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 se xing cattle spermatozoa. Clear hybridization signals were obtained for ever y spermatozoon using a modified sperm decondensation protocol and fluoresce nce in situ hybridization (FISH). The procedure was evaluated using the est ablished 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 wi th the X-Y paint set. The FISH method is a simple, reliable and robust proc edure for assessing the effectiveness of separation of X and Y spermatozoa.