取样后动物胚胎的冷冻保存技术研究进展

doi:10.11843/j.issn.0366-6964.2025.10.004

摘要/Abstract

摘要：

取样后胚胎冷冻保存技术是胚胎植入前遗传学检测(preimplantation genetic testing，PGT)的重要组成部分，在胚胎性别控制、基因筛查、基因组选择及辅助生殖领域具有重要应用价值。然而，胚胎取样操作可能导致透明带损伤、细胞数量减少及表观遗传异常等问题，限制了冷冻保存效率。近年来，通过优化取样方法(如极体活检、卵裂球活检及滋养外胚层活检)、改进培养基成分(如添加L-精氨酸、谷胱甘肽等)以及优化冷冻程序(如玻璃化冷冻技术)，显著提高了取样后胚胎的冷冻保存效率。此外，非侵入性技术(如胚胎培养液中的游离DNA分析)和表观遗传修饰(如DNA甲基化调控)为减少胚胎损伤提供了新思路。本文综述了取样后胚胎冷冻保存的损伤机制及保护措施，重点探讨了透明带修复、表观遗传调控及冷冻效率提升的最新研究进展。通过总结现有技术的优势与不足，旨在为开发高效、安全的取样后胚胎冷冻保存技术提供理论依据和技术参考，推动其在畜牧业和辅助生殖领域的应用与推广。

关键词: 胚胎取样, PGT, 冷冻保存, 冷冻效率

Abstract:

Post-sampling embryo cryopreservation technology is a critical component of preimplantation genetic testing (PGT), holding significant application value in embryo sex selection, genetic screening, genomic selection, and assisted reproduction. However, embryonic sampling procedures may lead to zona pellucida damage, reduced cell count, and epigenetic abnormalities, thereby limiting cryopreservation efficiency. Recent advancements, including optimized sampling methods (e.g., polar body biopsy, blastomere biopsy, and trophectoderm biopsy), improved culture medium formulations (e.g., supplementation with L-arginine and glutathione), and refined cryopreservation protocols (e.g., vitrification techniques), have markedly enhanced the cryopreservation efficiency of post-sampling embryos. Furthermore, non-invasive techniques (e.g., analysis of cell-free DNA in embryo culture medium) and epigenetic modifications (e.g., DNA methylation regulation) offer novel approaches to minimize embryonic damage. This review summarizes the mechanisms of cryopreservation-induced damage in post-sampling embryos and protective strategies, with a focus on cutting-edge advancements in zona pellucida repair, epigenetic regulation, and cryopreservation efficiency. By evaluating the strengths and limitations of existing technologies, this review aims to provide theoretical foundations and technical references for developing safe and efficient cryopreservation protocols, thereby promoting their application and adoption in livestock breeding and assisted reproductive technologies.

Key words: embryo sampling, PGT, cryopreservation, freezing efficiency

中图分类号:

S814

董建华, 杨柏高, 张笑梦, 冯肖艺, 宋浩然, 刘阳, 王子卓, 王彦博, 李崇阳, 吕丽华, 赵学明. 取样后动物胚胎的冷冻保存技术研究进展[J]. 畜牧兽医学报, 2025, 56(10): 4796-4806.

DONG Jianhua, YANG Baigao, ZHANG Xiaomeng, FENG Xiaoyi, SONG Haoran, LIU Yang, WANG Zizhuo, WANG Yanbo, LI Chongyang, LÜ Lihua, ZHAO Xueming. Research Progress on Cryopreservation Technology of Animal Embryos after Sampling[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(10): 4796-4806.

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物种 Species	推荐活检阶段 Recommendation for biopsy stage	取样细胞类型 Sample cell type	技术特点 Technical characteristics	参考文献 Reference
牛Cattle	囊胚期	滋养外胚层(TE)	激光辅助透明带打孔，取5~8个细胞；玻璃化冷冻技术成熟。	[40]
小鼠Mouse	卵裂期(8细胞)	卵裂球	显微操作针机械取样，单细胞活检；冻存耐受性差，需改良冷冻液。	[41]
人类Human	囊胚期	滋养外胚层(TE)	激光脉冲精准切割，取5~10个细胞；玻璃化冷冻后存活率可达90%以上。	[42]
猪Pig	囊胚期	滋养外胚层(TE)	透明带厚且弹性差，需高强度激光或酶解法辅助打孔；冷冻保存技术落后。	[43]
绵羊Sheep	囊胚期	滋养外胚层(TE)	激光辅助取样，活检后短期培养(5~8 h)可提升冷冻存活率。	[44]
山羊Goat	囊胚期	滋养外胚层(TE)	透明带较薄，激光打孔后机械吸取；冷冻耐受性中等。	[45]
兔Rabbit	卵裂期(8细胞)	卵裂球	胚胎体积较大(120~150 μm)，操作相对容易；冻存耐受性中等。	[46]
猴Monkey	囊胚期	滋养外胚层(TE)	非侵入性cfDNA分析为主，避免伦理争议；激光取样技术成熟。	[47]
斑马鱼Zebrafish	卵裂期(16-32细胞)	卵裂球	胚胎透明，易于观察；显微注射技术成熟。	[48]

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