Updated project metadata. Cryoinjury and protein changes are a consequence of cryopreservation and may have a negative impact on sperm quality regarding motility, viability and fertilizing ability. However, potential proteomic changes in rabbit semen throughout the cryopreservation process have never been investigated previously. The aim of the present study was to compare the whole proteome of fresh and cryopreserved rabbit extracellular fluid of semen. Comparative analysis and identification of proteins were performed using 2-dimensional difference in-gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization mass (MALDI TOF/TOF) spectrometry. We found that 28 proteins differed in abundance between fresh and frozen extracellular fluid.