State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources; Animal Reproduction Institute, Guangxi University, 100 Daxue Road, Nanning, Guangxi, PR China.
Anim Reprod Sci. 2011 Jun;126(1-2):32-6. doi: 10.1016/j.anireprosci.2011.04.021. Epub 2011 May 11.
Flow-cytometry sorting technology has been successfully used to separate the X- and Y-chromosome bearing spermatozoa for production of sex-preselected buffalo. However, an independent technique should be employed to validate the sorting accuracy. In the present study, X-chromosomes of bovine were micro-dissected from the metaphase spreads by using glass needles. Then X-chromosomes were then amplified by PCR and labelled with Cy3-dUTP for use as a probe in hybridization of the unsorted and sorted buffalo spermatozoa -chromosome. The results revealed that 47.7% (594/1246) of the unsorted buffalo spermatozoa were positive for X- chromosome probe, which was conformed to the sex ratio in buffalo (X:Y spermatozoa=1:1); 9.6% (275/2869) of the Y-sorted buffalo spermatozoa and 86.1% (1529/1776) of the X-sorted buffalo spermatozoa showed strong X-chromosome FISH signals. Flow cytometer re-analysis revealed that the proportions of X- and Y-bearing spermatozoa in the sorted X and Y semen was 89.6% and 86.7%, respectively. There were no significant differences between results assayed by flow-cytometry re-analysis and by FISH in this study. In conclusion, FISH probe derived from bovine X- chromosomes could be used to verify the purity of X and Y sorted spermatozoa in buffalo.
流式细胞术分选技术已成功应用于分离携带 X 和 Y 染色体的精子，以生产性别预选的水牛。然而，应该采用独立的技术来验证分选的准确性。本研究中，使用玻璃针从小牛中期分裂相中微切割 X 染色体。然后通过 PCR 扩增 X 染色体，并使用 Cy3-dUTP 标记作为未分选和分选水牛精子染色体杂交的探针。结果表明，47.7%（594/1246）的未分选水牛精子呈 X 染色体探针阳性，这与水牛的性别比例（X：Y 精子=1:1）相符；9.6%（275/2869）的 Y 分选水牛精子和 86.1%（1529/1776）的 X 分选水牛精子显示出强烈的 X 染色体 FISH 信号。流式细胞仪重新分析表明，分选的 X 和 Y 精液中携带 X 和 Y 染色体的精子比例分别为 89.6%和 86.7%。流式细胞仪重新分析和 FISH 检测的结果之间没有显著差异。总之，来自牛 X 染色体的 FISH 探针可用于验证水牛 X 和 Y 分选精子的纯度。
