The OF collection was performed as previously described by Kusama et al. (2018). Briefly, the oviductal lumen of the two oviducts from the same animal was flushed in the direction from the ampulla to the isthmus with 5 mL phosphate-buffered saline (PBS) at 4°C and using a catheter (24G BD Insyte™, 381212, Becton Dickinson Infusion Therapy Systems, Inc., Sandy, Utah, United States) adapted to a 10 mL syringe. The fluid collected after the oviduct flushing with PBS was processed to isolate EVs.

For the collection of UF, the uterus was irrigated in the direction from the caudal uterine horn to the utero-tubal junction with 10 mL PBS at 4°C using a 10 mL syringe. Immediately after collection, the flushing medium from both uterine horns was placed into Petri dishes and embryos were isolated from the obtained flushes under stereomicroscope to ensure pregnancy and were assessed for morphological quality and developmental stage classified as morula or blastocyst. The fluid collected after the uterus flushing with PBS was collected and processed to isolate EVs.

The presence and morphology of EVs were determined by Transmission Electron Microscopy (TEM). Samples were processed according to the protocol described by Théry et al. (2006) with the following modifications. A 10 µL aliquot of the vesicle suspension was placed on a Formvar-Carbon-Coated grid for 30 s at room temperature. The vesicle-coated grids were washed once with distilled water for 1 min and then stained with 10 µL of 2% uranyl acetate for 1 min for negative contrast. The samples were air-dried at room temperature for 20 min. Photographs were taken using a JEOL1011 electron microscope slide at 80 kV (Jeol, Japan). Digital images were taken at 59,000–97,000 magnification.

The size distribution of EVs was measured by Dynamic Light Scattering (DLS) according to Sabín et al. (2007) and using a Malvern Autosizer 4,800 (Malvern Instruments, Malvern, United Kingdom) equipped with a solid state He-Ne laser at a wavelength of 488 nm. The intensity of the scattered light was measured at 25°C. A 10-μL aliquot of the vesicle suspension was diluted up to 1 mL in PBS and transferred to a disposable solvent-resistant cuvette specific for the DLS analysis. Data were acquired and analyzed using the PCS software (version 1.61, Rev. 1, Malvern Instruments, Malvern, United Kingdom) in the automatic acquisition mode. We chose to use the intensity values as they best represent the data of the pure sample, as they assume the least error (criterion taken from Malvern Instruments INC). In addition, particle intensity classification provides more accurate data on the size of the EVs.

EVs quantitation was performed using the EXOCET Exosome Quantitation Kit (System Biosciences, SBI) which is an enzymatic, colorimetric assay designed as a direct measurement of esterase activity known to be within exosomes.

Fresh EVs pooled pellets in a concentrated solution (corresponding to a protein concentration of 1–2 μg/μL) were resuspended with kit lysis Buffer at (1:4, v:v) to a total volume of 100 μL per reaction. EVs lysates were incubated at 37°C for 5 min and centrifuged at 1,500 g for 5 min to remove cell debris. 50 μL of transferred supernatants and standards were added to clear microtiter wells and mixed with 50 μL of reaction buffer (buffer A + buffer B) up to a total 100 µL volume in a 96 well plate (Nunclon Delta, Thermo Scientific). Replicates of each sample were made fourfold, and the microtiter plate was incubated for 20 min at room temperature. Optical density was read using a spectrophotometric plate reader (Apollo 11 LB913, Berthold Technologies GmbH & Co., TN, United States) at 405 nm. Finally, quantitate results were obtained by calculating the standard curve, previously calibrated by Nano Sight analysis, and plotting the sample readings on the standard curve. Quantitative results were represented in number of particles per mL.

