利用流式细胞仪建立牦牛X、Y精子分选体系的研究

doi:10.11843/j.issn.0366-6964.2021.02.012

Abstract

Abstract: The main objective of this study was to optimize the technical conditions of yak (Bos grunniens) sperm sorting and establish an efficient yak X, Y sperm sorting technology system. Yak semen suspension was prepared, and sperm cells were incubated with different amounts of DNA dye Hoechst33342 and Allura Red. Yak X and Y sperms were separated by flow cytometer, and the sorting conditions were optimized by comparing sorting efficiency, sorted sperm motility and developmental potential. Furthermore, the purity of sorted sperm was detected by single sperm RT-PCR, and the motility of sorted sperm was detected by sperm analysis system. Then, the sorted sperm were fertilized in vitro with matured oocytes. The developmental potential of the sorted sperm was evaluated, and the sex of early embryo was detected by PCR of SRY fragment. The results showed that the accuracy and efficiency of 10 μL Hoechst33342 staining for 40 min group was significantly better than those of other groups, and the motility of sorted X and Y sperms was similar to that of 20 μL Hoechst33342 staining for 20 min group, which was significantly higher than that of other groups (P<0.05). The addition of 5 μmol·L^-1 Allura Red had no significant effect on the purity of sorted sperms (P>0.05), but significantly improved the sperm motility after sorting (P<0.05). After sorting, X and Y sperms were fertilized with oocytes in vitro, there was no significant difference in fertilization rate and the developmental potential of early embryos between the sorted group and unsorted group(P>0.05), and the sex ratio of embryos was consistent with the purity of sorted sperms. In summary, the method of sorting yak X， Y sperm by flow cytometer was established and optimized in this study, and adding Allure Red could improve the sperm motility after sorting, which laid a foundation for the preparation and production of yak sex controlled semen.

Key words: yak, flow cytometer, sorting, sperm motility, embryo sex

CLC Number:

S823.85

XIONG Xianrong, ZHANG Yan, XIONG Yan, MIN Xingyu, WU Jinbo, HAO Zhenyu, ZHANG He, LI Jian. The Establishment of X, Y Sperm Sorting System in Yak by Flow Cytometer[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(2): 399-407.

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The Establishment of X, Y Sperm Sorting System in Yak by Flow Cytometer

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