绵羊卵母细胞小群培养条件优化

doi:10.11843/j.issn.0366-6964.2025.12.023

摘要/Abstract

摘要：

旨在优化绵羊卵母细胞小群(5枚/组)体外培养体系，改善其成熟质量及后续发育潜能。本研究通过设置不同培养体积(50 COCs/500 μL、5 COCs/500 μL、5 COCs/50 μL)及添加外源BMP15与GDF9蛋白(200 ng·mL^-1)，评估颗粒细胞扩展指数及其相关HAS2基因表达、卵丘和卵母细胞凋亡相关基因(BAX、BCL2、C-Myc)表达、卵母细胞氧化还原稳态(GSH、ROS、GPX-1、GSR)及胚胎发育指标(卵裂率、囊胚率、囊胚平均细胞数)，并进一步优化体外成熟培养时长(18 h、21 h、24 h)，确立优化方案。结果表明，添加BMP15和GDF9显著提升卵丘扩展指数(P < 0.01)，上调HAS2基因表达，改善氧化还原稳态(GSH水平升高，ROS降低)；500 μL培养液结合21 h成熟方案使小群培养卵母细胞体外受精后的48 h卵裂率达到70.23%±2.78%及囊胚率35.33%±1.53%，而21 hIVM组较24 h IVM组BAX表达量和BAX/BCL2比值显著降低。5 COCs /500 μL小群培养效果优于5 COCs/50 μL，联合添加BMP15和GDF9蛋白可降低细胞凋亡并提升胞内氧化还原能力，体外成熟时长由24 h缩短至21 h可通过降低细胞凋亡并促进囊胚OCT4、CCNB1基因表达并且提高囊胚平均细胞数。

关键词: 绵羊, 卵母细胞, 小群培养

Abstract:

This investigation aimed to optimize the in vitro culture system for small cohorts of ovine COCs(5 per group), and enhance in vitro maturation quality and subsequent developmental potential. Experimental variables included culture volume (50 COCs/500 μL, 5 COCs/500 μL, 5 COCs/50 μL, ) supplemented with exogenous BMP15 and GDF9 proteins (200 ng·mL^-1), and maturation durations tested (18, 21, 24 h). Comprehensive evaluations encompassed cumulus expansion index, HAS2 gene expression profiles, apoptosis-related gene expression (BAX, BCL2, C-Myc) in cumulus-oocyte complexes, oocyte redox homeostasis parameters (GSH, ROS, GPX-1, GSR), and embryonic developmental indexes (cleavage rate, blastocyst rate, mean cell numbers/blastocyst). Addition of the combination of BMP15 and GDF9 into maturation medium significantly enhanced cumulus expansion indexes (P < 0.01), upregulating HAS2 expression and improving redox balance through elevated GSH levels and reduced ROS accumulation. The optimized protocol employing 500 μL culture medium with 21 h maturation achieved superior outcomes, yielding cleavage rate of 70.23%±2.78% and blastocyst rate of 35.33%±1.53% at 48 h after in vitro fertilization of oocytes cultured in small group. Notably, the 21-hour maturation group exhibited significantly lower BAX expression and BAX/BCL2 ratios compared to the 24-hour group. Culturing 5 COCs in 500 μL microdrops yielded better results than culturing 5 COCs in 50 μL microdrops. Co-supplementation with BMP15 and GDF9 proteins reduced apoptosis and enhanced intracellular redox capacity. Shortening the in vitro maturation (IVM) duration from 24 h to 21 h reduced apoptosis, promoted OCT4 and CCNB1 gene expression in blastocysts, and increased the average blastocyst cell count.

Key words: ovine, oocyte, small group cultivation

中图分类号:

S826.3

胡伯欣, 高程远, 刘从, 陈茹曼, 朱捷, 田树军. 绵羊卵母细胞小群培养条件优化[J]. 畜牧兽医学报, 2025, 56(12): 6204-6218.

HU Boxin, GAO Chengyuan, LIU Cong, CHEN Ruman, ZHU Jie, TIAN Shujun. Optimization of Culture Conditions for Small Groups of Ovine Oocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6204-6218.

