The present study was designed to determine the effect of three different extenders on ram sperm quality during a freeze–thawing procedure using flow cytometric and microscopic evaluations. Several in vitro qualitative analyses of postthawed sperm parameters including motility and velocity parameters, plasma membrane functionality, total abnormality, capacitation status, acrosome integrity, mitochondrial activity and apoptosis features were considered. In the breeding season, seven ejaculates from each Zandi ram were collected routinely twice a week. Following semen collection, samples were pooled and equally divided into three aliquots. Each aliquot was diluted and frozen with one of the following extenders: (1) Tris-based extender containing 1.5% (w/v) soybean lecithin (TSL), as a chemically defined extender, (2) Bioxcell, a commercial soybean lecithin-based extender, and (3) Tris-based extender containing 20% (v/v) egg yolk (TEY). The results of the present study indicated no differences in total [TSL (55.8 2.02%) vs TEY (50.2 2.02%; P < 0.05)] and progressive motility of spermatozoa [TSL (26.2 1.36%) vs Bioxcell (22.4 1.36%; P < 0.05)]. Semen freezing by means of TSL resulted in a higher percentage of live spermatozoa (39.42 1.81%) compared with TEY (29.17 1.81%; P < 0.05), and a higher percentage of functional plasma membrane (50.8 192%) compared with TEY (44 1.92%) and Bioxcell (38.8 1.92%; P < 0.05). The effect of extenders on sperm capacitation status showed that the percentage of post-thawed capacitated spermatozoa was higher in TEY (61.9 1.48%) compared with that in TSL (56.6 1.48%; P < 0.05). The evaluation of post-thawed spermatozoa indicated that the percentage of live spermatozoa with active mitochondria was higher in TSL (53.05 2.31%) compared with Bioxcell (45.92 2.31; P < 0.05) and the percentage of intact acrosome spermatozoa was higher in TSL (84.55 2.51%) compared with TEY (74.91 2.51%; P < 0.05). The use of TSL and Bioxce
