Comparative profiling of extracellular vesicles and miRNA cargo from in vivo- and in vitro-derived bovine embryos during blastulation and hatching

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Gutiérrez-Reinoso, Ioanna Martinez-Hormaza, Yat S Wong, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8248877/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Apr, 2026 Read the published version in Journal of Animal Science and Biotechnology → Version 1 posted 9 You are reading this latest preprint version Abstract Background: Extracellular vesicles (EVs) play a central role in embryo–maternal communication, transporting bioactive molecules that reflect the embryo's physiological state and developmental competence. This study examined how embryonic origin and developmental stage shape the characteristics and molecular content of EVs secreted by bovine embryos. Results: IVV embryos exhibited higher developmental competence and secreted larger EVs whose concentrations remained stable across developmental windows. In contrast, IVP embryos released smaller and more abundant vesicles, particularly during hatching, indicating origin- and stage-specific regulation of EV output. Distinct miRNA profiles clearly separated both embryo types. EVs from IVV embryos were enriched in miRNAs associated with implantation and lineage specification (e.g., miR-124, miR-125, miR-181), whereas EVs from IVP embryos contained higher levels of miRNAs linked to stress response, apoptosis, and the unfolded protein response (e.g., miR-23b, miR-92a, miR-409). Consistently, functional enrichment analyses revealed that IVV-derived miRNAs targeted pathways related to immune modulation and purinergic signaling, while IVP-derived miRNAs were associated with calcium transport and endoplasmic reticulum stress pathways. Together, these differences point to divergent regulatory programs shaped simultaneously by embryonic origin and developmental progression. Conclusions: Embryonic origin and developmental stage modulate both the biophysical properties and molecular signatures of embryo-secreted EVs. The distinct miRNA landscapes identified in IVV and IVP embryos reflect contrasting developmental trajectories and support the potential of EV-derived miRNAs as non-invasive biomarkers of embryo quality and developmental competence. bovine embryo extracellular vesicles microRNA in vitro fertilization blastulation hatching embryo–maternal communication Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Assisted reproductive technologies (ART) are crucial to the bovine industry because they accelerate the propagation of genetically superior livestock. Recently, a shift has occurred from in vivo-produced (IVV) to in vitro-produced (IVP) embryos as the preferred strategy for maximizing genetic gains in cattle (1). Despite notable advances in IVP protocols and the optimization of culture media to enhance gamete and embryo competence, the cryotolerance and post-transfer pregnancy rates of IVP embryos remain lower than those of IVV embryos (2, 3). The in vitro environment exerts a profound influence on early embryonic development, rendering them less competent than their in vivo counterparts. Morphological selection of blastocysts often fails to predict implantation potential, despite its widespread adoption for its simplicity and non-invasiveness. Even blastocysts considered of the highest quality (Grade 1) according to IETS grading (4) may exhibit compromised implantation capacity (5). Most pregnancy losses occur during the peri-implantation period (6-8). The in vitro environment impacts epigenetic and gene expression patterns, effects that may not be evident at the blastocyst stage but manifest later during embryo-maternal recognition processes (9-11), due to the compromised signaling required for successful maternal recognition (12, 13). In bovines, interferon tau (IFN-τ) secreted by the embryo is the primary signal for maternal recognition of pregnancy. Despite the critical role of IFN-τ in pregnancy recognition, there is evidence that earlier signals secreted by the embryo may also prepare the maternal side for implantation (14, 15). Pre-implantation embryos release extracellular vesicles (EVs), which are internalized by endometrial cells and participate in early embryo-maternal communication (16, 17). EVs are nanometer-sized particles surrounded by a lipid bilayer and are naturally secreted into the extracellular environment by various cell types, including embryonic cells (18, 19). During their biogenesis, EVs are loaded with diverse molecules, including proteins, lipids, RNAs, and DNA, initially described as mechanisms for cellular waste disposal (20). However, EVs are now recognized as key mediators