中国畜禽种业 ›› 2026, Vol. 22 ›› Issue (5): 88-95.doi: 10.19543/j.cnki.1673-4556.20260428.006

• 动物繁殖生理 • 上一篇    下一篇

猪胚胎冷冻保存技术研究进展

刘志国1(), 黄雷2, 文一龙1, 牟玉莲1()   

  1. 1.中国农业科学院北京畜牧兽医研究所/畜禽生物育种全国重点实验室,北京 100193
    2.中国农业科学院农业基因组研究所/农业农村部畜禽生物组学重点实验室/岭南现代农业科学与技术广东省实验室深圳分中心,广东 深圳 518120
  • 收稿日期:2025-11-27 出版日期:2026-05-26 发布日期:2026-06-17
  • 通讯作者: 牟玉莲 E-mail:liuzhiguo@caas.cn;mouyulian@caas.cn
  • 作者简介:刘志国(1986—),男,副研究员,河北唐山人,研究方向:动物遗传育种与繁殖,E-mail:liuzhiguo@caas.cn
  • 基金资助:
    畜禽生物育种全国重点实验室(XQSWYZQZ-QNYC1);中国农业科学院科技创新工程(ASTIP-IAS05)

Advances in cryopreservation of porcine embryos

Zhiguo Liu1(), Lei Huang2, Yilong Wen1, Yulian Mu1()   

  1. 1.State Key Laboratory of Animal Biotech Breeding, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193
    2.Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Livestock and Poultry Multi-omics of MARA, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 518120, Guangdong
  • Received:2025-11-27 Online:2026-05-26 Published:2026-06-17
  • Contact: Yulian Mu E-mail:liuzhiguo@caas.cn;mouyulian@caas.cn

摘要:

猪胚胎冷冻保存技术是猪种质资源保护、高效利用以及基因编辑猪新品种(系)创制的关键手段之一,但由于猪胚胎脂质含量高且对低温极度敏感,其冷冻胚胎的解冻存活率以及后续发育潜能仍然与新鲜胚胎存在较大差距,尚未形成能支撑规模化应用的技术体系。该文系统介绍了猪胚胎冷冻保存技术的重要性以及常规缓慢冷冻法的局限性,重点讨论了玻璃化冷冻技术的研究现状和主要研究方向,阐述了猪玻璃化冷冻技术中冷冻保护剂改良、冷冻流程优化方面的研究现状以及利用高通量测序技术在转录组与蛋白质组水平解析猪胚胎低温损伤分子机制的最新突破性进展,最后指出未来应结合人工智能模型、代谢重编程及自动化装载设备,进一步提升冷冻胚胎的发育潜能,为地方猪遗传资源的长期保存与产业化应用奠定坚实基础。

关键词: 猪胚胎, 胚胎冷冻, 玻璃化冷冻

Abstract:

Porcine embryo cryopreservation technology serves as a key approach for the conservation and efficient utilization of pig germplasm resources, as well as for the creation of new gene-edited pig breeds. However, due to the high lipid content in porcine embryos and their extreme sensitivity to low temperatures, the post-thaw survival rate and subsequent developmental potential of cryopreserved embryos still lag significantly behind those of fresh embryos. A technical system capable of supporting large-scale applications has yet to be established. This article systematically introduces the importance of porcine embryo cryopreservation and the limitations of conventional slow-freezing methods. It focuses on discussing the current research status and main directions of vitrification cryopreservation technology, elaborating on recent advances in improving cryoprotectants and optimizing freezing protocols for porcine vitrification. Furthermore, it highlights the latest breakthroughs in using high-throughput sequencing technologies to elucidate the molecular mechanisms of cryo-induced damage in porcine embryos at the transcriptomic and proteomic levels. Finally, it proposes that future efforts should integrate artificial intelligence models, metabolic reprogramming, and automated loading devices to further enhance the developmental potential of cryopreserved embryos, thereby laying a solid foundation for the long-term preservation and industrial application of local pig genetic resources.

Key words: Porcine embryo, Embryo freezing, Vitrification

中图分类号: 

  • S828

表1

猪胚胎的冷冻效果比较"

胚胎Embryo冷冻方法Methods解冻存活率Survival rate移植产仔率Farrowing rate参考文献References
囊胚Blastocyst常规缓慢冷冻法31.0%±10.2%[1]
囊胚BlastocystOPS65%55%[18-20]

表2

常用胚胎玻璃化冷冻技术方案汇总"

技术方案Methods

冷冻保护剂

Cryoprotective agent

操作步骤

Operation

参考文献

References

OPS

冷冻液1:TCM199+20% FBS+ 7.5% EG+7.5% DMSO

冷冻液2:TCM199+20% FBS + 18% EG + 18% DMSO + 0.6 M 蔗糖

(1)将收集到的体内或体外胚胎在TCM199+20% FBS液中清洗

(2)胚胎首先在冷冻液1中处理3 min

(3)将胚胎转入冷冻液2中

(4)在转入冷冻液2后的30 s内,将2~6枚胚胎吸入OPS管末端

(5)直接将OPS管插入液氮中保存

[18]
SOPS

冷冻液1:TCM199 HEPES+20% FBS +7.5% DMSO +7.5% EG

冷冻液2:TCM199 HEPES+20% FBS + 16% DMSO +16% EG+ 0.4 M 蔗糖

(1)将5~7枚胚胎置于冷冻液1中平衡3 min

(2)将胚胎转移至冷冻液2中处理1 min

(3)将胚胎置于小于1 μL 的冷冻液2液滴中,利用毛细作用装入SOPS细管

(4)立即将装有胚胎的细管水平插入液氮中

[21-22]
Cryotop

冷冻液1:TCM199+20% FBS + 10% EG + 10% DMSO

冷冻液2:TCM199+20% FBS + 20% EG + 20% DMSO + 0.5 M 蔗糖

(1)将胚胎放入冷冻液1处理30 s

(2)转入冷冻液2处理20 s

(3)使用极细的拉细玻璃管将胚胎吸出,以“点样”方式将胚胎和极少量的保护液(<0.1 μL)放置在Cryotop尼龙条的顶端

(4)迅速将尼龙条端直接浸入液氮中

(5)在液氮液面下为Cryotop套上专用的塑料保护外套,防止液氮流动引起胚胎脱落或污染

[37-40]

图1

猪胚胎冷冻保存技术发展历程"

表3

不同玻璃化冷冻载体性能对比表"

载体

Carrier

冷却速度

Cooling rate/(℃·min-1)

装载体积

Loading volume/μL

特点

Features

参考文献References
传统细管Straw~2000250降温速率慢,易形成冰晶,猪囊胚冻融后的胚胎平均存活率在31%[1,7,21]
OPS>200001~2通过减小内径提升换热效率,猪胚胎解冻存活率显著提高[21]
SOPS~30000<1换热更快,但每次仅能装载4~6个胚胎[21,38]
Cryotop>40000<0.1换热效率极高,支持20枚胚胎同步冷冻[37-40]
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[1] 翟亚莹, 张子敬, 吕世杰, 朱肖亭, 张格阳, 朱进华, 李峥, 于翔, 王红利, 施巧婷, 闫祥洲, 王二耀. 牛胚胎冷冻技术及冷冻保护剂研究进展[J]. 中国畜禽种业, 2022, 18(10): 34-36.
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