147 Profiling boar semen quality through near-infrared spectroscopy and proteomic tools

Artificial insemination (AI) is the leading reproductive tool used in the swine industry to introduce and maintain superior genes; however, the AI breeding method is still facing challenges due to the lack of reliable predictors for semen quality. Seminal plasma (SP), a protective biofluid for spermatozoa, may constitute a great reservoir for possible detection of noninvasive predictors (e.g. proteins). Various extrinsic and intrinsic factors affect sperm characteristics (motility and morphology), which may cause peculiarities in corresponding seminal plasma. Near-infrared spectroscopy (NIRS) is a nondestructive tool capable of analysing biochemical changes in biofluids. It could be used to predict semen quality effortlessly and rapidly. Here, we tested the NIRS tool on SP derived from phenotypically different boar semen and conducted proteomic analyses for additional molecular characterisation. Single fresh semen was harvested from 75 commercial Duroc boars (Prestage Farms). Semen samples (n = 75) were classified as Passed (>70%) or Failed (<70%) based on their motility and normal morphology (≥70%) cut-off criteria. Individual samples were centrifuged to isolate SP and stored at −80°C until analysis. NIRS absorbance (750–2500 nm) was measured using ASD FieldSpec^®3 portable spectrometer/1 mm quartz cuvettes. Multivariate analysis of NIRS spectra was conducted using Unscrambler^®X v. 10.5 (CAMO Analytics), and both principal component analysis (PCA) and linear discriminant analysis (PCA-LDA) were used in the transformed spectra containing the water information (1300–1600 nm), followed by nanoLC-MS/MS proteomic analyses. The threshold of significance was set at P < 0.05. Failed and Passed SP showed different NIRS aquaphotomics spectrum profiles. Differences were observed in the C1, C5, and C12 (nm) water bands, revealing high water molecule associations with kosmotropic, bulk water, and chaotropic solutes in water asymmetrical stretching vibrations (ν3) and water dimers (S0, S1). The PCA-LDA analysis revealed high accuracy (92.2%), sensitivity (94.2%), and specificity (90.3%) in the calibration process, which led to the identification of three sub-groups of SP samples with Shared, Passed-specific, or Failed-specific chemicals. Comparative proteomic analyses of specific sub-groups showed significant differentially expressed proteins associated with single fertilisation (e.g. β-hexosaminidase, PSP1). In addition, subsets of proteins appeared unique to each sub-group, and those belonging to the Passed-specific were associated with sperm maturation, capacitation, and resilience to storage (e.g. Ezrin, Lipocalin). In conclusion, the use of near-infrared spectroscopy in combination with aquaphotomics identified chemical differences between Passed and Failed boar semen, which were further confirmed through proteomic analyses. Ongoing studies are investigating the nature of such proteins to serve as potential biomarkers of boar semen quality.

Research was supported by the USDA-ARS project #6066-31000-015-00D.