FRACTIONATION OF BOVINE SPERMATOZOA FOR SEX SELECTION - A RAPID IMMUNOMAGNETIC TECHNIQUE TO REMOVE SPERMATOZOA THAT CONTAIN THE H-Y-ANTIGEN

A study was conducted to rapidly fractionate bovine spermatozoa on the basis of cell-surface H-Y antigen (i.e., Y chromosome-bearing spermat ozoa). A novel, rapid immunomagnetic method was developed for removal of spermatozoa that bound to anti-H-Y IgG. Fluorescent labeling and fl ow cytometry were used to measure the efficiency with which spermatozo a binding to anti-H-Y were removed by the immunomagnetic technique. Wa shed bovine spermatozoa (n=7 bulls) were treated with a mouse monoclon al IgG antibody to H-Y antigen(MoAb 12/49). Fluorescent labeled goat a ntibody against mouse IgG was added to label those spermatozoa with ce ll-surface H-Y antigens. Supermagnetized polymer beads coated with an anti-antibody to the MoAb 12/49 were then added to the spermatozoa. Af ter 20 min of incubation, spermatozoa were exposed for 2 min to a magn et, causing the magnetized particles to adhere to the sides of the tub e. Nonmagnetized spermatozoa in the supernatent were aspirated and ana lyzed for fluorescent label by flow cytometry. Approximately 50% of sp ermatozoa not subjected to immunomagnetic separation were fluorescent labeled, and about one-half of the spermatozoa were observed microscop ically to be bound to the magnetized polymer beads prior to magnetic s eparation (P<0.05). Following magnetic separation, only 1.2% (P<0.05) of the spermatozoa in the magnetic supernatent were fluorescent labele d. Assuming that only Y chromosome-bearing spermatozoa have cell-surfa ce H-Y antigens, the present immunomagnetic fractionation removed almo st all of the Y chromosome-bearing spermatozoa, leaving a population t hat was greater than 98% X chromosome-bearing spermatozoa.