In the field of mammalian reproduction, the success rate of fertilization largely relies on spermatozoa fertility potential. Total proteins change to a large degree during gametogenesis to activate gametes. There are some techniques that are most popular for proteomic studies of fertilization, including SDS-PAGE, 2D-DIGE, Western Blotting and Immunocytochemistry, as well as Mass Spectrometry. Proteins and their cofactors play important roles at different stages of gametogenesis, fertilization and embryo development. However, insufficiencies in the construction mode of fertility hinder the techniques in determination the sperm fertility. This study focused on identifying and tracking crucial proteins for fertility based on comparison between high- and lowertility sperms by means of 2D-DIGE and immunoblotting. We identified 18 proteins that varied significantly, including Outer dense fiber 2 (ODF2) of sperm tails and Manganous superoxide dismutase (MnSOD). Differences in these proteins suggest that posttranslational modification and deoxydation of sperm proteins might be associated with fertility.