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

Abstract

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

CLC Number:

S823.3

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

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