The proteomic characterization of ram sperm during cryopreservation analyzed by the two-dimensional electrophoresis coupled with mass spectrometry.

The aim of this study was to analyze the effects of the cryopreservation process on the protein profile of ram sperm using two-dimensional electrophoresis (2-DE) coupled with mass spectroscopy. Semen was collected from five rams and cryopreserved in a Tris-based extender supplemented with glycerol and egg yolk as the main cryoprotectants. The fresh and post-thaw sperm total proteins were extracted and purified, followed by the 2-DE. The differential proteins in the stained gel were determined by mass spectrometry. The results indicated that there were 39 differential proteins between fresh sperm and frozen-thawed sperm. Among these proteins, the abundance of 28 proteins in fresh sperm was higher than those in post-thaw sperm (P < 0.05). However, 11 proteins in post-thaw sperm were up-regulated instead. The gene ontology (GO) analysis showed that most of differential proteins were implicated in cellular process, metabolism and regulation of the biological process. The networks of protein-protein interaction indicated a strong interaction among these differential proteins, which may be involved in sperm metabolism, acrosomal function, sperm motility, and reducing ROS level. In conclusion, the cryopreservation process modifies the proteome of ram sperm, which may be directly associated with ram sperm cryodamage, consequently influencing their fertility. Additionally, these differential proteins can be used as biomarkers for evaluation of frozen ram semen quality.