Validation of sperm sexing in the cattle (Bos taurus) by dual colour fluorescence in situ hybridization

Separation of X- and Y-bearing sperm cells, together with artificial insemination using sex-specific semen, makes it possible to pre- determine the sex of calves. This has the potential to considerably improve cattle breeding, genetic resource management and particularly the efficiency of dairy and meat production. However, the broad use of sexed semen will depend on availability, price, fertilizability and in particular the actual sorting purity of sperm doses. To validate the accuracy of sperm sexing in Bos taurus, we have developed a simple, fast and reliable dual colour fluorescence in situ hybridization (FISH) test, where Y-bearing spermatozoa are identified by a DNA fragment hybridizing to a large pericentromeric repetitive DNA block on the bovine Y chromosome (locus DYZ1, Yp13-q12). To avoid an underestimation of Y signals, we used a second DNA probe identifying a large subcentromeric block of complex repetitive DNA on the bovine autosome 6 (locus D6Z1, 6q12-15) as a positive control. Bovine sperm were fixed with methanol:acetic acid and denatured by simply immersing in 3 M NaOH, yielding consistent hybridization results and good preservation of sperm morphology. The FISH protocol was evaluated on unsorted sperm as well as on sperm samples sexed using the Beltsville technology, which separates X- and Y-bearing spermatozoa by staining with Hoechst 33342 and flow sorting according to their DNA content (Johnson et al. 1987).