图/表 16

图 1

表 1

图 2

表 2

图 3

图 4

表 3

图 5

图 6

表 4

图 7

图 8

图 9

图 10

图 11

图 12

参考文献 40

1	VIANA J H M . Development of the world farm animal embryo industry over the past 30 years[J]. Theriogenology, 2024, 230, 151- 156. doi: 10.1016/j.theriogenology.2024.09.012
2	孙克佳. 活体采卵与小群培养对牛羊体外胚胎生产的影响[D]. 保定: 河北农业大学2022.
	SUN K J. The impact of ovum pick-up and small group culture on in vitro embryo production in cattle and sheep[D]. Baoding: Hebei Agricultural University, 2022. (in Chinese)
3	PRIEGO-GONZÁLEZ A , MUNOZ-MACEDA A , CERDEIRA-LOZANO J , et al. Successful ultrasound-guided ovum pick-up (OPU) and subsequent in vitro embryo production in a domestic cat[J]. Theriogenology, 2024, 229, 47- 52. doi: 10.1016/j.theriogenology.2024.08.017
4	DU Y , XIA Y , XU J , et al. Effects of donor age and reproductive history on developmental potential of ovum pickup oocytes in Japanese Black cattle (Wagyu)[J]. Theriogenology, 2024, 221, 25- 30. doi: 10.1016/j.theriogenology.2024.03.012
5	SIMMONS R , TUTT D A , GUVEN-ATES G , et al. Enhanced progesterone support during stimulated cycles of transvaginal follicular aspiration improves bovine in vitro embryo production[J]. Theriogenology, 2023, 199, 77- 85. doi: 10.1016/j.theriogenology.2023.01.003
6	WIECZOREK J , KOSENIUK J , SKRZYSZOWSKA M , et al. L-OPU in goat and sheep-different variants of the oocyte recovery method[J]. Animals (Basel), 2020, 10 (4): 658.
7	VEGA D A , NARVÁEZ H J . Oocyte quality in adapted Bos taurus taurus cows[J]. Anim Biotechnol, 2023, 34 (9): 4675- 4679. doi: 10.1080/10495398.2023.2185248
8	GHAFARI F , SALAVATI M , PIERCY R J , et al. Beneficial effects of melatonin on canine oocyte nuclear maturation via reduction of oxidative stress[J]. Reproduction, 2025, 169 (4): e240388.
9	MISHRA A , REDDY I J , GUPTA P S , et al. Expression of apoptotic and antioxidant enzyme genes in sheep oocytes and in vitro produced embryos[J]. Anim Biotechnol, 2017, 28 (1): 18- 25. doi: 10.1080/10495398.2016.1193743
10	HUANG K , LI C , GAO F , et al. Epigallocatechin-3-gallate promotes the in vitro maturation and embryo development following IVF of porcine oocytes[J]. Drug Des Devel Ther, 2021, 15, 1013- 1020. doi: 10.2147/DDDT.S295936
11	MAHMOODI M , CHERAGHI E , RIAHI A . Correction to: The effect of wharton's jelly-derived conditioned medium on the in vitro maturation of immature oocytes, embryo development, and genes expression involved in apoptosis[J]. Reprod Sci, 2023, 30 (11): 3400. doi: 10.1007/s43032-023-01356-z
12	TANG Y , LU S , WEI J , et al. Growth differentiation factor 9 regulates the expression of estrogen receptors via Smad2/3 signaling in goat cumulus cells[J]. Theriogenology, 2024, 219, 65- 74. doi: 10.1016/j.theriogenology.2024.02.021
13	JIAO Y , JIANG T , LIN Q , et al. Molecular characterization of the follicular development of BMP15-edited pigs[J]. Reproduction, 2023, 166 (4): 247- 261. doi: 10.1530/REP-23-0034
14	NISHIO M , HOSHINO Y , SATO E . Effect of droplet size and number of oocytes examined on mouse oocyte quality in in vitro maturation[J]. J Mamm Ova Res, 2011, 28 (1): 53- 60. doi: 10.1274/jmor.28.53
15	EBRAHIMI M , MARA L , SUCCU S , et al. The effect of single versus group culture on cumulus-oocyte complexes from early antral follicles[J]. J Assist Reprod Genet, 2025, 42 (3): 961- 976. doi: 10.1007/s10815-025-03404-w
16	GILCHRIST R B , LANE M , THOMPSON J G . Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality[J]. Hum Reprod Update, 2008, 14 (2): 159- 177. doi: 10.1093/humupd/dmm040
17	DU C , DAVIS J S , CHEN C , et al. FGF2/FGFR signaling promotes cumulus-oocyte complex maturation in vitro[J]. Reproduction, 2021, 161 (2): 205- 214. doi: 10.1530/REP-20-0264
18	MORIKAWA R , LEE J , MIYANO T . Effects of oocyte-derived growth factors on the growth of porcine oocytes and oocyte-cumulus cell complexes in vitro[J]. J Reprod Dev, 2021, 67 (4): 273- 281. doi: 10.1262/jrd.2021-026
19	CHENG M , CHEN X , HAN M , et al. miR-155-5p improves oocyte maturation in porcine cumulus cells through connexin 43-mediated regulation of MPF activity[J]. Theriogenology, 2024, 214, 124- 133. doi: 10.1016/j.theriogenology.2023.10.013
20	STOCKER W A , WALTON K L , RICHANI D , et al. A variant of human growth differentiation factor-9 that improves oocyte developmental competence[J]. J Biol Chem, 2020, 295 (23): 7981- 7991. doi: 10.1074/jbc.RA120.013050
