Artificial insemination with cryopreserved semen enables affordable, large-scale dissemination of gametes with superior genetics. However, cryopreservation can cause functional and structural damage to spermatozoa that is associated with reactive oxygen species (ROS) production, impairment of sperm motility and decreased fertilizing potential, but little attention has been paid to protein changes. The goal of this study was to investigate the oxidative modifications (measured as carbonylation level changes) of bull spermatozoa proteins triggered by the cryopreservation process. Flow cytometry and computer assisted sperm analysis were used to evaluate changes in viability, ROS level and motility of spermatozoa. Western blotting, in conjunction with two-dimensional electrophoresis (2D-oxyblot) and matrix assisted laser desorption/ionization time-of-flight/time-of-flight spectrometry, was employed to identify and quantify the specifically carbonylated spermatozoa proteins. Cryopreservation decreased motility and viability but increased the number of ROS-positive cells. We identified 11 proteins (ropporin-1, outer dense fiber protein 2, glutathione S-transferase, triosephosphate isomerase, capping protein beta 3 isoform, actin-related protein Ml, actin-related protein T2, NADH dehydrogenase, isocitrate dehydrogenase, cilia- and flagella-associated protein 161, phosphatidylethanolamine-binding protein 4) showing differences in protein carbonylation in response to cryopreservation. The identified proteins are associated with cytoskeleton and flagella organization, detoxification and energy metabolism. Moreover, almost all of the identified carbonylated proteins are involved in capacitation. Our results indicate for the first time that cryopreservation induces oxidation of selected sperm proteins via carbonylation. We suggest that carbonylation of sperm proteins could be a direct result of oxidative stress and potentially lead to disturbances of capacitation-involved proteins or could indicate cryopreservation-induced premature capacitation. (C) 2017 Published by Elsevier Inc.