SAMPLING FACTORS INFLUENCING ACCURACY OF SPERM KINEMATIC ANALYSIS

Sampling conditions that influence the accuracy of experimental measur ement of sperm head kinematics were studied by computer simulation met hods. Several archetypal sperm trajectories were studied. First, mathe matical models of typical flagellar beats were input to hydrodynamic e quations of sperm motion. The instantaneous swimming velocities of suc h sperm were computed over sequences of flagellar beat cycles, from wh ich the resulting trajectories were determined. In a second, idealized approach, direct mathematical models of trajectories were utilized, b ased upon similarities to the previous hydrodynamic constructs. In gen eral, it was found that analyses of sampling factors produced similar results for the hydrodynamic and idealized trajectories. A number of e xperimental sampling factors were studied, including the number of spe rm head positions measured per flagellar beat, and the time interval o ver which these measurements are taken. It was found that when one fla gellar beat is sampled, values of amplitude of lateral head displaceme nt (ALH) and linearity (LIN) approached their actual values when five or more sample points per beat were taken. Mean angular displacement ( MAD) values, however, remained sensitive to sampling rate even when la rge sampling rates were used. Values of MAD were also much more sensit ive to the initial starting point of the sampling procedure than were ALH or LIN. On the basis of these analyses of measurement accuracy for individual sperm, simulations were then performed of cumulative effec ts when studying entire populations of motile cells. It was found that substantial (double digit) errors occurred in the mean values of curv ilinear velocity (VCL), LIN, and MAD under the conditions of 30 video frames per second and 0.5 seconds of analysis time. Increasing the ana lysis interval to 1 second did not appreciably improve the results. Ho wever, increasing the analysis rate to 60 frames per second significan tly reduced the errors. These findings thus suggest that computer-aide d sperm analysis (CASA) application at 60 frames per second will signi ficantly improve the accuracy of kinematic analysis in most applicatio ns to human and other mammalian sperm.