DETERMINATION OF ACTIVABLE PROACROSIN ACROSIN IN BOVINE SPERM USING AN IRREVERSIBLE ISOCOUMARIN SERINE-PROTEASE INHIBITOR/

The activable proacrosin/acrosin levels in bovine sperm were examined using fluorescent staining and flow cytometry. The proportion of sperm with active acrosin were determined using the biotinylated isocoumari n serine protease inhibitor, Bi-Aca-Aca-OMe-IC (BIG). The presence of bound inhibitor on sperm was then determined by secondary labeling wit h avidin fluorescein conjugate. The proportion of sperm with activable proacrosin/acrosin was assessed by using detergent treatment to expos e the active acrosin in intact sperm. The difference between untreated and detergent-treated aliquots was used to estimate the proportion of sperm with activable proacrosin/acrosin. In the 24-h stored samples f rom six bulls, the mean proportion of sperm with activable proacrosin/ acrosin was 78.8 +/- 2.8%, whereas the mean proportion with exposed ac rosin after cryopreservation of these samples was 55.8 +/- 4.1%. Signi ficant differences (p < 0.05) were found among bulls in the proportion of sperm with activable proacrosin/acrosin both before and after cryo preservation. Activable proacrosin/acrosin levels in samples of cryopr eserved sperm from five bulls were not correlated with fertility. Thes e results do indicate, however, that the irreversible isocoumarin seri ne protease inhibitor BIC can be used to determine the proportion of s perm cells that retain activable proacrosin/acrosin after cryopreserva tion and thawing.