Trehalose modifies the protein profile of ram spermatozoa during cryopreservation

As a magical oligosaccharide, trehalose has been revealed to enhance the post-thaw quality of stock semen. However, information regarding the cryoprotective mechanism of trehalose during cryopreservation has not yet been determined. This study was designed to observe the effects of trehalose on the proteome of ram frozen spermatozoa by applying the isobaric tag for relative and absolute quantification (iTRAQ) strategy combined with parallel reaction monitoring (PRM). A total of 1269 proteins were identified. Among them, there were 21 differentially expressed proteins (DEPs), with 9 up-regulated proteins and 11 down-regulated proteins in spermatozoa frozen with trehalose. These DEPs were primarily located in nucleus, cytoplasm, and extracellular region. The Gene Ontology (GO) enrichment analysis demonstrated the involvement of the DEPs in signal transduction, ion binding, oxidoreductase activity, response to stress, and catabolic processes. Based on the STRING analysis, tight functional correlations were observed between 6-phosphogluconate dehydrogenase, fructose-bisphosphate aldolase A isoform 1, 14-3-3 protein epsilon, tyrosine-protein kinase Fer, and beta-hexosaminidase subunit alpha precursor. Furthermore, 10 DEPs were verified using PRM, confirming the accuracy of the iTRAQ data acquired in this study. In conclusion, trehalose can modify the protein profile of ram spermatozoa during cryopreservation, which may be associated with its cryoprotective effects. Additionally, trehalose may function on frozen spermatozoa through antioxidation, involvement in glycolysis, and increment of spermatozoa tolerance to various stresses.