The objectiveof this work was to develop a bovine spermatozoid sex selection method by usingPercoll density gradient centrifugation. It was used frozen semen of bulls keptin semen collection regime. Fraction X or Y was separated by centrifugationin three different Percoll density gradient formed by 1 to 12 layers with densitiesvarying from 1.004 g/mL to 1.123 g/mL. Solutions with different densities wereprepared by mixing, at different proportions, Hank's culture medium and a stocksolution composed of NaCl 1.5 M and Percoll (1:9, v/v). On each gradient, itwas put 50 × 106 spermatozoids thawed in Hank's medium and centrifugedat 250 X g for 30 min in a horizontal rotor, at 25°C. The spermatozoidsin the superior and inferior fractions were treated with Mustard Quinacrineand analyzed (200 spermatozoids) for the presence of F-body. Of the spermatozoidsfound in the sediment of the two gradients, composed of 8 and 12 fractions withdensities varying from 1.050 to 1.120 g/mL and 1.044 to 1.123 g/mL, respectively,it was identified 25% with the F-body and the other 75% were probably X chromosome-bearingspermatozoids. Increase in percentage of the X-spermatozoid after density gradientcentrifugation will allow this system of spermatozoid sexing to be used in largescale in commercial production of meat and milk as well as for progeny testsin bovines.
