title: Microisolation and microcloning of bovine X-chromosomes for identification of sorted buffalo (Bubalus bubalis) spermatozoa.
abstract: 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.
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources; Animal Reproduction Institute, Guangxi University, 100 Daxue Road, Nanning, Guangxi, PR China.
authors.forename: Xin-jie
authors.initials: XJ
authors.lastname: Zhuang
authors.forename: Yang-qing
authors.initials: YQ
authors.lastname: Lu
authors.forename: Ming
authors.initials: M
authors.lastname: Zhang
authors.forename: Sheng-sheng
authors.initials: SS
authors.lastname: Lu
authors.forename: Ke-huan
authors.initials: KH
authors.lastname: Lu
mesh_terms: D000818:Animals; D002020:Buffaloes / Q000502:physiology*; D002469:Cell Separation; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D008677:Metaphase; D016133:Polymerase Chain Reaction / Q000662:veterinary; D015203:Reproducibility of Results; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology*; D014960:X Chromosome / Q000235:genetics*; D014998:Y Chromosome / Q000235:genetics