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The predictability of semen quality of mature sires from measurements at an early age is not well established. The aim of the present study was to determine age-dependent changes in the quality of bull semen for artificial insemination (AI). Semen was collected and frozen from each of six Swedish Red and White (SRB) dairy AI bulls when they were 1 and 4 years old. Three batches were randomly selected from each bull and age group. From each batch, semen was analysed immediately after thawing [post-thaw (PT), control] as well as after washing/resuspension (W) and after a swim-up procedure (SU). The analyses comprised subjective and computerized (computer-aided sperm analysis, CASA) measurements of motility as well as sperm concentration, morphology and membrane integrity. When semen was analysed, PT, overall sperm motility (CASA), concentration of motile spermatozoa and membrane integrity improved when sires were older. After SU, there was a similar improvement in membrane integrity and concentration of motile spermatozoa, but linear motility decreased. No significant differences between ages were recorded after W-treatment. The above findings indicate that SU is not only superior to W-treatment in differentiating semen quality among bulls but also reveals age-dependent changes. Improved motility and membrane integrity suggest increased viability of spermatozoa at 4 years of age in the SRB sires examined here.

A significant proportion of mammalian fertilization is mediated through the proteomic composition of the sperm surface. These protein constituents can present as biomarkers to control and regulate breeding of agricultural animals. Previous studies have addressed the bovine sperm cell apical plasma membrane (PM) proteome with nitrogen cavitation enrichment. Alternative workflows would enable to expand the compositional data more globally around the entire sperm's surface. We used a cell surface biotin-labeling in combination with differential centrifugation to enrich sperm surface proteins. Using nano-LC MS/MS, 338 proteins were confidently identified in the PM-enriched proteome. Functional categories of sperm-egg interaction, protein turnover, metabolism as well as molecular transport, spermatogenesis, and signal transduction were represented by proteins with high quantitative signal in our study. A highly significant degree of enrichment was found for transmembrane and PM-targeted proteins. Among them, we also report proteins previously not described on bovine sperm (CPQ, CD58, CKLF, CPVL, GLB1L3, and LPCAT2B) of which CPQ and CPVL cell surface localization was further validated. A descriptive overview of the bovine sperm PM integral and peripheral proteins is provided to complement future studies on animal reproduction and its relation to sperm cell surface. All MS data have been deposited in the ProteomeXchange with identifier PXD001096 (http://proteomecentral.proteomexchange.org/dataset/PXD001096).