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Abstract Seminal plasma (SP) is rich in extracellular vesicles (EVs), which are still poorly studied, especially in livestock species. To better understand their functional role in both spermatozoa and endometrial epithelial cells, proper characterization of EVs is an essential step. The objective was to phenotypically characterize porcine seminal EVs (sEVs) using cryogenic electron microscopy (cryo-EM), which allows visualization of EVs in their native state. Porcine ejaculates are released in fractions, each containing SP from different source. This allows characterization sEVs released from various male reproductive tissues. Two experiments were performed, the first with SP from the entire ejaculate (n:6) and the second with SP from three ejaculate fractions (n:15): the first 10 mL of the sperm-rich ejaculate fraction (SRF-P1) with SP mainly from the epididymis, the remainder of the SRF (SRF-P2) with SP mainly from the prostate, and the post-SRF with SP mainly from the seminal vesicles. The sEVs were isolated by size exclusion chromatography and 1840 cryo-EM sEV images were acquired using a Jeol-JEM-2200FS/CR-EM. The size, electron density, complexity, and peripheral corona layer were measured in each sEV using the ImageJ software. The first experiment showed that sEVs were structurally and morphologically heterogeneous, although most (83.1%) were small (less than 200 nm), rounded, and poorly electrodense, and some have a peripheral coronal layer. There were also larger sEVs (16.9%) that were irregularly shaped, more electrodense, and few with a peripheral coronal layer. The second experiment showed that small sEVs were more common in SRF-P1 and SRF-P2, indicating that they originated mainly from the epididymis and prostate. Large sEVs were more abundant in post-SRF, indicating that they originated mainly from seminal vesicles. Porcine sEVs are structurally and morphologically heterogeneous. This would be explained by the diversity of reproductive organs of origin.

Abstract Background Porcine seminal plasma (SP) is endowed with a heterogeneous population of extracellular vesicles (sEVs). This study evaluated the immunophenotypic profile by high-sensitivity flow cytometry of eight sEV subpopulations isolated according to their size (small [S-sEVs] and large [L-sEVs]) from four different SP sources, namely three ejaculate fractions (the first 10 mL of the sperm rich fraction [SRF-P1], the remaining SRF [SRF-P2], and the post-SRF [PSRF]) and entire ejaculate (EE). Methods Seminal EVs were isolated using a size exclusion chromatography-based protocol from six SP pools (five ejaculates/pool) of each SP source and characterized using complementary approaches including total protein (BCA™assay), particle size distribution (dynamic light scattering), morphology (transmission electron microscopy), and purity (albumin by Western blot). Expression of CD9, CD63, CD81, CD44 and HSP90β was analyzed in all sEV subpopulations by high-sensitivity flow cytometry according to MIFlowCyt-EV guidelines, including an accurate calibration, controls, and discrimination by CFSE-labelling. Results Each sEV subpopulation exhibited a specific immunophenotypic profile. The percentage of sEVs positive for CD9, CD63, CD81 and HSP90β differed between S- and L-sEVs (P

Seminal plasma contains a heterogeneous population of extracellular vesicles (sEVs) that remains poorly characterized. This study aimed to characterize the lipidomic profile of two subsets of differently sized sEVs, small (S-) and large (L-), isolated from porcine seminal plasma by size-exclusion chromatography and characterized by an orthogonal approach. High-performance liquid chromatography–high-resolution mass spectrometry was used for lipidomic analysis. A total of 157 lipid species from 14 lipid classes of 4 major categories (sphingolipids, glycerophospholipids, glycerolipids, and sterols) were identified. Qualitative differences were limited to two cholesteryl ester species present only in S-sEVs. L-sEVs had higher levels of all quantified lipid classes due to their larger membrane surface area. The distribution pattern was different, especially for sphingomyelins (more in S-sEVs) and ceramides (more in L-sEVs). In conclusion, this study reveals differences in the lipidomic profile of two subsets of porcine sEVs, suggesting that they differ in biogenesis and functionality.

The present work approaches the level of the tax culture of the merchants of the municipal market of the city of San Lorenzo. One way to strengthen the tax culture (understood as the set of values, beliefs and attitudes shared by a society with respect to taxation and the laws that govern it, which leads to permanent compliance with tax duties), is that people get knowledge about the issues and understand the importance of your tax responsibilities. Through the investigation, it was possible to detect the lack of knowledge regarding the taxes that merchants must pay in the municipal market, which drives to look for mechanisms so that they can access a constant training and by this formalize the activities they carry out. With the results obtained, it is evident the need for the Tax Administration to promote the Tax Education of the merchants through educational talks to inform and strengthen the knowledge that strengthens the Tax Culture and is gradually formalized. The central axes for the promotion of the tax culture are Information, Training and Awareness, so that mechanisms can be created to formalize economic activities and create a tax culture for municipal market merchants. The research in its inception was exploratory, resorting to documentary bibliographic modality. The results obtained show or prove the lack of knowledge about the taxes that merchants must pay in the municipal market of San Lorenzo.