of intercellular communication, mediating the transfer of bioactive molecules to recipient cells through their molecular cargo (21, 22). EVs have been linked to several physiological processes, including gamete and embryo development. Pre-implantation embryos from various species, including humans, pigs, mice, and cattle, secrete EVs (23-25). The characteristics (size, concentration, and molecular cargo) of EV populations vary according to the origin, developmental stage, and quality of the embryo (26). The differential molecular composition of embryonic EVs reflects the embryo's developmental competence and may modulate embryo-maternal communication. Although the number of studies is still limited, emerging evidence suggests that embryo-maternal communication mediated by EVs may differ depending on whether the embryo was produced in vitro or in vivo (17, 27-29). However, our understanding of how EVs derived from IVV or IVP produced embryos influence the establishment of a receptive maternal environment and proper implantation remains scarce. This study aimed to characterize EV populations derived from in vivo- and in vitro-produced bovine embryos at distinct developmental stages to elucidate their role in embryo–maternal communication. It was hypothesized that both the origin and developmental stage exert significant influences on population characteristics and miRNA content, potentially affecting molecular pathways involved in embryo-maternal communication and implantation. Understanding these differences was anticipated to offer novel insights into the mechanisms underlying the diminished success rates observed in in vitro-produced embryos and to inform the development of enhanced strategies for embryo selection and culture. MATERIALS AND METHODS All experiments were conducted at the University of Concepción, Campus Chillán, except for transmission electron microscopy, which was carried out at the Advanced Microscopy Facility of the Pontificia Universidad Católica de Chile. Experimental Design Two experiments were conducted to characterize EV populations during specific windows of early embryonic development to elucidate how the characteristics of EVs secreted by bovine embryos are influenced by their origin (IVV or IVP) and developmental stage (blastulation and hatching) (Figure 1). For both experiments, embryos were produced through in vivo and in vitro procedures. In Experiment 1, corresponding to the blastulation stage (Days 5–7), a total of 30 in vivo (IVV) and 60 in vitro (IVP) embryos were obtained. In Experiment 2, which focused on the hatching stage (Days 7–9), 30 IVV and 30 IVP embryos were produced. Experiment 1: Evaluation of Characteristics and miRNA Profiles of EVs Secreted During Blastulation in In Vitro and In Vivo Bovine Embryos. IVV 5-7 Group: Morulae obtained via superovulation and artificial insemination. IVP 5-7 Group: Morulae generated through in vitro fertilization. Experiment 2: Evaluation of Characteristics and miRNA Profiles of EVs Secreted During Hatching in In Vitro and In Vivo Bovine Embryos. IVV 7-9 group: Blastocysts obtained via superovulation and artificial insemination. IVP 7-9 group: Blastocysts generated through in vitro fertilization. In both experiments, the culture media of the blastocysts on Day-7 and Day-9 were collected and stored at -80°C for subsequent EV and content analysis. EV characterization included size and concentration assessment via nanoparticle tracking analysis (NTA), as well as surface marker detection (CD9, CD40, CD63, CD81) using cytometry and transmission electron microscopy (TEM). Additionally, miRNA profiles within the EVs were evaluated through sequencing. All blastocysts were transferred to a new drop of EV-depleted SOF medium and maintained in individual culture until day 11 to assess their post-hatching developmental capacity. Only conditioned media from these competent embryos were used for the analysis. The IETS classification system was applied at each stage of embryo development. Only embryos graded 1-2 on days 5 and 9 of development were considered for further study. Morphological evaluation was performed under blind conditions, in accordance with IETS guidelines. After classification, embryos were randomly and blindly assigned to the corresponding experimental groups. Subsequently, embryos that showed linear growth and a diameter greater than 270 µm on day 11 were considered competent (24). Bovine Embryo In Vitro Production Cumulus-oocyte complexes (COCs) were obtained from ovaries collected from slaughtered beef cattle at a local abattoir (Frigosur, Chillán, Chile). The collection, maturation, fertilization, and culture procedures followed protocols described by (25, 30). COCs were aspirated