Protein extraction from 50 μL of the EVs suspension was carried out adding RIPA Lysis Buffer System (Santa Cruz Biotechnology Inc., Dallas, TX, United States) at 1:1 (v:v). The mixtures were pipetted up and down to resuspend the pellet for 5 min, incubated 30 min on ice, placed in an ice-cold sonication bath for 10 s at 20% amplitude and were centrifuged at 14,000 g for 5 min at 4°C to remove cell debris (Subedi et al., 2019). The supernatants were transferred to new tubes and the protein concentration was determined using the Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, United States).

Equal protein amount of each sample (40 µg) was mixed with reducing Laemmli-buffer and was resolved on 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (10% Mini-PROTEAN^® TGX™ Precast Protein Gels, Bio Rad^®) and transferred to a 0.45 µm polyvinylidene difluoride nitrocellulose (PVDF) membrane (Immobilon®-P Transfer Membranes). Membranes were washed with distilled water and unspecific unions were blocked with Tris buffered saline containing 0.1% Tween 20 (T-TBS; P-1379, Sigma-Aldrich^®, Madrid, Spain) supplemented with 5% skimmed milk for 1 h at room temperature. After that, membranes were incubated with the primary antibodies anti-HSP70 (Sigma Aldrich; Cat # H5147-2ML, 1:1.000) and anti-CD63 (Santa Cruz Biotechnology; Cat # sc-5275, 1:100) in blocking solution overnight at 4°C. After washing with fresh T-TBS, membranes were incubated for 1 h in goat anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (HRP) in blocking solution (Du et al., 2016). Positive immunoreactive bands were detected by an enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific, Waltham, MA, United States) and chemiluminescence was detected with Amersham™ Imager 600 (GE Healthcare) equipment.

The EVs suspensions were thawed at 4°C and subsequently subjected to digestion. This digestion process took place in a solution containing 100 µL of a buffer consisting of 50 mM ammonium bicarbonate with a pH of 8.5. To aid in the digestion, a small amount (0.01%, v:v) of ProteaseMax (Promega, WI, United States) was added. In addition, the samples were reduced by introducing 20 mM of dithiothreitol (DTT) and incubating the mixture for 20 min at 56°C. Following this, the samples were alkylated by adding 100 mM of iodoacetic acid (IAA) and allowing it to react for 30 min at room temperature while keeping the environment dark. Once the alkylation was completed, the digestion process was initiated by adding Trypsin Gold Proteomics Grade (Promega) to the mixture at a ratio of 1:100 (w/w). The digestion was carried out for a duration of 3 h at 37 °C. To halt the reaction, 0.1% formic acid was added, and the resulting mixture was filtered through a filter with a pore diameter of 0.2 µm. Finally, the samples were dried using an Eppendorf Vacuum Concentrator 5301.

The separation and analysis of the digested peptides from the samples were performed using an HPLC/MS system. This system consisted of an Agilent 1290 Infinity II Series HPLC (Agilent Technologies, Santa Clara, CA, United States) equipped with an Automated Multisampler module and a High-Speed Binary Pump. The HPLC system was connected to an Agilent 6550 Q-TOF Mass Spectrometer, and the interface used was the Agilent Jet Stream Dual electrospray (AJS-Dual ESI). The experimental parameters for both the HPLC and Q-TOF components were configured in the MassHunter Workstation Data Acquisition software (Agilent Technologies, Rev. B.08.00).

To prepare the dry samples resulting from trypsin digestion, they were reconstituted in a 20 µL volume of resuspending buffer. This buffer, known as buffer A, was composed of a mixture of water, acetonitrile, and formic acid in the ratio of 94.9:5:0.1, respectively. The reconstituted samples were injected into an Agilent AdvanceBio Peptide Mapping HPLC column with dimensions of 2.7 µm × 100 × 2.1 mm, which was thermostatted at 50°C. The injection of the samples occurred at a flow rate of 0.4 mL/min. Subsequently, the column was washed with buffer A for a duration of 3 min, and the digested peptides were eluted using a linear gradient of buffer B (acetonitrile:water:formic acid, 97:2.9:0.1). This gradient started at 0% and gradually increased to 40% over a period of 40 min. After that, there was a linear gradient from 40% to 95% of buffer B for 8 min, followed by a 95% concentration of buffer B for 3 min. Finally, the column was equilibrated in the initial conditions for 6 min before each subsequent injection. Ten µg of albumin were injected to check the purity of the samples.

