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The tail beat frequency (TBF) of sperm was measured with a sperm-head fixation method which was based on the tendency of sperm to attach its head to the surface of a glass slide. Ferrous sulphate, a promotor of lipid peroxidation, inhibited TBF within 5 min of mixing with sperm. This inhibition can be reversed dose-dependently by superoxide dismutase, catalase, glutathione, and albumin. It was concluded that TBF could be a new pharmacological model for studying the effect of lipid peroxidation and antioxidant on sperm motility. Both enzymatic and non-enzymatic antioxidants can be screened with this method.

In a solution containing less than 5% seminal fluid, human sperm has a tendency to attach its head on the smooth surface of an object. Based on such a phenomenon, a simple method was developed to fix sperm head on the surface of glass slide. This method was demonstrated by studying caffeine effect on the tail movement of human sperm in phosphate buffered saline. By analysing the images recorded on a videocassette, caffeine, a well known stimulator of human sperm motility, was shown to induce an immediate increase of sperm TBF (tail beat frequency) and the amplitude of TBF increase was found to be larger in sperm with lower basal TBF value. This method can provide a flock of head-fixed motile sperm for a precise sperm tail movement assessment or for other purposes.

Human follicular fluid collected by laparoscopic oocyte pick-up during IVF was studied with a computer-assisted semen analyser to evaluate the effect of hFF on human sperm motility and velocity. Freshly ejaculated human sperm were washed with phosphate buffered saline and mixed with either PBS or hFF. At various incubation periods of time, hFF increased both sperm motility and velocity as compared with control (P less than 0.01). After incubation of sperm with hFF at 37 degrees C and 5% CO2 in air for 0, 1, 3, 6, and 12 h, the amplitude increase of motility were 49%, 77%, 330%, 2020%, and 3340% when individual control motility was considered to be 100%. The amplitude increase of curvilinear velocity were 43%, 51%, 67%, 152%, 278%, respectively. Comparison of the motility and velocity of the sperm treated with hFF between 0 and 12 h, showed that hFF preserved both motility and velocity in vitro (P less than 0.01). The stimulatory effect of hFF was retained after boiling at 100 degrees C for 30 min, or after being filtered through Amicon membrane cones, but it disappeared if the hFF had been pre-treated with chymotrypsin. However, hFF did not stimulate the motility and velocity of unwashed sperm in freshly ejaculated human semen. A non-dialyzable and heat-stable factor(s) with a molecular weight below 50,000 in hFF may improve and maintain the motility and velocity of washed human sperm. Whether this factor could be used to improve pregnancy rate in assisted reproduction awaits further investigation.

Comparing motility parameters with a trans-membrane migration method and a Hamilton-Thorn HTM-2000 computer assisted semen analyzer, we found that trans-membrane migration ratio (TMMR) correlated best with critical motility which indicated the fraction of fastest and straightest sperm in a semen sample. We also found that TMMR correlated better with progressive velocity than with track speed. It is concluded that nonprogressive sperm were not included in the estimation of TMMR and the trans-membrane migration method is most suitable for studying drug effect on straight and rapid sperm motility.

When testosterone concentrations in serum, saliva and seminal plasma were measured with a solid-phase enzymeimmunoassay in which antitestosterone antibody was bound to CNBr-Sepharose-4B, we found the ratios of testosterone in saliva and seminal plasma to that in serum were 2.5 +/- 0.2% and 5.19 +/- 0.42%. Testosterone level in saliva could be an index of free serum testosterone. In addition, we found a correlation between sperm motility and testosterone level in seminal plasma when sperm motility was measured with a transmembrane migration method. This enzymeimmunoassay could be applied to clinical studies of testosterone.

The stimulatory effects of two plasminogen activators, namely streptokinase and urokinase, were measured with a trans-membrane migration method. Both drugs induced maximal motility increase at a concentration of 200 international unit/ml; the amplitude of maximal motility increase ranged from 17% to 19% of control. Although their stimulatory effects were much less than those of calcium regulating agents, the clinical application of these two drugs for improving the successful rate of artificial insemination deserves further investigation because the action site is seminal plasma rather than sperm.

We developed a supravital stain of human sperm with fluorescent dyes. Either Hoechst 33,258 or fluorescein isothiocyanate could be used, the former stained sperm head while the later stained the whole sperm. Sperm vitality assessed with any of these two fluorescent dyes correlated well with that determined by eosin-nigrosin counterstain. When sperm vitality was compared with sperm motility measured with a transmembrane migration method, we found that many vital sperm were immotile because sperm vitality was higher than sperm motility in tested samples.