Detection of water buffalo sex chromosomes in spermatozoa by fluorescence in situ hybridization

In order to identify X- and Y-bearing spermatozoa in water buffalo by fluorescence in situ hybridization (FISH), some available probes of closely related species were examined. An X- and Y-specific probe set, made from flow sorted yak chromosomes, labelled in somatic metaphases of water buffalo the whole X and Y, respectively, except their centromere regions. A cattle Y-chromosome repeat sequence (BC1.2) showed strong signal on the telomere region of the buffalo Y-chromosome, demonstrating the evolutionary conservation of this locus in water buffalo. In hybridization experiments with spermatozoa from five buffaloes, the yak X-Y paint set demonstrated clear signals in more than 92% (46.8% X and 45.8% Y) of the cells. Using the cattle Y-chromosome specific BC1.2 probe, clear hybridization signal was detected in more than 48% of the cells. Statistical analysis showed that there was no significant difference between bulls or from the expected 50 : 50 ratio of X- and Y-bearing cells. The probes presented here are reliable to assess separation of X- and Y-bearing spermatozoa.