The forces that hold spermatozoa in rouleaux were present in living and dead spermatozoa. Rouleaux killed with 0.1 M NaOH or 0.1 M HCl dissociated immediately into single spermatozoa with damaged acrosomes, while rouleaux treated with 10% formalin, 95% ethyl alchohol, or picric acid remained intact. Rouleaux cultured in vitro dissociated more rapidly at 37°C than at 7°C. Treatment with trypsin resulted in rouleaux dissociating into single spermatozoa after 2 h of culturing at 37°C.

From this study it appears that more than one type of cohesive force is responsible for rouleaux maintenance. It is proposed that these forces must be weakened for rouleaux to dissociate into single spermatozoa prior to capacitation in vivo.