Variation in donkey sperm freezability (capacity to withstand freeze-thawing) between ejaculates is a limitation for sperm cryopreservation. Seminal plasma proteins are essential for sperm function and also related to individual differences in sperm freezability. A Tandem Mass Tag (TMT) peptide labeling combine with a LC-MS/MS approach was conducted to quantitatively identify the seminal plasma proteins differentially abundant in ejaculates with optimal freezability characteristics (GFE) compared with those with suboptimal freezability characteristics (PFE). A total of 866 proteins were identified, and 99 ejaculates were in larger abundance in GFE samples. Differentially abundant proteins (DAPs) were subjected to intensive bioinformatic analysis. The majority of DAPs were involved in metabolic processes, oxidation-reduction processes and biological regulation. Results from functional protein analysis suggested that proteins functioned in oxidoreductase activity and acid phosphatase activity. This is the first report where there were analyses of the proteome of seminal plasma from donkey ejaculates with different freezability and to identify candidate proteins that could be used to explore the molecular mechanism related to donkey sperm cryotolerance. In this study, there also was elucidation of biomarkers for the early identification and selection of donkeys with optimal semen freezability.
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