马卵母细胞玻璃化冷冻保存研究进展

doi:10.11843/j.issn.0366-6964.2025.09.002

摘要/Abstract

摘要：

卵母细胞的冷冻保存技术对于人类辅助生殖技术和畜牧生产具有重要的意义，该技术有助于优良品种的遗传资源保护和生物多样性的维持。然而，由于卵母细胞具有表面积与体积比值较小、细胞膜渗透性差等特性，其在冷冻过程中极易形成冰晶造成机械损伤、遭受氧化应激和冷冻保护剂毒性等影响，严重降低其解冻后的发育能力。与其他动物相比，马卵母细胞中丰富的脂滴含量降低了其低温耐受性，冷冻保存难度更大。尽管可以从玻璃化冷冻的未成熟卵母细胞中获得后代，但与新鲜卵母细胞相比，经玻璃化冷冻卵母细胞的发育能力还有待进一步提升。本文主要从玻璃化冷冻对马卵母细胞造成的损伤类型和影响马卵母细胞冷冻效率的因素等方面进行综述，为进一步建立和完善马卵母细胞玻璃化冷冻保存方案提供参考。

关键词: 马, 卵母细胞, 玻璃化冷冻, 冷冻损伤, 冷冻保护剂

Abstract:

Oocyte cryopreservation is of significant importance for human assisted reproductive technology and livestock production.This technology aids in the conservation of genetic resources from superior breeds and the maintenance of biodiversity.However, due to their small surface area-to-volume ratio and low membrane permeability, oocytes are highly susceptible to damage during freezing.This includes mechanical injury caused by ice crystal formation, oxidative stress, and the toxicity of cryoprotectants, all of which severely reduce their developmental competence after thawing. Compared to other animals, the high lipid content within equine oocytes reduces their cryotolerance, making cryopreservation considerably more challenging. Although offspring can be obtained from vitrified immature oocytes, the developmental competence of vitrified-thawed oocytes remains to be enhanced compared to their fresh oocytes.This article primarily reviews the types of damage caused by vitrification to equine oocytes and the factors influencing the cryopreservation efficiency of equine oocytes.It aims to provide a reference for further establishing and optimizing vitrification protocols for equine oocyte cryopreservation.

Key words: equine, oocytes, vitrification, cryodamage, cryoprotective agent

中图分类号:

S821.35

杨明颖, 王娜, 刘源壹, 李昕俞, 巴音那木拉, 石玉杰, 芒来, 杜明. 马卵母细胞玻璃化冷冻保存研究进展[J]. 畜牧兽医学报, 2025, 56(9): 4143-4155.

YANG Mingying, WANG Na, LIU Yuanyi, LI Xinyu, Bayanamar , SHI Yujie, MANG Lai, DU Ming. Research Progress on Vitrification Cryopreservation of Equine Oocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4143-4155.

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方法 Method	研究者 Investigator	年份 Year	物种 Species	成就 Event	参考文献 Reference
慢速冷冻 Slow freezing	Whittingham	1977	小鼠	首次保存卵母细胞获得后代	[8]
	Chen	1986	人	保存卵母细胞获得后代	[18]
	Al-Hasani等	1989	兔	保存卵母细胞获得后代	[19]
	Didion等	1990	猪	成功保存卵母细胞	[11]
	Fuku等	1992	牛	保存卵母细胞获得后代	[9]
	Hochi等	1994	羊	成功保存卵母细胞	[14]
	Canesin等	2020	马	成功保存卵母细胞	[20]
玻璃化冷冻 Vitrification freezing	Al-Hasani等	1989	兔	成功保存卵母细胞	[21]
	Kono等	1991	小鼠	首次保存卵母细胞获得后代	[22]
	Rubinsky等	1992	猪	成功保存卵母细胞	[23]
	Hamano等	1992	牛	保存卵母细胞获得后代	[24]
	Hochi等	1994	马	成功保存卵母细胞	[14]
	Nagy等	2009	人	成功保存卵母细胞	[25]
	Dattena等	2000	羊	保存卵母细胞获得后代	[26]

研究者 Investigator	年份 Year	分裂阶段 Splitting stage	冷冻组合 Frozen combination	发育能力 Developmental capacity	参考文献 Reference
Hochi等	1994	GV	10% EG+10.0 mol·L^-1 Sucrose	成熟率15.8%	[14]
Maclellan等	2002	MII	ED/EDFS，Three-step Sucrose	存活率73%	[15]
Tharasanit等	2006	GV	10% EG+0.3 mol·L^-1Sucrose	成熟率35%，卵裂率8%	[27]
Tharasanit等	2006	GV	10% EG+10% DMSO	卵裂率34%，囊胚率16%	[28]
Tharasanit等	2006	MII	20% EG+20% DMSO+0.3 mol·L^-1Sucrose	卵裂率27%，囊胚率4%	[28]
Maclellan等	2010	MII	Commercial Cryotop medium	卵裂率83%，囊胚率40%，妊娠率26%	[29]
Nowak等	2014	MII	EquiPro-VitKit (Minitube) commercial medium	存活率63%，卵裂率10%	[30]
Canesin等	2021	GV	FBS+PG+EG+T；30 s	成熟率42%，卵裂率80%，囊胚率10%	[16]
De Coster等	2020	GV	BS+PE 40%+0.3 mol·L^-1Galactose	成熟率41.2%，卵裂率65.5%，囊胚率9.4%	[31]
Ortiz-Escribano等	2018	GV	HS+15% EG+15% DMSO+0.5 mol·L^-1Sucrose	成熟率25.3%，卵裂率30%，囊胚率5%	[7]
Canesin等	2018	GV	EP/EPT	成熟率21%，卵裂率85%，囊胚率15%	[32]

