牛胚胎性别鉴定荧光定量PCR方法的优化与应用

doi:10.11843/j.issn.0366-6964.2021.05.016

畜牧兽医学报 ›› 2021, Vol. 52 ›› Issue (5): 1307-1316.doi: 10.11843/j.issn.0366-6964.2021.05.016

牛胚胎性别鉴定荧光定量PCR方法的优化与应用

冯春涛^1,5,6,7, 顾文源^1,3, 王志仙^1,5, 赵增元^1,5,7, 朱宏波^1,5, 倪俊卿^4,5, 褚素乔², 余文莉^1,5,6,7*, 李树静^1,5,6,7*

1. 石家庄天泉良种奶牛有限公司, 石家庄 050200;
2. 石家庄市畜牧技术推广站, 石家庄 050000;
3. 河北省动物疫病预防控制中心, 石家庄 050035;
4. 河北省畜牧良种工作总站, 石家庄 050020;
5. 河北省牛产业技术研究院, 石家庄 050200;
6. 河北省奶牛良种繁育工程技术研究中心, 石家庄 050200;
7. 河北省第三批"巨人计划"创新创业团队, 石家庄 050200

收稿日期:2020-10-19 出版日期:2021-05-23 发布日期:2021-05-22
通讯作者: 余文莉,主要从事胚胎生物技术研发和推广,E-mail:anboyuwenli@126.com;李树静,主要从事胚胎生物技术研发和推广,E-mail:embroychina@163.com
作者简介:冯春涛(1982-),男,河北唐山人,硕士,高级畜牧师,主要从事胚胎生物技术研发和推广,E-mail:fenchuntao@163.com;顾文源(1992-),女,河北石家庄人,博士,兽医师,主要从事畜禽遗传改良、疫病防控等技术研究和推广,E-mail:447009688@qq.com。
基金资助:
河北省重点研发计划项目（19226608D）；河北省重点研发计划项目（18226615D）；石家庄市科学技术研究与发展计划（201500452A）

Optimization and Application of Fluorescence Quantitative PCR Method for Sex Identification of Bovine Embryos

FENG Chuntao^1,5,6,7, GU Wenyuan^1,3, WANG Zhixian^1,5, ZHAO Zengyuan^1,5,7, ZHU Hongbo^1,5, NI Junqing^4,5, CHU Suqiao², YU Wenli^1,5,6,7*, LI Shujing^1,5,6,7*

1. Shijiazhuang Tianquan Elite Dairy Co., Ltd., Shijiazhuang 050200, China;
2. Shijiazhuang Animal Husbandry Technology Extension Station, Shijiazhuang 050000, China;
3. Hebei Provincial Center for Animal Disease Control and Prevention, Shijiazhuang 050035, China;
4. Hebei Livestock Breeding Station, Shijiazhuang 050020, China;
5. Hebei Province Research Institute of Cattle Industry Technology, Shijiazhuang 050200, China;
6. Hebei Province Dairy Breeding Engineering Research Center, Shijiazhuang 050200, China;
7. Hebei Province Innovation and Entrepreneurship Team of the 3rd Batch Giant Plan, Shijiazhuang 050200, China

Received:2020-10-19 Online:2021-05-23 Published:2021-05-22

摘要/Abstract

摘要： 旨在建立一种可检测牛早期胚胎性别的复合探针体系，减少常规PCR方法电泳所带来的污染，提高鉴定准确率，降低鉴定成本。本研究以SRY为牛胚胎性别鉴定的目标基因，以进口YCD-PCR性别鉴定试剂盒为对照组（n=9 543），荧光定量PCR的单引物分别扩增体系（荧光单扩，FSPSA，n=6 570）和双引物混合扩增体系（荧光双扩，FDPMA，n=22 238）分别做试验组，从性别鉴定效率、无反应率、雌雄胚胎百分率和比值、移植妊娠率、雌性胚胎母犊率、不同细胞取样数下的性别鉴定结果和鉴定成本等方面进行各组间比较。结果表明，荧光单扩组的雌性胚胎百分率和性别鉴定效率极显著高于其他两组（P<0.01），无反应率极显著低于其他两组（P<0.01），雌雄胚胎比值与其他两组差异较大（1.03∶1 vs 1∶1.03和1∶1.02），雌性胚胎母犊率极显著低于荧光双扩组和对照组（P<0.01）；荧光双扩组的雌性胚胎百分率、雄性胚胎百分率和雌性胚胎母犊率均与对照组无显著差异（P>0.05），雌雄胚胎比值与对照组相似（1∶1.03和1∶1.02），无反应率极显著低于对照组（P<0.01），性别鉴定效率极显著高于对照组（P<0.01）。取样细胞4~6组和7~10组的性别鉴定效率极显著高于1~3组和SD（separated & died cells）组（P<0.01），而1~3组和SD组之间及4~6组和7~10组之间差异不显著（P>0.05）。移植妊娠率4~6细胞组最高（49.68%），但各取样组间无显著差异（P>0.05）。荧光双扩法与对照组相比，鉴定成本下降了46.76%。结果显示，荧光双扩方法产母犊准确率最高，鉴定成本较低，取样4~6细胞时的移植妊娠率最高，是一种更可行的牛胚胎性别鉴定技术，更有利于产业化推广和应用。