21	FERRER-RODA M , PARAMIO M T , VILA-BELTRÁN J , et al. Effect of BMP15 and GDF9 in the IVM medium on subsequent oocyte competence and embryo development of prepubertal goats[J]. Theriogenology, 2025, 234, 164- 173. doi: 10.1016/j.theriogenology.2024.12.015
22	WANG L , GAO J , MA J , et al. Effects of hyperhomocysteinemia on follicular development and oocytes quality[J]. iScience, 2024, 27 (11): 111241. doi: 10.1016/j.isci.2024.111241
23	JIAO Y , BEI C , WANG Y , et al. Bone morphogenetic protein 15 gene disruption affects the in vitro maturation of porcine oocytes by impairing spindle assembly and organelle function[J]. Int J Biol Macromol, 2024, 267 (Pt 1): 131417.
24	LI W , LIU Z , WANG P , et al. The transcription factor RUNX1 affects the maturation of porcine oocytes via the BMP15/TGF-β signaling pathway[J]. Int J Biol Macromol, 2023, 238, 124026. doi: 10.1016/j.ijbiomac.2023.124026
25	GIUSTARINI D , MILZANI A , DALLE-DONNE I , et al. How to increase cellular glutathione[J]. Antioxidants (Basel), 2023, 12 (5): 1094. doi: 10.3390/antiox12051094
26	ASHIBE S , MIYAMOTO R , KATO Y , et al. Detrimental effects of oxidative stress in bovine oocytes during intracytoplasmic sperm injection (ICSI)[J]. Theriogenology, 2019, 133, 71- 78. doi: 10.1016/j.theriogenology.2019.04.012
27	DAVOODIAN N , KADIVAR A , MEHRBAN H . Supplementation of media with gamma-oryzanol as a novel antioxidant to overcome redox imbalance during bovine oocyte maturation in vitro[J]. Reprod Domest Anim, 2024, 59 (1): e14503. doi: 10.1111/rda.14503
28	KIM M J , KANG H G , JEON S B , et al. The antioxidant betulinic acid enhances porcine oocyte maturation through Nrf2/Keap1 signaling pathway modulation[J]. PLoS One, 2024, 19 (10): e0311819. doi: 10.1371/journal.pone.0311819
29	READER K L , STANTON J L , JUENGEL J L . The role of oocyte organelles in determining developmental competence[J]. Biology (Basel), 2017, 6 (3): 35.
30	LI J , BALBOULA A Z , ABOELENAIN M , et al. Effect of autophagy induction and cathepsin B inhibition on developmental competence of poor quality bovine oocytes[J]. J Reprod Dev, 2020, 66 (1): 83- 91. doi: 10.1262/jrd.2019-123
31	ROBERT C . Nurturing the egg: the essential connection between cumulus cells and the oocyte[J]. Reprod Fertil Dev, 2021, 34 (2): 149- 159. doi: 10.1071/RD21282
32	YUAN Y , SPATE L D , REDEL B K , et al. Quadrupling efficiency in production of genetically modified pigs through improved oocyte maturation[J]. Proc Natl Acad Sci U S A, 2017, 114 (29): E5796- E5804.
33	ZHANG M , ZHANG J , WANG D , et al. C-X-C motif chemokine ligand 12 improves the developmental potential of bovine oocytes by activating SH2 domain-containing tyrosine phosphatase 2 during maturation†[J]. Biol Reprod, 2023, 109 (3): 282- 298. doi: 10.1093/biolre/ioad079
34	MARTINEZ C A , CUELLO C , PARRILLA I , et al. Exogenous melatonin in the culture medium does not affect the development of in vivo-derived pig embryos but substantially improves the quality of in vitro-produced embryos[J]. Antioxidants (Basel), 2022, 11 (6): 1177. doi: 10.3390/antiox11061177
35	TONG J , SHENG S , SUN Y , et al. Melatonin levels in follicular fluid as markers for IVF outcomes and predicting ovarian reserve[J]. Reproduction, 2017, 153 (4): 443- 451. doi: 10.1530/REP-16-0641
36	CADENAS J , PORS S E , KUMAR A , et al. Concentrations of oocyte secreted GDF9 and BMP15 decrease with MⅡ transition during human IVM[J]. Reprod Biol Endocrinol, 2022, 20 (1): 126. doi: 10.1186/s12958-022-01000-6
37	TAWEECHAIPAISANKUL A , JIN JX , LEE S , et al. The effects of canthaxanthin on porcine oocyte maturation and embryo development in vitro after parthenogenetic activation and somatic cell nuclear transfer[J]. Reprod Domest Anim, 2016, 51 (6): 870- 876. doi: 10.1111/rda.12748
38	REN X , YUN X , YANG T , et al. Epifriedelanol delays the aging of porcine oocytes matured in vitro[J]. Toxicon, 2023, 233, 107256. doi: 10.1016/j.toxicon.2023.107256
39	HUANG K , LI C , GAO F , et al. Epigallocatechin-3-Gallate promotes the in vitro maturation and embryo development following IVF of porcine oocytes[J]. Drug Des Devel Ther, 2021, 15, 1013- 1020. doi: 10.2147/DDDT.S295936
40	MASSOUD G , SPANN M , VAUGHT K C , et al. Biomarkers assessing the role of cumulus cells on IVF outcomes: a systematic review[J]. J Assist Reprod Genet, 2024, 41 (2): 253- 275. doi: 10.1007/s10815-023-02984-9

