185 HIGH-THROUGHPUT PROTEOMIC ASSESSMENT OF FROZEN–THAWED BOAR SPERMATOZOA

Abstract

Cryopreservation is a tool of choice for seedstock constitution of genetically superior males. Its successful application in swine AI industries is limited because of the poor freezability of boar semen. Indeed, a subset of boars exists that can be successfully frozen–thawed for AI, whereas another group appears highly cryosusceptible, and therefore unusable for long-term semen storage. The reasons for such differences are unknown, and the full characterisation of the protein composition of boar spermatozoa will help determine potential cryosensitive proteins. Here, we performed high-throughput proteomic analyses of boar spermatozoa and compared the proteome profiles of ‘good’ and ‘poor’ freezer boars. Eight commercially proven fertile boars were selected based on conception rates after AI using fresh semen. Semen from 3 independent ejaculations was collected from 4 good and 4 poor freezer boars and frozen in 5-mL straws for the study. Frozen–thawed semen was diluted in the thawing solution and centrifuged through a discontinuous Percoll gradient (90/45) to remove seminal plasma, freezing extender, somatic cells, and dead sperm cells. Purified motile spermatozoa were washed 3 times with cold PBS and pooled in pellets of 3 × 108 spermatozoa per boar. Protein samples were digested with trypsin and prepared for LC-MS/MS analysis. Peptides yielding probability scores lower than 0.05 were subjected to protein identification, and the significance of differentially expressed protein was fixed at P < 0.05. More than 3000 proteins were identified in each group of spermatozoa. Proportions of 63 and 61% total proteins were exclusively detected in good and poor freezer boars, respectively. Many of the identified proteins were related to different cellular compartments and important molecular mechanisms related to sperm function, such as cell death regulation, macromolecule metabolism, and energy-related pathways. Approximately 5% of total proteins, representing 163 to 182 individual proteins, were detected at higher levels in both semen groups. Half of these highly abundant proteins were differentially expressed between good and poor freezer boars. Only 8 appeared partially annotated and 11 were predicted. The remaining list of fully annotated proteins included candidates such as transferrin, albumin, and fascin3, which were significantly (P < 0.05) abundant in good freezer boars, and outer dense fibre (ODF) 2, protamine (PRM) 2, and calmodulin (CALM) 1, which were significantly (P < 0.05) abundant in poor freezer boars. Overall, the results indicate that boar spermatozoa contain large amount of proteins whose susceptibility to cryopreservation and implications for sperm function are still to be characterised. Our findings are particularly important for 1) the search for potential biomarkers of semen freezability, and 2) improvement of semen freezing-thawing extenders for boars and other species with similar issues.