In in vitro fertilisation (IVF), sperm preparation as critical part and influencing the sperm quality is especially dependent on the chosen technique itself and incubation parameters including temperature and CO2.

In this study, we compared firstly density-gradient centrifugation technique (DGC) to the adapted DGC using the sperm pellet of 80% fraction (DGC/80P) in order to improve the sperm yield. Secondly, this study led to evaluate different sperm incubation conditions based on temperature effect (room temperature (RT = 23°C) versus 35°C) and in the other hand, with or without 5% CO2 during 24 hrs. Based on evaluating sperm conventional parameters and the DNA damage using TUNEL assay, our result showed that DGC/80P increased sperm quality compared to DGC with 25% of improvement.

For temperature incubation effect after 24 hrs, 35°C increased the DNA damage and decreased the sperm quality while RT could improve sperm motility by 38%.

Moreover, the sperm incubation with 5% CO2 after 24 hrs realised a negative impact on sperm parameters and its DNA damage.

Indeed, for current IVF practice, a good sperm quality can be maintained for several hours at room temperature, while the sperm preparation is processed using the DGC/80P without CO2.