猪卵母细胞形态及质量对体细胞核移植胚胎发育的影响

doi:10.16431/j.cnki.1671-7236.2025.12.023

摘要/Abstract

摘要： 【目的】试验旨在建立一套基于猪MⅡ期卵母细胞胞质颜色及第一极体形态的分级和评价标准，系统分析不同等级卵母细胞的形态学特征及其对胚胎发育能力的影响，从而提高成熟优质卵母细胞的筛选效率，提升体细胞核移植(SCNT)胚胎的发育潜力，为畜牧业中SCNT技术的应用提供科学依据。【方法】采集体外成熟的猪MⅡ期卵母细胞，根据胞质颜色(黑色致密或白色颗粒感)及第一极体形态(完整圆形或碎裂长条形)将卵母细胞分为A、B、C、D 4个等级。利用显微镜测量卵母细胞胞质直径，采用phalloidin-FITC染色检测微丝分布与表达，通过BODIPY染色评估脂滴大小与数量，并采用α-tubulin-TRITC染色观察纺锤体形态及异常率。通过电激活进行孤雌激活(PA)培养，计算卵裂率、囊胚率及凋亡率。采用近交系猪胎儿成纤维细胞作为供体，构建SCNT胚胎，评价各等级卵母细胞发育能力。【结果】A级猪卵母细胞占比最高(28.60%)，胞质直径(117.57 μm)显著大于C、D级卵母细胞(P＜0.05)；微丝表达适中(85.85 A.U.)，纺锤体异常率最低(24.56%)；PA和SCNT胚胎的囊胚率(52.37%和30.80%)最高，且囊胚细胞数量最多(63.73个)，均显著优于D级卵母细胞(P＜0.05)。B级猪卵母细胞的胞质直径最大(117.67 μm)，显著优于C、D级卵母细胞(P＜0.05)；微丝表达和小脂滴占比最高(88.72 A.U.和44.07%)，但囊胚凋亡率最低(87.12 A.U.)；PA胚胎的卵裂率和囊胚率显著低于A级卵母细胞(P＜0.05)。C、D级猪卵母细胞的胞质颜色较浅，脂滴较大，纺锤体异常率较高，PA及SCNT胚胎的囊胚率明显降低。【结论】通过对猪MⅡ期卵母细胞进行形态学分级，结合多种细胞质量指标和发育能力评价，证实胞质颜色致密、第一极体完整的A级卵母细胞具备较高的发育潜力。该形态学质量分级体系为猪卵母细胞的筛选提供了可靠、非侵袭性的评价标准，有助于优化SCNT克隆技术的应用效果，推动猪克隆技术在畜牧生产中的发展。

关键词: 猪卵母细胞; 形态学; 胚胎发育; 体细胞核移植; 孤雌胚胎

Abstract: 【Objective】 This study aimed to establish a classification and evaluation system for porcine MⅡ-stage oocytes based on cytoplasmic color and the morphology of the first polar body,and systematically analyze the morphological characteristics of oocytes at different grades and its effect on embryonic developmental competence,with the goal of improving the selection efficiency of high-quality matured oocytes and enhancing the developmental potential of somatic cell nuclear transfer (SCNT) embryos,so as to provide a scientific basis for the application of SCNT technology in livestock production. 【Method】 In vitro matured porcine MⅡ-stage oocytes were collected and categorized into four grades (A,B,C,and D) based on cytoplasmic appearance (dense black or pale granular) and the morphology of the first polar body (intact and round or fragmented and elongated).Cytoplasmic diameters of oocytes were measured under a microscope,the microfilament distribution and expression were assessed using phalloidin-FITC staining,the lipid droplet size and number were evaluated by BODIPY staining,and the spindle morphology and abnormality rates were observed using α-tubulin-TRITC staining.Parthenogenetic activation (PA) was performed by electrical stimulation,followed by assessments of cleavage rate,blastocyst rate,and apoptosis rate.SCNT embryos were generated using inbred porcine fetal fibroblasts as nuclear donors to evaluate developmental competence across the oocyte grades. 【Result】 Grade A porcine oocytes accounted for the highest proportion (28.60%),with the larger cytoplasmic diameter (117.57 μm),which was significantly higher than that of grades C and D oocytes (P＜0.05).The expression of microfilament in grade A oocytes was moderate (85.85 A.U.),and the spindle abnormality rate was the lowest (24.56%).The blastocyst rates of PA and SCNT embryos in grade A oocytes were the highest (52.37% and 30.80%),which were significantly higher than that in grade D oocytes (P＜0.05).The cytoplasmic diameter of grade B porcine oocytes were the highest (117.67 μm),which was significantly higher than that of grades C and D oocytes (P＜0.05).The expression of microfilament and the proportion of small lipid droplets in grade B oocytes were the highest (88.72 A.U.and 44.07%),while the blastocyst apoptosis rate was the lowest (87.12 A.U.).The cleavage rate and blastocyst rate of PA embryos in grade B oocytes were significantly lower than that in grade A oocytes (P＜0.05).In contrast,grades C and D porcine oocytes exhibited paler cytoplasm,larger lipid droplets,higher spindle abnormality rates,and significant lower blastocyst rate of PA and SCNT embryos. 【Conclusion】 Through morphological grading of porcine MⅡ-stage oocytes,combined with multiple cellular quality and developmental indicators evaluations,it was confirmed that grade A oocytes with dense cytoplasm color and intact the first polar body possessed higher developmental potential.The morphological classification system provided a reliable and non-invasive criterion for selecting high-quality porcine oocytes,optimized the efficiency of SCNT cloning technology,and promoted the advancement of porcine cloning technology in livestock breeding.

Key words: porcine oocytes; morphology; embryonic development; somatic cell nuclear transfer; parthenogenetic embryos

中图分类号:

S814.8

李卓, 王培, 郭建雄, 张雄, 赵红芳, 角德灵, 贾宝瑜, 卿玉波, 成文敏, 奎华, 魏红江. 猪卵母细胞形态及质量对体细胞核移植胚胎发育的影响[J]. 中国畜牧兽医, 2025, 52(12): 5785-5796.

LI Zhuo, WANG Pei, GUO Jianxiong, ZHANG Xiong, ZHAO Hongfang, JIAO Deling, JIA Baoyu, QING Yubo, CHENG Wenmin, KUI Hua, WEI Hongjiang. Effects of Porcine Oocyte Morphology and Quality on the Development of Somatic Cell Nuclear Transfer Embryo[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(12): 5785-5796.

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