This study investigated the effects of long-term (24 h) in-vitro sperm incubation at room temperature (RT; 23 C) versus
testis temperature (35 C) on various sperm-quality parameters. Semen samples (n = 41) were prepared both by density-gradient centrifugation
(DGC) and the swim-up technique in order to compare the influence of sperm preparation on sperm quality after incubation.
Progressive motility and morphology were significantly higher after incubation at RT compared with 35 C (P < 0.001 and
P < 0.01, respectively). The proportions of acrosome-reacted, apoptotic and dead spermatozoa were significantly lower in samples
incubated for 24 h at RT compared with 35 C (P < 0.001, P = 0.01 and P < 0.001, respectively). The number of motile, morphologically
normal, non-acrosome-reacted and nonapoptotic spermatozoa recovered after sperm preparation was significantly higher in
DGC compared with swim-up samples (P < 0.001). However, spermatozoa prepared by swim-up showed better survival after incubation
compared with DGC-prepared spermatozoa, especially when incubated at 35 C. In conclusion, this study indicates a significantly
better and longer preservation of sperm quality when incubation is performed at RT. These findings may convince
laboratories to change the routinely used sperm storage conditions in order to maximize the quality of the prepared sperm sample.