关键词: 牛, PCR, 荧光定量PCR, 胚胎性别鉴定

Abstract: This study aimed to establish a compound probe system for indentifying the sex of bovine early embryos, reduce the pollution caused by electrophoresis of conventional PCR method, improve the identification accuracy and reduce the identification cost. In this study, using SRY as the target gene for identifying the sex of bovine embryos, the imported YCD-PCR sex identification kit was used as the control group (n=9 543), and the single primer separate amplification system (FSPSA, n=6 570) and the double primer mixed amplification system (FDPMA, n=22 238) of fluorescence quantitative PCR were used as the experimental groups, respectively. The comparison among the groups was conducted in the aspects of the efficiency of sex identification, non-reaction rate, percent of female and male embryos, ratio of female and male embryos, pregnant rate of embryo transfer，female calf rate of female embryos, results of sex identification under the different number of sampled cells and cost of sex identification to optimize the fluorescence quantitative PCR technology for bovine early embryo sex identification. The results showed that， in FSPSA group, the percent of female embryos and the efficiency of sex identification were significantly higher than those in the other two groups (P<0.01), the non-reaction rate were significantly lower than those in the other two groups (P<0.01), and the ratio of female and male embryos was obviousty different from that of the other two groups (1.03∶1 vs 1∶1.03 and 1∶1.02), and the female calf rate of female embryos was significantly lower than that of the FDPMA group and control group (P<0.01); In the FDPMA group, the percent of female embryos, percent of male embryos and the female calf rate of female embryos were not significantly different from those in the control group(P>0.05), and the ratio of female and male embryos was similar to that in the control group (1∶1.03 and 1∶1.02), but the non-reaction rate was significantly lower than that in the control group (P<0.01), and the efficiency of sex identification was significantly higher than that of the control group (P<0.01). The efficiency of sex identification of the groups sampled separately 4-6 cells and 7-10 cells was significantly higher than that of the groups sampled 1-3 cells and separated or/and died cells(SD) (P<0.01), however, there were no significant differences between 1-3 cells group and SD group and between 4-6 cells group and 7-10 cells group(P>0.05). The pregnancy rate of embryo transfer was the highest in the 4-6 cells group (49.68%), but there was no significant difference among all the sampled groups(P>0.05). Compared with the control group, the identification cost of the FDPMA group was reduced by 46.76%. In conclusion, the double primer mixed amplification of fluorescent quantitative PCR technology is more feasible and conducive to the industrial promotion and application of sex identification of bovine embryos because of the highest calving accurate rate of female embryos, the highest pregnancy rate of embryo transfer when sampled 4-6 cells, and the lower cost of sex identification.

Key words: bovine, PCR, fluorescence quantitative PCR, embryo sex identification

中图分类号:

S823.3

冯春涛, 顾文源, 王志仙, 赵增元, 朱宏波, 倪俊卿, 褚素乔, 余文莉, 李树静. 牛胚胎性别鉴定荧光定量PCR方法的优化与应用[J]. 畜牧兽医学报, 2021, 52(5): 1307-1316.

FENG Chuntao, GU Wenyuan, WANG Zhixian, ZHAO Zengyuan, ZHU Hongbo, NI Junqing, CHU Suqiao, YU Wenli, LI Shujing. Optimization and Application of Fluorescence Quantitative PCR Method for Sex Identification of Bovine Embryos[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1307-1316.

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牛胚胎性别鉴定荧光定量PCR方法的优化与应用

Optimization and Application of Fluorescence Quantitative PCR Method for Sex Identification of Bovine Embryos

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