An X-Y paint set and sperm FISH protocol that can be used for validation of cattle sperm separation procedures.

X and Y chromosome paints were developed from sorted yak chromosomes for sexing cattle spermatozoa. Clear hybridization signals were obtained for every spermatozoon using a modified sperm decondensation protocol and fluorescence in situ hybridization (FISH). The procedure was evaluated using the established Beltsville sperm sexing technology, which separates spermatozoa by flow cytometry into X- and Y-bearing fractions. Close agreement was found between the assessment of sperm separation by flow cytometry and by FISH with the X-Y paint set. The FISH method is a simple, reliable and robust procedure for assessing the effectiveness of separation of X and Y spermatozoa.