SELECTIVE ENRICHMENT OF BOVINE X-SPERMATOZOA AND Y-SPERMATOZOA BY PERCOLL DENSITY GRADIENT CENTRIFUGATION

A method is described for enrichment of X and Y spermatozoa of cattle by means of Percoll density gradient centrifugation. The gradient was made up of ten steps with densities ranging from 1.034 to 1.068 g/ml. Spermatozoa were centrifuged at 6-8-degrees-C with 500 g for ten minutes. The highest and lowest of the resulting 4-5 fractions were recentrifuged. Fractionated spermatozoa retained their full capability of in vitro fertilization. Identification of Y spermatozoa by in situ hybridization, using Y-specific DNA fragments, and by means of the f-body assay revealed significant alterations in ratios of X to Y spermatozoa in the highest (66.1 +/- 2.5 % and 62.8 +/- 2.1 %) and lowest fractions (34.1 +/- 10.3 % and 27.4 +/-3.0 %). Early experiments for in vitro insemination with the fractionated spermatozoa resulted in changed sex ratios among embryos produced: 75 % male embryos (n=20; spermatozoa of the upper fraction) and 90% female embryos (n=20; spermatozoa of the lower fraction).