Short Communication IXth International Conference on Boar Semen Preservation 2019

Shotgun proteome analysis of seminal plasma proteins differentiate boars by reproductive performance (#4.1)

Kara R Stewart 1 , Theresa Casey 1 , Kayla Mills 1 , Uma K Uma Aryal 1 , Amanda M Minton 2
  1. Purdue University, West Lafayette, INDIANA, United States
  2. The Maschhoffs, Carlyle, Illinois, US

Modern (commercial or terminal) boars are selected for improved growth rate, feed efficiency and carcass characteristics. Performance traits are typically inversely related to fertility traits; hence the great variation in reproductive performance among boars. Seminal plasma proteins are essential for normal sperm function and transport. Proteomics analysis using two-dimensional (2D) gel electrophoresis support that seminal plasma protein profiles reflect differences in reproductive performance. However, the identification of potential fertility biomarkers has been limited by the lack of robustness of the 2D gel approach. The objective of this study was to use shotgun LC-MS/MS proteome analysis to investigate whether differences in boar fertility phenotype can be differentiated by seminal plasma protein expression. Following 50 single sire matings, commercial boars were categorized into one of four phenotypes: high farrowing rate and total born (HH; n=9), high farrowing rate with low total born (HL; n=10), low farrowing rate and total born (LL; n=9), and low farrowing rate with high total born (LH; n=4). Semen from the first acceptable ejaculate (>75% motile and normal) from boars in the stud was shipped overnight to Purdue University where seminal plasma was extracted and stored in -20 ºC until fertility data was available. Samples were thawed on ice, proteins precipitated with acetone, reconstituted with 8M urea, reduced, and digested with trypsin and Lys-C mix. Dried peptides were re-suspended in 3% acetonitrile and 0.1% formic acid and analyzed using gel-free, label-free shotgun LC-MS/MS in the Q Exactive Orbitrap HF mass spectrometer coupled with the Dionex UltiMate 3000 RSLC Nano System. Data were analyzed using MaxQuant software (v. against UniProt sus scrofa protein database. LFQ intensities were used for protein relative abundance measurement and downstream analysis.  There were 523 proteins measured in at least one sample across all animals (n=32). Among those, 318 were considered high confidence proteins, with 227 expressed commonly among the phenotype groups. Functional annotation analysis of commonly expressed proteins in DAVID 6.8 found enrichment of proteins in secretoryglycoprotein, glycan degradation, lysosome, adhesion proteins, and transferrin categories. There were 103 proteins at least 2-fold different in mean LFQ between LL and the HH group.  Among the 48 proteins 2-fold more abundant in LL versus HH were multiple proteins associated with inflammation including heat shock protein A2 (HSPA2), alpha-2-macroglobulin (A2M), S100 calcium binding protein A12(S100A12), and microseminoprotein beta(MSMB), with HSPA2 and A2M linked to infertility in humans. Lactotransferrin (LTF), defensin beta 1 (DFB1), complement factor I (CFI) and complement C3 (C3) were 2-fold or more abundant in HH versus LL group, all of which have antimicrobial functions. These findings support that seminal plasma protein profiles are distinct between boars with different fertility and presence of inflammatory biomarkers may be predictive of reproductive performance.