A fluorescent staining technique, the B-body test, is utilized to ascertain the proportion of male and female spermatozoa in separated semen. This test is also used to monitor progress of Thermal Convection Counterstreaming Sedimentation and Forced Convection Galvanization processes for separating heavier electropositive (female) and lighter electronegative (male) spermatozoa into the two constituent fractions. Biological field tests using separated semen with 1,115 cows resulted in 510 progeny showing close correlation in the percentage of male versus female and B-body positive in the male spermatozoa fractions, while a control group with nonseparated semen, usually 46% B-body positive, yielded 55% male offspring and 72.0% pregnancy. Thus, the accuracy of the separation techniques has been established to the extent of producing more females with heavier and electropositive and more males with the lighter and electronegative spermatozoa fractions. However, the pregnancy rate slopes downward as the positive electropotential and density of the spermatozoa are successively increased.