Establishment Of Proteomics Methods Of Bovine Spermatozoa And Initial Analysis Of The Differential Proteins Between The Fresh And Frozen-thawed Spermatozoa

The fertilizability would be injuried no matter using what cryoprotective reagents and freezing methods, accompanied with the lose of proteins, the change of expression and function in the study of domestic livestock frozen semen. The proteomics technique platform of bovine common frozen spermatozoa was established by exploring the different sample preparation methods, the components of rehydration solution and optimizing the 2D electrophoresis protocols in this research. The differential proteins between the same batch of fresh and frozen-thawed spermatozoa were also explored, for illuminating the cryodamage mechanism by revealing the change of structure and function. The results showed:(1)compared with the urea-guanidine hydrochloride-based method, the TRIzol-based method was much more suitable for extracting the spermatozoa proteins. (2) the technique system with the high qualitative and stable 2D image had been obtained by combining the heat TRIzol-based method with the 24cm (pH3-10)Lstrips, optimizing the isoelectric focusing protocols and the staining methods.(3)the differential in-gel electrophoresis showed that 20 protein spots were absent in quality, 2 spots decreased and 10 spots increased significantly in quantity after frozen-thawed.(4)the in-gel digestion protocols which could digest the silver-staing spots for mass spectrum was established.(5)the positive control BSA and one protein spot in the gel have been identified successly.As a intermediate result, the 2D platform established in the research and the differential proteins found between fresh and frozen spermatozoa combined with the results of mass spectrum will provide better basis for revealing the cryodamage mechanism of spermatozoa cryopreservation. Simultaneously, this research will provide the theoretical evidence for the cryopreservation of semen of other domestic livestock such as swine and sheep. This research also gave the theoretical and practical importance for studying the differential proteomics of the X, Y spermatozoa.