利用流式细胞仪建立牦牛X、Y精子分选体系的研究  

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

作　　者：熊显荣[1,2] 张雁 熊燕[2] 闵星宇 吴锦波 郝振宇 张贺 李键 XIONG Xianrong;ZHANG Yan;XIONG Yan;MIN Xingyu;WU Jinbo;HAO Zhenyu;ZHANG He;LI Jian(College of Animal &Veterinary Sciences, Southwest Minzu University, Chengdu 610041, China;Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization of Ministry of Education, Chengdu 610041, China;Animal Husbandry Science Institute of Aba Autonomous Prefecture, Aba 623000, China)

机构地区：[1]西南民族大学畜牧兽医学院,成都610041 [2]青藏高原动物遗传资源保护与利用国家教育部重点实验室,成都610041 [3]四川省阿坝州畜牧科学研究所,阿坝623000

出　　处：《畜牧兽医学报》2021年第2期399-407,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家“十三五”重点研发专项(2018YFD0502304);西南民族大学中央高校基本科研业务费专项资金项目(2020PTJS15005)。

摘　　要：旨在探索与优化牦牛精子分选条件,建立高效的牦牛X、Y精子分选技术体系。本研究制备了牦牛精液细胞悬液,采用不同量的DNA染料Hoechst33342和诱惑红共孵育精子细胞。利用流式细胞仪分选牦牛X、Y精子,通过比较分选效率、分选后精子活力及发育潜能,优化分选条件。运用RT-PCR检测分选精子的纯度,利用精子分析系统检测分选后精子的活力。将分选后的精子与体外成熟的卵母细胞进行体外受精,统计分选后精子的发育潜力,并采用SRY片段的PCR法检测早期胚胎的性别。结果显示,10μL Hoechst33342染色40 min后的分选效果最佳,其分选产物的准确度和分选效率显著优于其他组,分选后的X、Y精子活力与20μL Hoechest33342染色20 min组相当,显著高于其他组(P<0.05)。添加5μmol·L^-1的诱惑红对分选的纯度无显著影响(P>0.05),但能显著提高分选后精子的活力(P<0.05)。分选后X、Y精子分别与卵母细胞进行体外受精,受精率和囊胚形成率与未分选组差异不显著(P>0.05),且胚胎性别比例与分选后精子纯度吻合。综上,本研究建立并优化了流式细胞仪分选牦牛X、Y精子的方法,添加诱惑红有助于改善分选后牦牛精子的活力,为后期牦牛性控精液的制备及生产奠定了基础。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 10μL 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 20μL Hoechst33342 staining for 20 min group,which was significantly higher than that of other groups(P<0.05).The addition of 5μmol·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.

关 键 词：牦牛 流式细胞仪 分选 精子活力 胚胎性别 

分 类 号：S823.85[农业科学—畜牧学]