Secretory extracellular vesicles (EVs) are membrane-enclosed microparticles that mediate cell to cell communication in proximity to, or distant from, the cell of origin. Cells release a heterogeneous spectrum of EVs depending on their physiologic and metabolic state. Extracellular vesicles are generally classified as either exosomes or microvesicles depending on their size and biogenesis. Extracellular vesicles mediate temporal and spatial interaction during many events in sexual reproduction and supporting embryo-maternal dialogue. Although many omic technologies provide detailed understanding of the molecular cargo of EVs, the difficulty in obtaining populations of homogeneous EVs makes difficult to interpret the molecular profile of the molecules derived from a miscellaneous EV population. Notwithstanding, molecular characterization of EVs isolated in physiological and pathological conditions may increase our understanding of reproductive and obstetric diseases and assist the search for potential non-invasive biomarkers. Moreover, a more precise vision of the cocktail of biomolecules inside the EVs mediating communication between the embryo and mother could provide new insights to optimize the therapeutic action and safety of EV use.

Persistent post-breeding induced endometritis (PPBIE) is considered a major cause of subfertility in mares. It consists of persistent or delayed uterine inflammation in susceptible mares. There are many options for the treatment of PPBIE, but in this study, a novel approach aimed at preventing the onset of PPBIE was investigated. Stallion semen was supplemented with extracellular vesicles derived from amniotic mesenchymal stromal cells (AMSC-EVs) at the time of insemination to prevent or limit the development of PPBIE. Before use in mares, a dose–response curve was produced to evaluate the effect of AMSC-EVs on spermatozoa, and an optimal concentration of 400 × 10⁶ EVs with 10 × 10⁶ spermatozoa/mL was identified. At this concentration, sperm mobility parameters were not negatively affected. Sixteen susceptible mares were enrolled and inseminated with semen (n = 8; control group) or with semen supplemented with EVs (n = 8; EV group). The supplementation of AMSC-EVs to semen resulted in a reduction in polymorphonuclear neutrophil (PMN) infiltration as well as intrauterine fluid accumulation (IUF; p < 0.05). There was a significant reduction in intrauterine cytokine levels (p < 0.05) for TNF-α and IL-6 and an increase in anti-inflammatory IL-10 in mares in the EV group, suggesting successful modulation of the post-insemination inflammatory response. This procedure may be useful for mares susceptible to PPBIE.

Background: During epididymal transit spermatozoa acquire specific morphological features which enhance their ability to swim in a progressive manner and interact with the oocytes. At the same time, sperm cells undergo specific molecular rearrangements essential for the fertilizing sperm to drive a correct embryo development. To assess epigenetic sperm changes during epididymal maturation, the caput, corpus and cauda epididymis sperm tracts were isolated from eight bulls and characterized for different sperm quality parameters and for CpG DNA methylation using Reduced Representation Bisulfite Sequencing (RRBS) able to identify differentially methylated regions (DMRs) in higher CpG density regions. Results: Caput sperm showed significant variation in motility and sperm kinetics variables, whereas spermatozoa collected from the corpus presented morphology variation and significant alterations in variables related to acrosome integrity. A total of 57,583 methylated regions were identified across the eight bulls, showing a significantly diverse distribution for sperm collected in the three epididymal regions. Differential methylation was observed between caput vs corpus (n = 11,434), corpus vs cauda (n = 12,372) and caput vs cauda (n = 2790). During epididymal transit a high proportion of the epigenome was remodeled, showing several regions in which methylation decreases from caput to corpus and increases from corpus to cauda. Conclusions: Specific CpG DNA methylation changes in sperm isolated from the caput, corpus, and cauda epididymis tracts are likely to refine the sperm epigenome during sperm maturation, potentially impacting sperm fertilization ability and spatial organization of the genome during early embryo development.

