Proteomic Analysis of Fresh and Liquid-Stored Boar Spermatozoa

SIMPLE SUMMARY: Male gametes can be stored for a long period of time for the purpose of preserving genetic material. Cryopreservation and liquid preservation are two main storage procedures commonly used for boar semen. There is evidence in the literature suggesting that cryopreservation changes the profile of proteins that are linked to the motility and function of spermatozoa. It was postulated that they can affect motility, capacitation, oocyte binding ability, and the acrosome reaction of spermatozoa. On the other hand, little is known about changes in protein levels in sperm that occur during liquid storage. Therefore, the objective of this study was to investigate whether liquid storage also causes an alteration in the proteomic profile of stored spermatozoa. A comparative proteomic approach was used to analyze protein samples from fresh spermatozoa and spermatozoa stored for three days at 15–17 °C. Results obtained show that liquid preservation causes quantitative changes in the boar sperm proteome with the over-expression of three out of four proteins in the liquid-stored sperm. Our findings can help elucidate the events involved in liquid preservation. ABSTRACT: In this study comparative proteomics was used to define changes in the expression of the spermatozoa proteins during liquid storage. Semen from eight boars was analyzed on the day of collection and after liquid preservation at 15–17 °C for three days. Sperm parameters (concentration, motility, morphology, vitality) and percentage of non-capacitated and acrosomal-reacted spermatozoa were determined. Sperm proteins were extracted and separated by two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and proteomic profiles were computationally compared to highlight differentially expressed protein spots that were, in turn, identified by mass spectrometry. The intensities of four spots were significantly different between fresh and liquid stored sperm. Namely: ATP citrate lyase, chaperonin containing T-complex polypeptide 1 (TCP1) subunit ε and probable phospholipid-transporting ATP-ase were over-expressed in liquid stored sperm, whereas cytosolic non-specific dipeptidase was over-expressed in fresh sperm. These differentially expressed proteins could be used as plausible biomarkers for the evaluation of boar semen quality and spermatozoa survival after liquid storage and could help to address problems associated with sperm preservation.