引物Primer	引物序列(5′→3′) Primer sequence	产物长度/bp Products length
β-actin	F: GTCACCAACTGGGACGACA R: AGGCGTACAGGGACAGCA	208
C-Myc	F: GGATAGTGGAAATACGGGCT R: GTGGTAGAAGTTCTCCTCCT	181
BAX	F: AGCGAGTGTCTGAAGCG R: CCCAGTTGAAGTTGCCGT	145
BCL2	F: ATGACTTCTCTCGGCGCTAC R: CTCCACACACATGACCCCTC	176
GDF9	F: AGCTAGGACTGCGTTGGAAT R: AGGTCTGTGTCTCCCACCTA	133
BMP15	F: TTGTCAGTGAGCTGGTGGAT R: TGCAGGACTGGGCAATCATA	202
OCT4	F: GGAAAGGTGTTCAGCCA R: ATTCTCGTTGTTGTCAGC	109
GPX-1	F: AACGTAGCATCGCTCTGAGG R: CAAACTGGTTGCACGGGAAG	115
GSR	F: CTGGAAGAGTTGCCTCGCC R: TCATTATTGATGTCTTAGAACCCAGG	103
HAS2	F: CCTCATCATCCAAAGCCTG R: ACATTTCCGCAAATAGTCTG	218
CCNB1	F: CACAGGATACACAGAGAATG R: CTTGATGGCGATGAATTTAG	143

组别 Group	卵子数 Number of oocytes	IVF 24 h卵裂率/% IVF 24 h cleavage rate	IVF 48 h卵裂率/% IVF 48 h cleavage rate	7 d囊胚率/% 7 d blastocyst rate
50 COCs /500 μL	350	70.5±1.7^a	76.3±1.5^a	38.8±2.2^a
5 COCs /500 μL	220	53.8±2.3^b	61.9±2.7^b	23.0±1.9^b
5 COCs /50 μL	220	55.8±2.5^b	64.0±0.8^b	23.7±1.2^b

蛋白浓度 Protein concentration	组别Group
蛋白浓度 Protein concentration	5 COCs /500μL	5 COCs /50μL
0 ng·mL^-1 BMP15+0 ng·mL^-1 GDF9	66.23±1.37^c	65.33±1.52^d
100 ng·mL^-1 BMP15	68.33±3.51^b	67.67±1.57^cd
200 ng·mL^-1 BMP15	74.33±1.16^b	72.34±3.21^bc
100 ng·mL^-1 GDF9	68.67±3.06^b	69.00±3.61^bcd
200 ng·mL^-1 GDF9	72.67±2.08^b	70.33±1.15^bc
100 ng·mL^-1BMP15+100 ng·mL^-1 GDF9	73.33±1.53^b	73.67±3.06^b
200 ng·mL^-1 BMP15+200 ng·mL^-1 GDF9	82.67±3.06^a	82.33±4.16^a