The mass spectrometer was operated in the positive mode. The nebulizer gas pressure was set at 35 psi, while the drying gas flow rate was maintained at 14 L/min at a temperature of 300°C. The sheath gas flow rate was set at 11 L/min with a temperature of 250°C. The capillary spray, nozzle, fragmentor, and octopole RF Vpp voltages were configured at 3500 V, 100 V, 360 V, and 750 V, respectively. Profile data were collected for both MS and MS/MS scans using the extended dynamic range mode at a scan rate of 4 GHz. The mass range for both MS and MS/MS scans was set between 50–1700 m/z, and the scan rates were 8 spectra/sec for MS and 3 spectra/sec for MS/MS. Auto MS/MS mode was used, and precursor selection was based on abundance with a maximum of 20 precursors selected per cycle. A ramped collision energy was employed with a slope of 3.68 and an offset of −4.28. The exclusion of the same ion was applied after two consecutive spectra.

The data processing and analysis were conducted using the Spectrum Mill MS Proteomics Workbench software (Rev B.06.00.201, Agilent Technologies, Santa Clara, CA, United States). The raw data were extracted using default conditions, which included the identification of unmodified or carbamidomethylated cysteines, a range of [MH]+ 50–10,000 m/z, a maximum precursor charge of +5, and a minimum signal-to-noise MS (S/N) ratio of 25. The software also utilized specific parameters for peak finding, such as the identification of 12C signals.

For the MS/MS search, an appropriate and updated protein database was employed, and the search criteria included variable modifications (carbamidomethylated cysteines, STY phosphorylation, oxidized methionine, and N-terminal glutamine conversion to pyroglutamic acid), a tryptic digestion allowing for up to 5 missed cleavages, the use of an ESI-Q-TOF instrument, a minimum matched peak intensity of 50%, a maximum ambiguous precursor charge of +5, monoisotopic masses, a peptide precursor mass tolerance of 20 ppm, a product ion mass tolerance of 50 ppm, and the calculation of reversed database scores. The validation of peptide and protein data was performed using auto thresholds.

TEM observations confirmed the presence of EVs in porcine oviductal and uterine flushings (Figures 2A, B). The presence of two populations of EVs in the fluid was observed as two different peaks detected in sows from the four experimental groups, either in OF or UF (Figures 2C, D). Diverse populations of EVs ranging in size (142.40–297.76 nm) were observed. These EVs appeared as round or cup-shaped membrane-surrounded vesicles in their native stage. We observed a population of small EVs (30–150 nm) resembling exosomes and a population of large EVs (>150 nm) resembling microvesicles. Very large EVs (>1,000 nm) were considered as aggregates of multiple EVs.

The size of the oEVs and uEVs was analyzed and compared and there were no statistically significant differences between groups of sows (p > 0.05; Table 1). In general terms, oEVs tended to be larger (ranging from 215.52 to 297.76 nm) than uEVs (ranging from 142.840 to 202.92 nm); however, there were no differences in size between groups of sows (Table 1).

The immunoblotting revealed that EVs were present both in the OF and UF of all sows in study (Figure 2) since EVs protein markers HSP70 (2E) and CD63 (2F) were detected in AI- and C-sows.

The results of the quantitation of oEVs particles were similar for all experimental groups (Table 1), ranging from 278.36 to 414.79 × 10⁹ particles/mL (p > 0.05). However, the concentration of uEVs particles were lower (p < 0.05) in AI-F1 and AI-F2 (321.92 ± 8.05 and 395.14 ± 32.00 × 10⁹ particles/mL) than in C (687.28 ± 85.19 × 10⁹ particles/mL). AI-F3 showed an intermediate concentration of uEVs particles with respect to the rest of groups (525.14 ± 32.81 × 10⁹ particles/mL; p > 0.05).

