Full identification of SP-proteins remains challenging, particularly in some livestock species such as porcine. This experimental study aims to provide an extensive proteomic analysis of boar SP and to generate a public accessible database of boar SP-proteome. A SP-pool from 33 entire ejaculates from 11 boars (3 ejaculates per boar) was analyzed to characterize the boar SP-proteome. Moreover, 20 ejaculates collected in fractions (P1: first 10 mL of sperm rich ejaculate fraction (SRF), P2: rest of SRF and P3: post-SRF) from 5 boars (4 ejaculates per boar) were analyzed to evaluate differentially expressed SP-proteins among portions. SP-samples were subjected to a combination of SEC, 1-D SDS PAGE and NanoLC-ESI-MS/MS followed by functional bioinformatics analysis.The identified proteins were quantified from normalized LFQ intensity data. A total of 33,557 spectra corresponding to 8,189 peptides and 536 SP-proteins were identified with ≥ 95% Confidence (Unused Score > 1.3) and a false discovery rate (FDR) ≤ 1%. Of the 536 SP-proteins, 409 were identified in Sus Scrofa taxonomy and 374 of them were Biological Significance: This proteomic study provides the major characterization of the boar SP-proteome with more than 250 proteins first reported. The boar SP-proteome is described so that a spectral library can be built for relative 'label free' protein quantitation 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 identification of biomarkers for sperm quality and fertility.
A complete characterization of the proteome of seminal plasma (SP) is an essential step to understand how SP influences sperm function and fertility after artificial insemination (AI). The purpose of this study was to identify which among characterized proteins in boar SP were differently expressed among AI boars with significantly different fertility outcomes. A total of 872 SP proteins, 390 of them belonging specifically to Sus Scrofa taxonomy, were identified (Experiment 1) by using a novel proteomic approach that combined size exclusion chromatography and solid-phase extraction as prefractionation steps prior to Nano LC-ESI-MS/MS analysis. The SP proteomes of 26 boars showing significant differences in farrowing rate (n = 13) and litter size (n = 13) after the AI of 10 526 sows were further analyzed (Experiment 2). A total of 679 SP proteins were then quantified by the SWATH approach, where the penalized linear regression LASSO revealed differentially expressed SP proteins for farrowing rate (FURIN, AKR1B1, UBA1, PIN1, SPAM1, BLMH, SMPDL3A, KRT17, KRT10, TTC23, and AGT) and litter size (PN-1, THBS1, DSC1, and CAT). This study extended our knowledge of the SP proteome and revealed some SP proteins as potential biomarkers of fertility in AI boars.
Boar bulk ejaculates are now being collected instead of usual sperm-rich fractions (SRF) for artificial insemination purpose. The present study evaluated the influence of holding boar sperm samples before freezing surrounded in their own seminal plasma (SP), from either fractions/portions or the entire ejaculate, on post-thawing sperm quality and functionality. Ejaculates collected as bulk (BE) or as separate (first 10 mL of SRF [P1] and rest of SRF [P2]) from 10 boars were held 24h at 15-17°C and then frozen. Some bulk ejaculate samples were frozen immediately after collections as Control. In addition, epididymal sperm samples from the same 10 boars were collected post-mortem and extended in SP from P1 (EP1), P2 (EP2) and post SRF (EP3), and also held 24h before freezing for a better understanding of the influence of SP on boar sperm cryopreservation. The sperm quality (motility, evaluated by CASA, and viability, evaluated by flow cytometry) and functionality (flow cytometry assessment of plasma membrane fluidity, mitochondrial membrane potential and intracellular generation of reactive oxygen species [ROS] in viable sperm) were evaluated at 30, 150 and 300 min post-thaw. Post-thawing sperm quality and functionality of P1 and P2 were similar but higher (p < 0.01) than BE samples. Control samples showed higher (p < 0.01) post-thaw sperm quality and functionality than BE samples. Post-thawing sperm quality and functionality of EP1 and EP2 were similar but higher (p < 0.05) than EP3. These results showed that boar sperm from BE are more cryosensitive than those from the SRF, particularly when held 24h before freezing, which would be attributable to the cryonegative effects exerted by the SP from post SRF.
Seminal extracellular vesicles (EVs) include exosomes (ø 40–120 nm) and microvesicles (MVs, ø 120–1000 nm), which would be involved in multiple functional reproductive roles. The study aimed to establish which EV subtypes are present in pig semen, using a high-resolution flow cytometer to explore differences in their tetraspanin expression profile. The EVs were isolated from 12 pig ejaculates using serial ultracentrifugation and characterized by dynamic light scattering and electron microscopy for size and morphology as well as for tetraspanin expression using flow cytometry with Carboxyfluorescein succinimidyl ester (CFSE) and antibodies against CD9, CD63 and CD81. Pig semen contained a heterogeneous EV-population regarding size and morphology. Flow cytometric analysis demonstrated that the proportion of EVs expressing CD63 and CD9 was higher in MVs ( P < 0.001 and P < 0.05, respectively) than in exosomes, while the opposite was true for CD81; higher ( P < 0.001) in exosomes than in MVs. In conclusion, (1) the new generation of flow cytometers are able to accurately identify EVs and to gate them in two size-different populations named exosomes and MVs. (2) Tetraspanins CD9, CD63 and CD81 are present in both seminal EVs, albeit with exosomes and MVs differing in expression profiles, suggesting dissimilar cargo and binding affinity.
The study attempted to clarify the role of total antioxidant capacity of seminal plasma (SP-TAC) on boar sperm survival and fertility after artificial insemination (AI). SP-TAC differed (P < 0.001) among boars (n° = 15) and, to a lesser degree, among ejaculates within male (4 ejaculates/boar). SP-TAC also differed (P < 0.001) among ejaculate fractions (43 ejaculates and 3 fractions per ejaculate), of which the sperm-peak portion of the sperm rich ejaculate fraction (SRF) had the highest SP-TAC. SP-TAC was not correlated with sperm quality (motility and viability) or functionality (intracellular ROS generation and lipid peroxidation) of liquid AI-semen samples stored at 17 °C for 72 h (90 AI-samples), but the decline in sperm quality was larger (P < 0.05) in ejaculates with low, compared with high SP-TAC (hierarchically grouped). The SP-TAC differences among ejaculate portions agree with sperm cryosurvival rates (14 ejaculates from 7 boars), showing sperm from sperm-peak portion better (P < 0.01) post-thaw quality and functionality than those from the entire ejaculate (mainly post-SRF). Boars (n° = 18) with high SP-TAC (hierarchically grouped) had higher (P < 0.05) fertility outcomes (5,546 AI-sows) than those with low SP-TAC. Measurement of SP-TAC ought to be a discriminative tool to prognosis fertility in breeding boars.
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