Enrichment of bovine X and Y spermatozoa by free-flow electrophoresis.

Conditions of separation of bovine spermatozoa according to their differences in electrophoretic mobility were studied using free-flow electrophoresis. Distribution of spermatozoa in the electric field depends on composition of buffer system, field intensities and temperature. Conditions are described for reproducible separation of anodically migrating spermatozoa into two distinct peaks both for cold-immobilized (10-13 degrees C) and for motile cells (25 degrees C). Electrophoresis with the buffer used at 25 degrees C and field intensities of 70-100 V/cm provided high percentages of motile spermatozoa (50-90%) in all fractions. However, the sperm distribution across the fractions was the result of superimposed passive electrophoretic motion towards the anode and the active galvanotactic migration towards the cathode. Separation of X- and Y-spermatozoa was verified by quantification of Y-spermatozoa by means of both in situ hybridization, using Y-specific DNA fragments, and the Y-specific fluorescence staining (f-body test). Y-spermatozoa could be enriched in anodic fractions (112 and 144% relative to control). X-spermatozoa were enriched in cathodic fractions (80 and 68% Y-spermatozoa, as compared to control).