The objective of this work was to develop a bovine spermatozoid sex selection method by using Percoll density gradient centrifugation. It was used frozen semen of bulls kept in semen collection regime. Fraction X or Y was separated by centrifugation in three different Percoll density gradient formed by 1 to 12 layers with densities varying from 1.004 g/mL to 1.123 g/mL. Solutions with different densities were prepared by mixing, at different proportions, Hank's culture medium and a stock solution composed of NaCl 1.5 M and Percoll (1:9, v/v). On each gradient, it was put 50 × 10(6) spermatozoids thawed in Hank's medium and centrifuged at 250 X g for 30 min in a horizontal rotor, at 25°C. The spermatozoids in the superior and inferior fractions were treated with Mustard Quinacrine and analyzed (200 spermatozoids) for the presence of F-body. Of the spermatozoids found in the sediment of the two gradients, composed of 8 and 12 fractions with densities 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-bearing spermatozoids. Increase in percentage of the X-spermatozoid after density gradient centrifugation will allow this system of spermatozoid sexing to be used in large scale in commercial production of meat and milk as well as for progeny tests in bovines.
Bos taurus; semen; sex selection
