Identification and functional analysis of bull (Bos taurus) cauda epididymal fluid proteome

Despite recent advances in bull epididymal fluid proteome research, significant numbers of proteins secreted to epididymal lumen remain unidentified. The objective of this study was to expand the number of identified cauda epididymal fluid proteins in bulls and to contextualize them in a broader view of their mutual interactions and involvement in biological processes and pathways, to fully elucidate the ways in which epididymal fluid proteins are involved in storage and maturation of spermatozoa in epididymis. We collected postmortem cauda epididymal fluid from 6 mature Holstein Friesian bulls. We performed the identification of proteins using 2-dimensional electrophoresis coupled with MALDI mass spectrometry. Analysis of functionality and pathway involvement of identified proteins was performed using Ingenuity Pathway Analysis software. We identified a total of 189 epididymal fluid proteins, out of which 100 were newly identified in bull epididymal fluid. We have combined our data with 2 previously performed bull epididymal fluid proteome identifications, yielding 280 proteins total, and analyzed it. The main canonical pathways involving epididymal proteins were glycolysis, gluconeogenesis, protein ubiquitination pathway, nuclear factor-erythroid 2-related factor 2-mediated oxidative stress response, and farnesoid X receptor/retinoid X receptor activation. The main biological functions potentially performed by epididymal fluid proteins included carbohydrate metabolism, cellular growth and proliferation, cell death and survival, and small molecule biochemistry. Overall, our results have pointed out multiple novel pathways in bull epididymal fluid that might take part in various aspects of maturation and protection processes of epididymal spermatozoa.