH4751)
  37. NEAA(Sigma,catalog number: M7145)
  38. EAA(Sigma,catalog number: B-6766)
  39. 四孔板(NUNC)
  40. 24孔板(NUNC)
  41. 六孔板(NUNC)
  42. 培养皿(NUNC)
  43. 1.0mm×0.6mm玻璃管(WPI)
  44. 1.0×0.8mm玻璃管(WPI)
  45. 2.5×2.0mm玻璃管(WPI)

仪器设备

  1. 培养箱(THERMO-3111)
  2. 超净工作台(瑞智净化)
  3. 倒置显微镜(OLYMPUS-IX51)
  4. 体式显微镜(NIKON-SMZ645)
  5. 显微镜加热台(THERMO)
  6. 2.5×2.0mm玻璃管(WPI)

实验步骤

  1. 在饲料中添加四烯雌酮(Regu-mate, Intervet, RM480150) 18-20mg/天进行发情周期抑制,根据母猪处于的发情周期不同分别给药4-14天使性成熟母猪发情周期的同步化。
  2. 在四烯雌酮给药的最后一天肌肉注射Estrumate (250 mg, Bayer)诱导发情。
  3. 最后一次四烯雌酮给药15-17小时后,肌肉注射1500 IU 的马绒毛膜促性腺激素(eCG: Sigma, G4877) 诱导卵子成熟。
  4. eCG注射82小时后,注射1000IU人绒毛膜促性腺激素(hCG: Intervet CH4751),促进排卵。hCG注射46-54小时后,从输卵管冲取回收成熟卵母细胞。获取受精卵需要在注射hCG 24h后查情,在发情中期选取体重大小适宜的健康公猪进行配种,配种后 24~36h通过输卵管冲取回收受精卵。
  5. 胚胎冲取操作如下:供体猪手术前应停食12-24小时,可适当饮水。对供体猪注射氯胺酮进行诱导麻醉,带上麻醉面罩,打开麻醉机,用异氟烷维持麻醉。采用常规外科手术方法在腹中线倒数第二对乳头处切口7—10 cm,牵引出一侧子宫角,并沿子宫角、输卵管取出该侧卵巢。用冲卵液从输卵管子宫端至伞端冲取回收成熟卵母细胞或受精卵。采卵完毕后,将输卵管、子宫复位,用37℃灭菌生理盐水湿润子宫,冲去凝血块。按腹膜、皮下筋膜、皮肤三层缝合。腹膜、肌肉缝合后,涂抹磺胺等消炎防腐药。皮肤缝合后,在伤口周围涂碘酒,并肌肉注射青链霉素防止创口感染发炎。

结果与分析

  1. 经过超数排卵后,一头供体母猪可获得20-30枚卵母细胞或者受精卵。

失败经验

  1. 受精卵冲取实验中,为了确保卵母细胞能够受精,可用健康公猪进行配种2-3次。

溶液配方

  1. 洗卵液PVA-TL HEPES
    称取6.6633 g NaCl,0.2386 g KCl,0.1680 g NaHCO3,0.0408 g NaH2PO4,0.1017 g MgCl2·6H2O,2.3830 g Hepes,0.0650 g Penicillin,0.0100 g phenol Red,0.2940 g CaCl2·2H2O,0.1000 g Polyvinyl alcohol,2.1860 g Sorbitol,0.0250 g Gentamicin,0.0220 g Sodium pyruvate,加998.132 mL MilliQ H2O后再加1.868 mL Na Lactate,溶解后调节PH 为7.2-7.4,渗透压为295-310 mOsm。 
  2. 胚胎操作液
    9.500 g TCM-199,0.050 g NaHCO3,0.750 g Hepes,0.050 g Penicillin,0.060 g Streptomycin,1.755 g NaCl,3.00 g BSA,1000 mL MilliQ H2O,溶解后调节PH为7.2-7.4溶解后调节PH为7.2-7.4,渗透压为295-310 mOsm。
  3. 胚胎培养液PZM-3
    108.00 mM NaCl
    10 mM KCl
    0.35 mM KH2PO4
    0.40 mM MgSO4·7H2O
    25.07 mM NaHCO3
    0.2 mM Na-Pyruvate
    2.0 mM Ca.(Lactate)2·5H2O
    1.0 mM L-Glutamine
    5.0 mM Hypotaurine
    10 ml/L NEAA
    20 mg/mL EAA
    0.05 mg/mL Gentamicin
    3 mg/ml BSA

致谢

  1. 感谢国家重点研发计划”干细胞与转化研究”专项提供经费支持。
  2. 已发表的使用本实验方案的文章“A Huntingtin Knockin Pig Model Recapitulates Features of Selective Neurodegeneration in Huntington's Disease”“Piglets cloned from induced pluripotent stem cells”。 
  3. 该实验方案摘自“Production of Cloned Pigs by Using Somatic Cells as Donors”。

参考文献

  1. 樊娜娜,杨东山,赖良学.(2009). 克隆猪技术及其在转基因猪研究中的应用. 生命科学, 21:652-657.
  2. Lai, L., Kolber-Simonds, D., Park, K. W., Cheong, H. T., Greenstein, J. L., Im, G. S., Samuel, M., Bonk, A., Rieke, A., Day, B. N., Murphy, C. N., Carter, D. B., Hawley, R. J. and Prather, R. S. (2002). Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning.Science 295(5557): 1089-1092.
  3. Lai, L. and Prather, R. S. (2003). Production of cloned pigs by using somatic cells as donors. Cloning Stem Cells 5(4): 233-241.
  4. Lai, L., Kang, J. X., Li, R., Wang, J., Witt, W. T., Yong, H. Y., Hao, Y., Wax, D. M., Murphy, C. N., Rieke, A., Samuel, M., Linville, M. L., Korte, S. W., Evans, R. W., Starzl, T. E., Prather, R. S. and Dai, Y. (2006). Generation of cloned transgenic pigs rich in omega-3 fatty acids. Nat Biotechnol 24(4): 435-436.
  5. Yang, D., Wang, C. E., Zhao, B., Li, W., Ouyang, Z., Liu, Z., Yang, H., Fan, P., O'Neill, A., Gu, W., Yi, H., Li, S., Lai, L. and Li, X. J. (2010). Expression of Huntington's disease protein results in apoptotic neurons in the brains of cloned transgenic pigs. Hum Mol Genet 19(20): 3983-3994.
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Copyright: © 2022 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:樊娜娜, 赵宇, 欧阳振, 李昊, 赖良学. (2022). 体内成熟卵母细胞和受精卵收集. // 实验动物胚胎操作实验手册. Bio-101: e1010966. DOI: 10.21769/BioProtoc.1010966.
How to cite: Pan, N. N., Zhao, Y, Ouyang, Z, Li, H. and Lan, L. X. (2022). Collection of in vivo Mature Oocytes and Fertilized Oocyte. // Embryo Manipulation Manual of Laboratory Animals. Bio-101: e1010966. DOI: 10.21769/BioProtoc.1010966.
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