Introduction: Semen cryopreservation is the most popular practice for semen production for artificial insemination and in vitro fertilization in cattle. The Seminal plasma contains extracellular vesicles (spEVs) which modulate sperm viability and function during oocyte fecundation. The study of spEVs in frozen-thawed semen doses may yield novel indicators for predicting bull fertility, but the presence of the semen extender may hinder molecular profiling of spEVs. The aim of this study was to provide extensive characterization of EVs isolated from seminal plasma before and after the cryopreservation process and the addition of a commercial animal protein-free semen extender to understand the potential influence of EVs originating from the extender in hindering the use of spEVs derived biomarkers for assessment of bull fertility.Methods: EVs were isolated from the seminal plasma (with or without the extender), from the cryopreserved straw devoid of spermatozoa, and from the extender using two different methods, ultracentrifugation (UC) and size exclusion chromatography (SEC), and characterized for their structure and composition.Results: Physical characterization of EVs showed that size and particle numbers were related to the method of isolation. spEVs were larger but less abundant (UC: 168.9 nm, n = 2.68 × 10⁹; SEC: 197.0 nm, n = 6.42 × 10⁹) compared to extender EVs (UC: 129.0 nm, n = 2.68 × 10¹¹; SEC: 161.8 nm, n = 6.47 × 10¹¹). Western blotting analysis (WB) confirmed the presence of typical EV markers in spEVS: the membrane bound CD9 (25 kDa) and the luminal markers Alix (96 kDa) and TSG101 (48 KDa). Although Transmission Electron Microscopy confirmed the presence of a lipid bilayer structure in all preparations, no specific EV markers were detected in the vesicles isolated from extender when the Single Molecule Array (SiMoa) was used. A total of 724 Bos taurus miRNAs were identified in at least one preparation. The percentage of miRNAs identified in EVs from the extender (0.05%−0.49% of the total reads) was lower than in the preparation containing spEVs (10.56%–63.69% of the total reads). Edge-R identified a total of 111 DE-miRNAs between EVs isolated from the extender by two methods. Among them, 11 DE-miRNAs (bta-miR-11980, bta-miR-11987, bta-miR-12057, bta-miR-1246, bta-miR-125b, bta-miR-181b, bta-miR-2340, bta-miR-2358, bta-miR-2478, bta-miR-2898, and bta-miR-345-3p) were also abundant in EVs isolated from seminal plasma preparations with extender.Conclusion: This study clearly demonstrates that the presence of the extender does not prevent the characterization of spEVs in cryopreserved semen. However, the molecular profiling of spEVs can be influenced by the isolation method used and by the presence of some miRNAs from the extender. Therefore, in such studies, it is advisable to characterize both spEVs and the vesicles isolated from the extender.

Beer and its components show potential for reducing hepatic steatosis in rodent models through multiple mechanisms. This study aimed to evaluate beer’s anti-steatotic effects in a high-fat diet (HFD)-induced mouse model of Metabolic dysfunction-Associated Liver Disease (MASLD) and to explore the underlying mechanisms. In the HFD group, steatosis was confirmed by altered blood parameters, weight gain, elevated liver lipid content, and histological changes. These markers were normalized in the HFD+beer group, reaching levels similar to the control (CTR) group. Protein carbonylation and lipid peroxidation levels were consistent across all groups, suggesting that the model represents an early stage of MASLD without oxidative stress. Transcriptomic and CpG methylation analyses revealed clear distinctions between the CTR and HFD groups. RNA sequencing identified 162 differentially expressed genes (DEGs) between the CTR and HFD groups, primarily related to inflammation and lipid regulation. Beer consumption modified the health of the HFD mice, affecting inflammation but not lipid homeostasis (CTR vs. HFD+beer, DEGs = 43). The CpG methylation analysis indicated that beer lowered methylation, impacting genes linked to lipid accumulation and inflammation. A cecal metabolite analysis suggested that beer improved short-chain fatty acid metabolism (SCFA). In summary, a moderate beer intake may mitigate MASLD by modulating lipid metabolism and SCFA pathways, likely through polyphenol activity.

Introduction: Breed epigenetic diversity was recently detected in pig muscle and cattle blood, probably as a result of long-term selection for morphological adaptive and quantitative traits, persisting after embryo epigenetic reprogramming.Methods: In our study, breed epigenetic diversity in the male germline from Holstein (H) and Montbéliarde (M) bulls was investigated using Reduced Representation Bisulfite Sequencing (RRBS) data publicly available at the NCBI database. Open-source Whole Genome Sequencing (WGS) data from H and M animals were used to estimate genetic diversity between the two breeds and, thus, correctly assess CpG positions with low frequencies or absence of SNPs.Results: Sperm epigenetic diversity was studied in 356,635 SNP-free CpG positions, and a total of 6,074 differentially methylated cytosines (DMCs) were identified. The analysis of the DMCs pattern of distribution revealed that DMCs: i) are partially associated with genetic variation, ii) are consistent with epigenetic diversity previously observed in bovine blood, iii) present long-CpG stretches in specific genomic regions, and iv) are enriched in specific repeat elements, such as ERV-LTR transposable elements, ribosomal 5S rRNA, BTSAT4 Satellites and long interspersed nuclear elements (LINE).Discussion: This study, based on publicly available data from two cattle breeds, contributes to the identification and definition of distinct epigenetic signatures in sperm, that may have potential implications for mammalian embryo development.