Four premises for sperm preservation were previously outlined. The present work tested 2 of these. The first premise was that sperm mobility phenotype affects procedural efficacy. Random bred roosters were phenotyped with the sperm mobility assay. A normal frequency distribution was observed with 35% (SD = 16.4) mobile sperm. Test subjects had values >51% (high) or between 19 and 35% (below average). Phenotypes were confirmed by repeated measure analysis. Ejaculates were pooled by phenotype. Sperm were washed by centrifugation through 12% (wt/vol) Accudenz. Washed sperm were suspended in Beltsville Poultry Semen Extender (BPSE) at 2 × 10 sperm/mL. Such sperm were stored at 10°C for 24 h. In the case of highly mobile sperm, an exponential decay was observed with a -intercept of 72% and an asymptote of 53%. In contrast, postwash values for below-average males decreased linearly from a -intercept of 31 to 17% after 24 h. A logistic decay was observed when sperm from high phenotype subpopulation males were extended with BPSE rather than washed before storage. Whereas -intercepts were equivalent between experiments, end points were not, that is, 53 vs. 17% mobile sperm. This difference was attributed to the extent of cytotoxic edema. The second premise tested was that the sperm mobility assay can predict the status of sperm cell mitochondria in response to sperm manipulation. Highly mobile sperm were washed and then suspended in either saline or glucose-free extender. Solution pH and osmolality were equivalent. Extender osmolality was controlled by replacing glucose with mannitol. Sperm were stressed by incubation at 2 × 10/mL at 20°C for 8 h. In each case, loss of sperm mobility approximated a logistic function. Whereas -intercepts were equivalent, the time at which loss of function was half maximal was prolonged with the extender ( < 0.01). This difference was attributed to a diminution of the process whereby energy-deprived sperm were rendered immobile by cellular edema. An a posteriori analysis was limited to pretreatment data from males categorized a priori with the high phenotype. Phenotype was independent of time ( = 0.81) during the 14-wk interval in which experiments were performed. In summary, extender efficacy was affected by sperm mobility phenotype as well as the means by which the extender was used. To date, such effects have not been addressed in attempts to preserve chicken sperm in vitro.