荣昌猪体内胚胎玻璃化冷冻程序优化研究

doi:10.11843/j.issn.0366-6964.2025.12.021

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (12): 6180-6190.doi: 10.11843/j.issn.0366-6964.2025.12.021

荣昌猪体内胚胎玻璃化冷冻程序优化研究

张亮¹(), 徐皆欢², 张凤鸣¹, 涂志¹, 王清³, 张力丹³, 张克勤³, 黄聪³, 戴建军²^,*(), 潘红梅¹^,*()

1. 重庆市畜牧科学院, 重庆 402460
2. 上海市农业科学院畜牧兽医研究所, 农业农村部畜禽资源(猪)评价利用重点实验室, 上海 201106
3. 国家生猪技术创新中心, 重庆 402460

收稿日期:2025-05-07 出版日期:2025-12-23 发布日期:2025-12-24
通讯作者: 戴建军,潘红梅 E-mail:zhang_liang1982@163.com;blackman0520@126.com;panhm_2118@163.com
作者简介:张亮(1982-)，男，重庆荣昌人，硕士，副研究员，主要从事地方猪种质资源保护利用研究，E-mail：zhang_liang1982@163.com
基金资助:
重庆市人民政府与中国农业科学院战略合作资助项目(23310)；国家重点研发计划课题(2021YFD1200303)；重庆市技术创新与应用发展专项重点项目(cstc2021jscx-dxwtBX0004)

Optimization of in Vivo Porcine Embryo Vitrification Protocol for Rongchang Pigs

ZHANG Liang¹(), XU Jiehuan², ZHANG Fengming¹, TU Zhi¹, WANG Qing³, ZHANG Lidan³, ZHANG Keqin³, HUANG Cong³, DAI Jianjun²^,*(), PAN Hongmei¹^,*()

1. Chongqing Academy of Animal Sciences, Chongqing 402460, China
2. Key Laboratory of Livestock and Poultry Resources (Pig) Evaluation and Utilization of Ministry of Agriculture and Rural Affairs, Institute of Animal Husbandry and Veterinary Science, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China
3. National Center of Technology for Pigs, Chongqing 402460, China

Received:2025-05-07 Online:2025-12-23 Published:2025-12-24
Contact: DAI Jianjun, PAN Hongmei E-mail:zhang_liang1982@163.com;blackman0520@126.com;panhm_2118@163.com

摘要/Abstract

摘要：

旨在优化荣昌猪体内胚胎冷冻方法，提高冷冻胚胎冻后囊胚孵化率。本试验使用50头荣昌猪经同期发情、定时输精和手术冲胚获得体内胚胎，通过比较不同冷冻载体(SOPS与Cryotop)，不同玻璃化冷冻保护剂(Medium-199、NCSU23与TL-PVA)，桑葚胚冻前脂质离心极化和解冻后胚胎透明带消化等处理，通过检测解冻后胚胎的存活率、继续发育能力和囊胚细胞数等，筛选最优的荣昌猪体内胚胎冷冻方法。荣昌猪经同期发情和手术冲胚后，平均获囊胚9.22枚·头^-1；结合体外培养，平均可生产囊胚12.16枚·头^-1。Cryotop冻胚存活率与发育率均显著高于SOPS(P < 0.05)；NCSU23组冻胚存活率和继续发育率最高，分别达85.91%±3.27%和80.61%±10.06%，优于Medium-199和TL-PVA；在解冻后囊胚细胞数方面，NCSU23组>Medium-199组>TL-PVA组；研究结果表明，NCSU23是荣昌猪胚胎的最适冷冻液。冷冻前离心极化脂质的桑葚胚，冷冻后未见继续发育。冷冻胚胎的透明带消化时间延长，透明带硬化阻碍了冻胚进一步扩张与孵化，通过消化透明带可促进冻胚扩张。荣昌猪体内胚胎生产结合体外培养可有效提升单位供体的囊胚获得数，平均生产囊胚12.16枚·头^-1。使用NCSU23冷冻保护剂和Cryotop玻璃化冷冻的方法，可获最高85.91%±3.27%的冻胚存活率和80.61%±10.06%的囊胚扩张/孵化率，且囊胚细胞数显著提高。解冻后消化透明带可促进解冻后的胚胎扩张。

关键词: 体内胚胎生产, 胚胎冷冻, 荣昌猪, 冷冻保护剂, 透明带硬化

Abstract:

