Spermatozoa need to undergo a physiological change called capacitation before pene­trating the egg. Capacitation is a temperature dependent phenomenon; variation in the incubation temperature could cause an alteration in events associated with this process.

Sperm incubation at room temperature produces a temporary blockage of capacitation related events. When maintained under these conditions, cells are in a “quiescent” state (non-capacitated), retaining their functional properties that can be expressed at body temperature of 37°C (1)

We therefore recommend density gradient preparation at room temperature. Remember, the best is to avoid temperature fluctuation and therefore, the best way to prepare and store the sample, is at room temperature.

Ref: Influence of temperature and sperm preparation on the quality of spermatozoa. Thijssen, Klerkx, Huyser, Bosmans, Campo, Ombelet. Reprod Biomed Online. 2014 Apr;28(4):436-42.