Modification of membrane cholesterol and desmosterol in chicken spermatozoa improves post-thaw survival and prevents impairment of sperm function after cryopreservation.

During cryopreservation, spermatozoa are subjected to cryodamage that leads to a decline in fertilisation ability. Due to the complex nature of this process, the initial trigger for cryodamage remains unknown. Recently, we demonstrated that cryopreservation induces early apoptotic changes characterised by phosphatidylserine (PS) translocation via sterol loss from the plasma membrane of chicken spermatozoa. This led us to hypothesise that sterol incorporation into membranes minimises cryodamage, thereby improving the quality of cryopreserved chicken spermatozoa. In the present study, treating spermatozoa with 1.5mgmL-1 cholesterol- and 3mgmL-1 desmosterol-loaded cyclodextrin (CLC and DLC respectively) increased post-thaw survival and motility. These effects appeared to be highly dependent the amount of sterol loaded into the spermatozoa. Localisation experiments confirmed the incorporation of exogenous cholesterol into the sperm head region. Detection of PS translocation showed that elevation of these sterols inhibited early apoptotic changes, thereby enhancing post-thaw survival. Furthermore, CLC and DLC treatment suppressed spontaneous acrosome reaction after cryopreservation, preserving the ability of spermatozoa to undergo acrosome reactions in response to physiological stimulation. These results demonstrate that loading sterols into chicken spermatozoa before cryopreservation enhances their quality by inhibiting early apoptotic changes and spontaneous acrosome reactions. The present study provides new mechanistic insight into cryodamage in chicken spermatozoa.