OPTIMIZATION OF A CONTINUOUS REAL-TIME COMPUTERIZED SEMEN ANALYSIS SYSTEM FOR RAM SPERM MOTILITY ASSESSMENT, AND EVALUATION OF 4 METHODS OFSEMEN PREPARATION

Sampling conditions that affect the biological validity of computer-as sisted analysis of ram sperm motion were examined using a continuous r eal-time computerized semen analysis system (Hobson Sperm Tracker). Se arch radius (SR, 10 settings) and minimum track point (MTP, 10 setting s) were varied factorially to evaluate their effects on the inclusion of sperm subpopulations within derived datasets. Low SR (< 12 mu m) or high MTP values (> 26 frames) precluded measurements of rapidly movin g cells, whereas high SR (> 17 mu m) and low MTP settings (<22 frames) led to erroneous tracking and poor data quality. Suitable settings fo r these set-up parameters were derived and tested for their biological consistency with four methods of preparing ram semen for computerized analysis.The preparation techniques tested were: centrifugation throu gh sucrose-based Ficoll and Percoll media, a swim-up technique and sim ple dilution in Tyrode's media. The 'selective' Percoll and swim-up me thods generated sperm populations with significantly higher linear vel ocities and a lower tendency to deviate from linear trajectories than from either the Ficoll method or dilution technique. Deleterious effec ts of centrifugation were evident, particularly on sperm survival in v itro over several hours. It is concluded that computer-assisted semen analysis provides useful information about the behaviour of ram sperma tozoa in vitro, but the measurement conditions must be defined careful ly.