Abstract: Heat Trizol method was used to prepare the global protein of bovine spermatozoa, and the global proteins of fresh sperm and frozen sperm was separated by the technique of 2-DE with linear IPG (pH 3 to pH 7, 24 cm). 839±34 protein spots were obtained from the sample of frozen spermatozoa, while 564±16 from the sample of fresh spermatozoa. 19 differential proteins between frozen and fresh sperm were detected after analysis and significance test with software of ImageMasterTM 2D Platinum, 9 of which were expressed up-regulated while one of which was down-regulated in the sample of frozen spermatozoa. In addition, 9 spots were found only in frozen spermatozoa. After enzymolysis and MS detection, mass spectrum information of 3 differential proteins were obtained, and the differential proteins were considered to be neutral sphingomyelinase(N- SMase) activation associated factor(FAN), glutathione-S-transferase(Mu5) and zinc ion binding structure of bovine heart cytochrome c oxidase in the fully oxidized state, which were related to cell stress and apoptosis. It was presumed that the increasing of these proteins significantly in frozen spermatozoa were involved with cryodamage of spermatozoa.

Key words: frozen and fresh spermatozoa; 2-DE; MS; cryodamage