ENRICHMENT OF BOVINE X-SPERMATOZOA AND Y-SPERMATOZOA BY FREE-FLOW ELECTROPHORESIS

Conditions of separation of bovine spermatozoa according to their diff erences in electrophoretic mobility were studied using free-flow elect rophoresis. Distribution of spermatozoa in the electric field depends on composition of buffer system, field intensities and temperature. Co nditions are described for reproducible separation of anodically migra ting spermatozoa into two distinct peaks both for cold-immobilized (10 -13 degrees C) and for motile cells (25 degrees C). Electrophoresis wi th the buffer used at 25 degrees C and field intensities of 70-100 V/c m provided high percentages of motile spermatozoa (50-90 %) in all fra ctions. However, the sperm distribution across the fractions was the r esult of superimposed passive electrophoretic motion towards the anode and the active galvanotactic migration towards the cathode. Separatio n of X- and Y-spermatozoa was verified by quantification of Y-spermato zoa by means of both in situ hybridization, using Y-specific DNA fragm ents, and the Y-specific fluorescence staining (f-body test). Y-sperma tozoa could be enriched in anodic fractions (112 and 144 % relative to control). X-spermatozoa were enriched in cathodic fractions (80 and 6 8 % Y-spermatozoa, as compared to control).