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Buruiana (1956) reported the presence of trypsin activity in the spermatozoa of several species, including the cock. Recently Stambaugh & Buckley (1969) demonstrated that the trypsin-like enzymatic activity ofrabbit sperm acrosomes could remove the zona pellucida of rabbit ova. The present study is concerned with the electrophoretic detection of multiple forms of trypsin-like enzymatic activity present in ejaculated spermatozoa of the domestic fowl, Gallus domesticus. Single-combed White Leghorn and Black Australorp cock semen was collected by abdominal massage, pooled, diluted with an equal volume of phosphate buffer (Wilcox, 1958), and centrifuged at 1200g for 10 min. The sperm deposit was washed three times with phosphate buffer, re-suspended in an equal volume of water containing glass beads and sonicated for 10 min with a model 185D sonifier (Heat Systems, Inc.) at an output setting of seven. The sonicated suspension was centrifuged at 48,000 g for 1 hr. Electrophoresis of 48,000 g supernatants and seminal plasma was performed with the Ortec Model 4200 flat-bed acrylamide electrophoresis system and Ortec Model 4100 pulsed constant power supply (Ortec, Inc.). Protein concentrations were determined by the method of Lowry, Rosebrough, Farr & Randall (1951). The technique of gradient gel preparation was similar to that described by Allen (1969). The gel consisted of successive layers of 4-5%, 6% and 8% acrylamide made up in 0-375 M-tris-sulphate buffer, pH 9. The upper and lower tank buffer was 0-0625 M-tris-borate, pH 9. Electrophoresis of 1-5 mg of protein was carried out for a total of 2\ hr at 4° C. The pulse rates utilized were 75 pulses/sec (pps) for 5 min, 150 pps for 75 min and 225 pps for the remainder ofthe2ihr. After electrophoresis, gels were incubated twice for 20 min at room tempera¬ ture in 0-1 M-succinic acid-tris buffer, pH 6, containing 1 mg/ml of Fast Blue Salt (Matheson, Coleman and Bell Chemicals) and 0-3 mg/ml of one of the following naphthylamide substrates (Fox Chemical Co.) : a-benzoyl-D,Larginine-jS-naphthylamide hydrochloride ( ), L-lysyl-/?-naphthylamide carbonate (L-Lys-/?-NA), L-arginyl-jS-naphthylamide hydrochloride (L-Arg-/?NA), L-lysyl-L-lysine-jS-naphthylamide carbonate (L-Lys-Lys-jß-NA) or N-

ELECTROPHORETIC DETECTION OF MULTIPLE FORMS OF TRYPSIN-LIKE ACTIVITY IN SPERMATOZOA OF THE DOMESTIC FOWL