Integrating Near-Infrared Spectroscopy and Proteomics for Semen Quality Biosensing

Abstract

Artificial insemination (AI) is a key breeding technique in the swine industry; however, the lack of reliable biomarkers for semen quality limits its effectiveness. Seminal plasma (SP) contains extracellular vesicles (EVs) that present a promising, non-invasive biomarker for semen quality. This study explores the biochemical profiles of boar SP to assess semen quality through near-infrared spectroscopy (NIRS) and proteomics of SP-EVs. Fresh semen from mature Duroc boars was evaluated based on sperm motility, classifying samples as Passed (≥70%) or Failed (<70%). NIRS analysis identified distinct variations in water structures at specific wavelengths (C1, C5, C12 nm), achieving high accuracy (92.2%), sensitivity (94.2%), and specificity (90.3%) through PCA-LDA. Proteomic analysis of SP-EVs revealed 218 proteins in Passed and 238 in Failed samples. Nexin-1 and seminal plasma protein pB1 were upregulated in Passed samples, while LGALS3BP was downregulated. The functional analysis highlighted pathways associated with single fertilization, filament organization, and glutathione metabolism in Passed samples. Integrating NIRS with SP-EV proteomics provides a robust approach to non-invasive assessment of semen quality. These findings suggest that SP-EVs could serve as effective biosensors for rapid semen quality assessment, enabling better boar semen selection and enhancing AI practices in swine breeding.

Keywords: NIRS; biomarkers; biosensing; extracellular vesicles; male fertility; pig; proteomics; semen quality; seminal plasma.

Figure 1

NIR absorbance and aquagram of boar seminal plasma (Passed vs. Failed): (A) PCA scores plot for Passed (n = 33) and Failed semen (n = 31); (B) PCA loadings showing the dominant peaks influencing the positive and negative trends in the scores plot: PC-1 = 74%, PC-2 = 15%; (C) Normalized absorbance of porcine seminal plasma; (D) Aquagram created with the key WABS from peaks between Passed and Failed SP.

Figure 2

Aquaphotomics of boar seminal plasma (Passed vs. Failed): (A) WAMACS barcode showing chemical shifts in Failed (red) and Passed (dark blue) groups compared to distilled water as the reference; (B) PCA-LDA plot for the calibration of Model 4 developed with the transformed absorbance (1300–1600 nm) from boar SP of Passed and Failed semen.

Figure 3

Morphological and molecular characterization of boar SP-EVs: (A) nanoparticle tracking analysis of Passed (blue bar) and Failed (red bar) SP-EVs particle size and concentration; (B) High-Resolution Transmission Electron Microscopy (HR-TEM) image showing a clear morphology of SP-EVs. The scale bar represents 400 nm.

Figure 4

Sperm co-incubation with labeled SP-EVs: (A) labeled SP-EVs with spermatozoa after centrifugation and the resultant sperm pellet (S1: Supernatant 1; S2: Supernatant 2; S3: Supernatant 3 and SP: Sperm Pellet); (B) labeled SP-EVs with spermatozoa (C: Control; P1: Failed SP-EV 1; P2: Failed SP-EV 2; G1: Passed SP-EV 1 and G2: Passed SP-EV 2); (C) confocal microscope imaging of spermatozoa taken two hours after co-incubation with labeled SP-EVs (green staining); (D) the sperm nuclei were counterstained with DAPI (blue staining)—The insert indicates control spermatozoa showing no sign of green fluorescence after incubation with or without unlabeled SP-EVs. The scale bar represents 10 μm.

Figure 5

Immunodetection of LGALS3BP in boar spermatozoa. (A) Western blotting indicated LGALS3BP protein detection in spermatozoa of Failed semen. Performing immunofluorescence, the negative control corresponded to spermatozoa labeled with a FITC-conjugated antibody in the absence of the anti-LGALS3BP (B). Immunofluorescence of LGALS3BP was detected in spermatozoa (FITC green staining—micrograph (C). Sperm nuclei are counterstained with DAPI (blue staining—micrograph (D)).

Figure 6

Protein–to–protein interactions (PPI) of differentially expressed SP-EV proteins: (A) Lectin (LGALS3BP); (B) Nexin-1 (PN-1); (C) seminal plasma protein pB1 (BSP1). The proteins are represented by nodes, and different-colored lines connect interacting partners.