The micro-sperm immobilization test: the use of only motile spermatozoa and studies of complement

A technique is described which shows how to obtain a suspension of only progressively motile spermatozoa, from semen, by active penetration into an overlying buffer layer. About 60% of the number of motile spermatozoa in the original semen sample can be obtained as an output in the removed buffer layer. With this method many more semen samples, in particular those with less than 70% motile spermatozoa, can be used than otherwise would have been possible. Moreover, with such a suspension of only motile spermatozoa the sensitivity of the micro-immobilization test is substantially improved, due to the absence of non-specific clumping, the minimization of specific tail-to-tail agglutination and an optimal end-point reading because of the absence of background immotility. As the complement source, a 1:8 dilution of rabbit or guinea-pig serum in human AB serum was found to be most suitable. The 1 microliter sperm suspension is initially mixed with 2 microliter of serum and incubated for 30 min, and then a further 2 microliter volume of complement is added, which is incubated for 2 hr with the serum-sperm mixture, and the test is read under an inverted microscope.