Swim-up (SU) is a technique that permits the obtaining of motile sperm. Based on the sperm motility of neat ejaculates, we analyzed functional parameters, cytoplasmic esterases, and mitochondrial dehydrogenases of ram sperm using calcein acetomethylester and resazurin, respectively. Active caspase-3 and -7 and chromatin structure were evaluated in ram sperm before and after the SU process. There were no changes in any of the viability parameters after SU in neat semen samples with less or more than 25% motility. The percentage of active caspase-3 and -7 decreased after SU (68.8 +/- 4.6 vs 54.2 +/- 6.0), whereas a small but significant increase of chromatin structural abnormalities was observed (DNA fragmentation index [DFI], 287.3 +/- 3.1 vs 297.2 +/- 2.4). For the first time, the location of active caspase-3 and -7 was described for ram sperm. Notably, we found active caspases in the implantation fossa region. The presence of active caspases in neat ejaculates and the diminished presence of active caspases in SU-processed ejaculates suggest a role for caspases in motility and possibly in male fertility. The results of this study indicate that the evaluation of more than one cell-function marker is necessary to appropriately evaluate sperm quality. Furthermore, in semen samples with low motility, a lower percentage of sperm with active caspases is obtained after SU, although these sperm present increased values of DFI.