研究者 Author	分裂阶段 Splitting stage	试验组 Experimental group	载体 Carrier	存活率/% Survival rate	成熟率/% Maturity rate	卵裂率/% Cleavage rate	囊胚率/% Blastocyst rate	妊娠率/% Pregnancy rate
Maclellan等^[15]	MII	对照组	Nylon	100	-	-	-	83
Maclellan等^[15]	MII	ED/EDFS	Nylon	73	-	-	-	12
Tharasanit等^[27]	GV	对照组	OPS	96	56	66	17	-
	GV	ED/EDS		63	54	34	1	-
	MII	ED/EDS		32	54	16	0	-
Maclellan等^[29]	MII	商业Cryotop培养基	Cryotop	72	86	83	40	26
Canesin等^[16]	GV	对照组	Stainless	-	73	93	19	-
Canesin等^[16]	GV	ED/EDS	Steel mesh	-	36	67	11	-
De Coster等^[31]	GV	对照组	MVD	-	58	76	20	-
De Coster等^[31]	GV	ED/EDS	MVD	-	48	30	0	-

作者 Author	分裂阶段 Splitting stage	载体 Carrier	平衡液VS1 Buffered solution	冷冻液VS2 Refrigerating fluid	解冻方案 Thawing solution	成熟率/% Maturity rate	卵裂率/% Cleavage rate	囊胚率/% Blastocyst rate	妊娠率/% Pregnancy rate
Canesin等^[32]	GV	不锈钢网	2%EG+2%PG+HM；40 s	17.5%EG+17.5%PG+0.3 mol·L^-1海藻糖；65 s	BM培养基一步解冻、标准逐步解冻	21	85	15	-
Agnieszka等^[58]	MII	Rapid-Ⅰ	EquiPro VitKit；10 min	EquiPro VitKit；30 s	EquiPro VitKit培养基一步解冻	-	10.2	10	-
Maclellan等^[36]	MII	Cryotop	7.5%EG+7.5%DMSO+HM；1 min	15%EG+15%DMSO+0.5 mol·L^-1蔗糖+HM；1min	标准逐步解冻	-	32	32	-
Ortiz-Escribano等^[7]	GV	Cryotop	10% EG+10%DMSO+HM；25 s	20% EG+20%DMSO+0.3 mol·L^-1蔗糖；15 s	标准逐步解冻	34	42	7	20
Angel等^[51]	GV	尼龙网	FBS/7.5%PG-7.5%EG；10 min	15%PG-15%EG；30 s	标准逐步解冻	42	80	10	-
Clérico等^[17]	GV	Cryotop	5% EG+5% DMSO；45 s	20% EG+20% DMSO+0.65 mol·L^-1；30 s	标准逐步解冻	54	46	9	33
Ducheyne等^[33]	GV	接种环	20% EG+20% DMSO+BS；25 s	10% EG+10% DMSO+0.5 mol·L^-1蔗糖；15 s	标准逐步解冻	33	-	-	-
Tharasanit等^[28]	GV	OPS	10% EG+10%DMSO+HM；30 s	20% EG+20% DMSO+0.5 mol·L^-1；5 s	0.3 mol·L^-1蔗糖一步解冻法	54	34	1	-
Tharasanit等^[28]	MII	OPS	10% EG+10% DMSO+HM；30 s	20% EG+20% DMSO+0.5 mol·L^-1；15 s	0.3 mol·L^-1蔗糖一步解冻法	-	16	-	-

类别 Category		物种 Species	浓度 Concentration	成果 Outcome	参考文献 Reference
抗氧化剂 Antioxidant	原花青素 B2(PB2)	水牛	10^-9 mol·L^-1	PB2改善水牛卵母细胞的发育能力和线粒体分布	[65]
	原花青素 B2(PB2)	小鼠	5 μg·mL^-1	PB2改善线粒体功能、提高囊胚率	[66]
	白藜芦醇(Res)	牛	2 μmol·L^-1	Res恢复DNA甲基化水平并显著改善玻璃化GV卵母细胞的成熟和发育能力	[67]
	白藜芦醇(Res)	小鼠	0.1 μmol·L^-1	Res修复玻璃化后胚胎的异常线粒体分布和线粒体功能障碍	[68]
	褪黑素(MT)	绵羊	1 μmol·L^-1	MT降低活性氧水平并促进胚胎抗氧化酶(如谷胱甘肽)的产生	[69]
	褪黑素(MT)	马	10 mmol·L^-1	MT降低线粒体相关的ROS和改善ICSI胚胎发育，卵裂率从45%提高至60%，囊胚率也从8%提升至14%	[20]
	虾青素(Ax)	牛	5.3 μmol·L^-1	Ax减少氧化应激改善囊胚质量	[70]
	虾青素(Ax)	猪	2.5 μmol·L^-1	Ax恢复玻璃化卵母细胞基因表达提高mRNA水平	[71]
细胞骨架稳定剂 Cytoskeleton stabilizer	紫杉醇(Taxol)	牛	1 μmol·L^-1	添加Taxol后线粒体分布正常率40%显著高于对照组22%	[72]
	紫杉醇(Taxol)	猪	10 mol·L^-1	添加Taxol后玻璃化线粒体分布正常率83.69%显著高于对照组48.5%	[56]
	细胞松弛素B(CB)	猪	7.5 μg·mL^-1	添加CB使卵母细胞卵裂率5.64%提高至20.91%，囊胚率由0.41%提高至5.00%	[73]
	细胞松弛素B(CB)	绵羊	7.5 μg·mL^-1	CB玻璃化组的受精率高于未添加CB组(57.0%vs40.7%)并提升胚胎质量	[74]