组别 Group	卵子数 Number of oocytes	IVF 24 h卵裂率/% IVF 24 h cleavage rate	IVF 48 h卵裂率/% IVF 48 h cleavage rate	7 d囊胚率/% 7 d blastocyst rate
5 COCs /500 μL	250	41.4±3.2^a	64.2±1.1^a	22.3±1.2^a
5 COCs /50 μL	250	44.0±2.5^a	63.0±3.0^a	22.5±1.2^a
5 COCs /500 μL+BMP15/GDF9	250	55.0±2.4^b	66.8±1.6^a	32.9±1.6^b
5 COCs /50 μL+BMP15/GDF9	250	59.0±2.4^b	68.0±1.0^a	31.8±1.4^b

[1]	刘丹妮, 种丽伟, 宫平, 魏佩玲, 柴婷, 耿天颖, 吴伟伟, 郑文新. 山羊感染绵羊肺炎支原体的病理学分析[J]. 畜牧兽医学报, 2026, 57(1): 423-431.
[2]	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.
[3]	王蕊, 衡诺, 胡樱凡, 王欢, 朱妮, 何维, 轩秀丽, 胡智辉, 熊铿, 巩建飞, 郝海生, 朱化彬, 赵善江. 不同等级牛卵丘-卵母细胞复合体体外成熟后卵母细胞发育能力差异的机制分析[J]. 畜牧兽医学报, 2025, 56(9): 4432-4451.
[4]	魏康康, 马贵, 李文迪, 田雨, 张令锴, 朱继红, 胡亚美. 单细胞测序技术在绵羊卵巢生长发育过程中的研究进展[J]. 畜牧兽医学报, 2025, 56(7): 3080-3087.
[5]	张嘉良, 黄畅, 杨永林, 杨华, 白文林, 马月辉, 赵倩君. 基于50K液相芯片的中国绵羊群体遗传结构与羊毛性状选择信号分析[J]. 畜牧兽医学报, 2025, 56(7): 3164-3176.
[6]	唐毓, 张颖, 杨镒峰, 薛海龙, 刘理想, 许保增. 甘氨酸提高水貂卵母细胞玻璃化冷冻保存效率的关键机制[J]. 畜牧兽医学报, 2025, 56(7): 3265-3277.
[7]	韩僖彤, 张楠, 张宁, 张家新. FLI通过增加糖代谢途径促进牛卵母细胞体外成熟[J]. 畜牧兽医学报, 2025, 56(6): 2778-2789.
[8]	乔利英, 王万年, 张莉, 庞志旭, 张思颖, 李一凡, 刘文忠. 基于基因组标记对绵羊品种分类的机器学习方法研究[J]. 畜牧兽医学报, 2025, 56(5): 2157-2167.
[9]	郭妍岩, 张羽欣, 陆瑞, 李玉鹏, 陈龙宾, 张金龙, 姚大为, 阮维斌, 张效生, 郭晓飞. 哺乳动物卵泡发育阶段颗粒细胞增殖与分化的研究进展[J]. 畜牧兽医学报, 2025, 56(4): 1484-1493.
[10]	闫瑞, 贾朝阳, 马静, 杨娟, 刘新峰, 陈强. 3D培养在家畜卵母细胞及胚胎培养的研究现状与应用前景[J]. 畜牧兽医学报, 2025, 56(4): 1494-1507.
[11]	马应天, 姜璐瑶, 李增开, 秦剑平, 赵建华, 贺玉芳, 宋宇轩, 张磊. 矢车菊素-3-芸香糖苷对奶绵羊精液冷冻保存效果的影响[J]. 畜牧兽医学报, 2025, 56(4): 1768-1778.
[12]	李艳娥, 梁友萍, 樊洁, 吴芳燕, 尧香悦, 李毛却乎, 次仁仓决, 郝桂英, 古小彬. 绵羊痒螨钙网蛋白对兔外周血单个核细胞Th1/Th2和Th17/Treg免疫平衡的影响[J]. 畜牧兽医学报, 2025, 56(4): 1910-1918.
[13]	杨杨, 李良远, 万鹏程, 卢守亮, 刘长彬, 杨华, 王立民, 代蓉, 周平. 绵羊季节性发情性状核心基因和关键lncRNA的筛选与分析[J]. 畜牧兽医学报, 2025, 56(3): 1264-1277.
[14]	何雨, 王翔宇, 狄冉, 储明星, 梁琛. BMP4/SMAD4通过下调GJA1基因表达影响绵羊卵巢颗粒间隙连接活性[J]. 畜牧兽医学报, 2025, 56(2): 679-688.
[15]	楚翼健, 崔久增, 李增开, 张磊, 褚婷婷, 黄艳平, 宋宇轩. 绵羊子宫内膜容受前期与容受期的阴道微生物比较研究[J]. 畜牧兽医学报, 2025, 56(2): 689-699.