The oEVs and uEVs protein concentration (protein cargo) was also compared in AI- and C-sows (Table 1). No statistical differences were found in the protein cargo of the total intact oEVs, ranging from 0.51 to 0.58 μg/μL (p > 0.05). In uEVs, the protein cargo of AI-F1 and AI-F2 (0.86 ± 0.06 and 0.86 ± 0.04 μg/μL) were significantly lower than AI-F3 and C (1.19 ± 0.09 and 1.30 ± 0.06 μg/μL).

Initially, the purity of the samples was confirmed since the samples for all the experimental groups only showed between 0.00% and 7.61% of the albumin’s spectra and between 0.00% and 0.66% of the albumin’s intensity. A total of 645 proteins were identified in oEVs and uEVs. To minimize the risk of false positives, only proteins with a minimum of two peptides and present in at least three of the five replicates were considered, resulting in 362 final proteins. The list of proteins identified in the different accumulative ejaculate fractions, including the relative number of peptides of each protein, is shown in Supplementary Tables S1–4 (Supplementary File S1) and Supplementary File S2.

Interestingly, a comparison of the oEVs and uEVs proteome of AI- and C-sows revealed four proteins common to all groups: 15S Mg (2⁺)-ATPase p97 subunit, annexin (ANXA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and tubulin alpha 1 chain (TUBA1C). These four proteins are involved in calcium signaling, tissue remodeling, immune modulation, and antioxidant defense systems. They exert biological functions that are regulated in the fallopian tube and endometrium. We decided to compare the oEVs and uEVs proteins from AI- and C-sows since it provides valuable insights into the molecular mechanisms involved in reproductive events and potentially influences the outcome of offspring development. The nature of the proteins and the possible differences are described below.

A total of 220 proteins were detected across all experimental groups. Figure 3 shows specific proteins from each experimental group and the common ones in two or more experimental groups. In addition, all proteins from oEVs are listed in Supplementary Tables S1, S2 (Supplementary File S1). A total of 139 proteins were detected in oEVs from AI-sows and 92 of them were detected in oEVs from AI-F1 group 31 of which were exclusive to this group. A total of 72 proteins were detected in oEVs from AI-F2 sows, 10 of which were exclusive to this group. Finally, 93 proteins were detected in oEVs from AI-F3 sows, 24 of which were exclusive to this group. In addition, the comparison between the three experimental groups identified 44 common proteins (Figure 3A). When C-sows were compared with AI-sows, 82 proteins were common to both groups. Of these, 57 proteins were exclusive to AI-sows and 81 proteins were exclusive to C-sows (Figure 3B).

The number and comparative overlap of the identified oEVs proteins in AI- and C-sows are illustrated in Figure 3C and listed in Supplementary Tables S1, S2 (Supplementary File S1). Eighty-one of the 220 proteins were exclusively present in C-sows: 14-3-3 protein theta (YWHAQ), cadherin 13 (CDH13), galectin-3 binding protein (LGALS3), and ubiquitin C (UBC). Twenty-three proteins were characteristically exclusive of AI-F1 sows: 14-3-3 protein zeta/delta (YWHAZ), 6 phosphogluconate dehydrogenase decarboxylating (PGD), peptidyl-prolyl trans isomerase E (PPIE), and tubulin alpha-1A chain (TUBA1A). In addition, 7 proteins were exclusive of AI-F2 sows: GTP-binding nuclear protein Ran (RAN), NIMA-related kinase 1 (NEK1), peroxiredoxin-2 (PRDX2), and syndecan-binding protein (SDCBP). Finally, 8 proteins were found exclusively in AI-F3 sows: Heat shock protein 70 kDa 1B (HSPA1B), haemoglobin subunit epsilon (HBE), profilin (PFN1), and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ).