This study aimed to optimize the in vivo embryo cryopreservation method for Rongchang pigs to improve the hatching rate of blastocysts after embryo freezing. The experiment used 50 Rongchang pigs to obtain in vivo embryos through synchronized estrus, artificial insemination, and surgical flushing. The effects of different cryo-carriers (SOPS and Cryotop), different vitrification cryoprotectants (Medium-199, NCSU23 and TL-PVA), lipid centrifugal polarization in pre-cryopreserved morula and zona pellucida (ZP) digestion of vitrified-warmed (V-W) embryos were compared, and the survival rate, continued development ability and blastocyst cell number of V-W embryos were determined to screen the optimal embryo V-W method in Rongchang pig. An average of 9.22 blastocysts per donator were obtained after embryo flushing, that raised to 12.16 blastocysts per donator combining with in vitro culture. The survival and development rate of V-W embryos using Cryotop were significantly higher than those of SOPS (P < 0.05). V-W embryos of NCSU23 group gained the highest survival rate (85.91%±3.27%) and development rate (80.61%±10.06%) comparing to Medium-199 and TL-PVA groups. The abilities of embryonic cell division were NCSU23>Medium-199>TL-PVA. This study proofed that NCSU23 was the most suitable cryoprotectant for Rongchang pig embryos. Lipids in morula were polarized by centrifugation before vitrification, while post-V-W embryonic development was not observed. The time cost of ZP digestion was prolonged after V-W, ZP hardening affected the expansion and hatching of V-W embryo, while ZP digestion offset this disadvantage and promoted the expansion of V-W embryo. In vivo embryo production combining with in vitro culture raised the efficiency to 12.16 blastocysts per donator. V-W embryos using Cryotop and NCSU23 reached the highest survival rate (85.91%±3.27%) and expansion and hatching blastocyst rate (80.61%±10.06%) and the highest level of embryonic cell division. The ZP digestion promoted V-W embryo expansion after warming.

Key words: in vivo embryo production, embryo freezing, Rongchang pig, cryoprotectant, zona pellucida hardening

中图分类号:

S828.3

张亮, 徐皆欢, 张凤鸣, 涂志, 王清, 张力丹, 张克勤, 黄聪, 戴建军, 潘红梅. 荣昌猪体内胚胎玻璃化冷冻程序优化研究[J]. 畜牧兽医学报, 2025, 56(12): 6180-6190.

ZHANG Liang, XU Jiehuan, ZHANG Fengming, TU Zhi, WANG Qing, ZHANG Lidan, ZHANG Keqin, HUANG Cong, DAI Jianjun, PAN Hongmei. Optimization of in Vivo Porcine Embryo Vitrification Protocol for Rongchang Pigs[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6180-6190.

图/表 8

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基础液 Base solution	试剂名 Reagent name	冷冻保护剂成分与含量Composition and content of cryoprotectant
基础液 Base solution	试剂名 Reagent name	二甲基亚砜/% Dimethyl sulfoxide	乙二醇/% Ethylene glycol	胎牛血清/% Fetal bovine serum	蔗糖/ (mmol·L^-1) Sucrose	牛血清白蛋白/ (mmol·L^-1) Bovine serum albumin	细胞松弛素B/ (μmol·L^-1) Cytochalasin B
Medium-199 with HEPES	冷冻液Ⅰ freezing solution Ⅰ	7.5	7.5	20.0	-	-	20.8
	冷冻液Ⅱ freezing solution Ⅰ	15.0	15.0	20.0	397.3	-	20.8
	解冻液Ⅰ thawing solution Ⅰ	-	-	20.0	300.0	-	20.8
	解冻液Ⅱ thawing solution Ⅱ	-	-	20.0	150.0	-	20.8
NCSU23	冷冻液Ⅰ freezing solution Ⅰ	7.5	7.5	20.0	-	13.3	20.8
	冷冻液Ⅱ freezing solution Ⅰ	15.0	15.0	20.0	397.3	13.3	20.8
	解冻液Ⅰ thawing solution Ⅰ	-	-	20.0	300.0	13.3	20.8
	解冻液Ⅱ thawing solution Ⅱ	-	-	20.0	150.0	13.3	20.8
TL-PVA	冷冻液Ⅰ freezing solution Ⅰ	7.5	7.5	-	-	-	20.8
	冷冻液Ⅱ freezing solution Ⅰ	15.0	15.0	-	397.3	-	20.8
	解冻液Ⅰ thawing solution Ⅰ	-	-	-	300.0	-	20.8
	解冻液Ⅱ thawing solution Ⅱ	-	-	-	150.0	-	20.8

组别 Group	IVC 6 h胚胎存活率/% Embryo survival rate(n≥30)	IVC 12 h扩张/孵化囊胚率/% Expansion/hatching blastocyst rate(n≥30)
新鲜胚胎对照Fresh embryo control	100.00±0.00^##	N/A
Medium-199冻胚Medium-199 frozen embryo	74.14±10.81	57.58±13.89
NCSU23冻胚NCSU23 frozen embryo	85.91±3.27	80.60±10.06
TL-PVA冻胚TL-PVA frozen embryo	62.41±7.42^*	19.09±8.67^***#

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荣昌猪体内胚胎玻璃化冷冻程序优化研究

Optimization of in Vivo Porcine Embryo Vitrification Protocol for Rongchang Pigs

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