Characterization of the porcine seminal plasma proteome comparing ejaculate portions

• SEC-1-D SDS PAGE & digestion in gel and solution brings out less abundant boar seminal plasma proteins.
• 374 proteins (Sus scrofa taxonomy, 99% confidence) identified in boar seminal plasma.
• More than 250 proteins first described in boar seminal plasma.
• 34 SP-proteins differentially expressed among ejaculate portions.
• A publicly accessible database of the boar SP-proteome created.

Full identification of boar seminal plasma (SP) proteins remains challenging. This study aims to provide an extensive proteomic analysis of boar SP and to generate an accessible database of boar SP-proteome. A SP-pool (33 entire ejaculates/11 boars; 3 ejaculates/boar) was analyzed to characterize the boar SP-proteome. Twenty ejaculates (5 boars, 4 ejaculates/boar) collected in portions (P1: first 10 mL of sperm rich ejaculate fraction (SRF), P2: rest of SRF and P3: post-SRF) were analyzed to evaluate differentially expressed SP-proteins among portions. SP-samples were analyzed using a combination of SEC, 1-D SDS PAGE and NanoLC-ESI-MS/MS followed by functional bioinformatics. The identified proteins were quantified from normalized LFQ intensity data. A total of 536 SP-proteins were identified, 409 of them in Sus scrofa taxonomy (374 validated with ≥ 99% confidence). Barely 20 of the identified SP-proteins were specifically implicated in reproductive processes, albeit other SP-proteins could be indirectly involved in functionality and fertility of boar spermatozoa. Thirty-four proteins (16 identified in S. scrofa taxonomy) were differentially expressed among ejaculate portions, 16 being over-expressed and 18 under-expressed in P1–P2 regarding to P3. This major proteome mapping of the boar SP provides a complex inventory of proteins with potential roles as sperm function- and fertility- biomarkers.Biological significanceThis proteomic study provides the major characterization of the boar SP-proteome with > 250 proteins first reported. The boar SP-proteome is described so that a spectral library can be built for relative ‘label free’ protein quantification with SWATH approach. This proteomic profiling allows the creation of a publicly accessible database of the boar SP-proteome, as a first step for further understanding the role of SP-proteins in reproductive outcomes as well as for the identification of biomarkers for sperm quality and fertility.

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