GO includes three orthogonal ontologies: biological process (BP), molecular function (MF), and cellular component (CC), although we only focused on BP, which is more relevant in our study. Identification of enriched pathways using GO analysis revealed 45 pathways related to oEVs proteins. In Figure 4, we have presented the results of the GO analysis, focusing on the ten most enriched pathways in artificially inseminated sows and non-inseminated control sows. All the enriched pathways involved in biological processes are listed in Supplementary File S3. The top 5 biological processes in F1 group included pathways involved in protein stabilization, protein folding, glycolytic process, proteasome-mediated ubiquitin-dependent protein catabolic process and positive regulation of RNA polymerase II transcriptional preinitiation complex assembly. We also identified enriched pathways in the F2 group that are involved in glycolytic process, vesicle-mediated transport, protein stabilization, protein folding and regulation of cell shape. However, the most enriched pathways in the sows inseminated with F3 were translation, protein stabilization, innate immune response, glycolytic process and DNA-templated transcription and initiation.

A total of 142 proteins were identified in uEVs. Figure 5 illustrates the exclusive proteins found in each group, as well as the shared proteins among the different experimental groups. The detailed lists of exclusive and common proteins can be found in Supplementary Tables S3, S4 (Supplementary File S1), respectively.

A group of 112 proteins were detected in uEVs proteins from AI-sows. Ninety-three proteins were detected in AI-F1 sows, being 34 of them, exclusive to this group. In uEVs from AI-F2 sows, a total of 69 proteins were detected, being 6 of them, exclusive to this group. Fifty-three proteins were detected in uEVs from AI-F3 sows, being 7 of them exclusive to this group.

The analysis of uEVs detected 38 proteins present in the three groups of AI-sows. In addition, 19 proteins were common in AI-F1 and AI-F2 sows, two proteins between AI-F1 and AI-F3 sows, and six proteins between AI-F2 and AI-F3 sows (Figure 5A). Comparing AI- with C-sows (Figure 5B), 85 proteins were exclusive to AI-sows, 30 to C-sows, and 27 were shared by the two groups.

The number and comparative overlap of the identified oEVs proteins in AI- and C-sows are illustrated in Figure 5C and listed in Supplementary Tables S3, S4 (Supplementary File S1). Thirty of the 142 proteins were characteristically exclusive of C-sows and included beta-2-microglobulin (B2M), clathrin heavy chain (CLTC), heat shock protein family A member 8 (HSPA8), and vitronectin (VTN). Thirty-one proteins were characteristically exclusive of AI-F1 sows, including 14-3-3 domain-containing protein (YWHAG), calmodulin-3 (CALM3), ezrin (EZR), and heat shock protein HSP90-alpha (HSP90AA1). In addition, six proteins were exclusive to AI-F2 sows: 14-3-3 protein zeta/delta (YWHAZ), adenylyl cyclase-associated protein (CAP1), heterogeneous nuclear ribonucleoprotein K (HNRNPK), and Na⁺-dependent phosphate cotransporter 2B (NPT2B). Finally, six proteins were found exclusively in AI-F3 sows: ATP-binding cassette subfamily G member 2 (ABCG2), pendrin (SLC26A4), proline-rich transmembrane protein 1B (PRRT1B), and STEAP family member 4 (STEAP4).

Identification of enriched pathways using GO analysis revealed 62 pathways in uEVs proteins. In Figure 6, we have presented the results of the GO analysis, focusing on the ten most enriched pathways in artificially inseminated sows and non-inseminated control sows. All the enriched pathways involved in biological processes are listed in (Supplementary File S4). The top 5 biological processes in F1 included protein stabilization, protein folding, glycolytic process, regulation of cell shape and protein localization to plasma membrane. The enriched pathways in F2 were also related to regulation of cell shape, protein folding, actin filament organization, protein stabilization and small GTPase mediated signal transduction. We also identified enriched pathways in the F3 group that are involved in regulation of cell shape, glycolytic process, actin filament organization, actin cytoskeleton organization and sensory perception of sound.