from antral follicles (3–6 mm diameter) using a 19-G needle. COCs were first transferred to manipulation medium (TCM199 supplemented with 4 mM bicarbonate, 18 mM HEPES, 10% fetal bovine serum (FBS), and 50 mg/mL gentamicin), pre-warmed to 38°C, for washing and morphological assessment. Selected COCs were matured in groups in 500 μL of TCM199-based medium supplemented with 0.6 mM glutamine, 0.2 mM pyruvate, 0.01 IU/mL FSH and LH, 1 μg/mL estradiol, 50 μg/mL gentamicin, 10 ng/mL epidermal growth factor (EGF), and 10% FBS. Cultures were incubated at 38°C in a humidified atmosphere of 5% CO₂ for 20-22 hours. Spermatozoa were obtained from commercial semen (Semex, Madison, WI, USA) and separated using a Percoll gradient method. Each matured oocyte was inseminated with 10,000 motile spermatozoa in 500 μL of fertilization medium (TALP-FIV) supplemented with 0.01 mg/mL heparin, 2 mM pyruvate, 50 mg/mL gentamicin, and 6 mg/mL fatty acid-free serum albumin (BSA). Fertilization was carried out at 39°C under 5% CO₂. Eighteen hours after fertilization, presumptive zygotes were mechanically denuded of cumulus cells by vortexing for 3 minutes in TCM199 medium containing HEPES and 0.3 mg/mL hyaluronidase. Zygotes were then cultured in groups of 25–30 in 500 μL of synthetic oviduct fluid (SOF) medium supplemented with 0.37 mM trisodium citrate, 2.77 mM myo-inositol, 10 ng/mL EGF, 2% FBS, and 3 mg/mL fatty acid-free BSA. For Experiment 1, on day 5 of development, Grade 1 morula-stage embryos were selected and cultured individually in 96-well plates containing 80 µl of EV-depleted SOF culture medium. Incubation was maintained until day 7 of development, after which the embryos were evaluated and classified based on morphological quality, and the culture medium was collected. In Experiment 2, on day 7, the embryos were classified. Grade I blastocysts were then selected and cultured individually in 96-well plates with 80 µl of EV-depleted SOF culture medium until day 9 of development. On day 9, the embryos were re-evaluated, classified by morphological quality, and the culture medium was collected for further analysis. In both cases, the blastocysts were transferred to fresh SOF medium for continued monitoring until day 11 of development. Incubation was carried out at 38.5°C under a gas mixture of 5% CO₂, 5% O₂, and 90% N₂, with a humidity of 85%. Bovine In Vivo Embryo Production The donor females underwent follicular population assessment via ultrasonography (Sonoscape X3V with a 5 MHz sector transducer). To synchronize the emergence of the follicular wave among the donors, a 1.38 g intravaginal progesterone device (CIDR®; Zoetis) combined with 2.5 mg of estradiol benzoate (Zoetis) administered intramuscularly and 50 mg of progesterone administered intramuscularly (31, 32). Hormonal stimulation for superovulation (SOV) began on Day 4 and continued until Day 7. Each donor in the control group received a total of 250 mg of NIH-FSH-P1 (Folltropin-V; Bioniche Animal Health, Belleville, Ontario, Canada), divided into eight intramuscular injections with decreasing doses from 50 mg to 10 mg, administered every 12 hours over 4 days. On Day 6, two doses of prostaglandin F2α (cloprostenol, Ciclase®, Zoetis), each 500 µg, were administered intramuscularly in the morning and evening, with a 12-hour interval. The CIDR devices were removed 12 hours before the last NIH-FSH-P1 injection on Day 7 (33). Following the onset of estrus, artificial insemination was performed at 12 hours and again at 48 hours after estrus detection. Embryos were collected from donor females on days 5 and 7 after the onset of estrus using a closed transrectal uterine lavage system (34). Uterine catheters of the Foley silicon type with two channels were used, along with commercial collection media ABT360 Complete Flush (35). In the IVV cultures, embryos were collected on day 5 (Experiment 1) and day 7 (Experiment 2), using a procedure similar to the in vitro method. The embryos were cultured under the same conditions, and the media and EVs were collected on the respective days for subsequent analysis. In both experiments, the blastocysts were transferred to fresh SOF medium for continued monitoring until day 11 of development. Incubation was maintained under the same conditions. EV Isolation and Characterization Isolation Culture media from each group were individually processed for EV purification using the protocol described by Théry, Amigorena (36) and validated in our laboratory by Mellisho, Velásquez (25). The procedure involves sequential centrifugation designed to remove cells and debris. Initially, samples were centrifuged at 200–700 × g for 10–20 minutes to remove cellular debris. The supernatant was then subjected to ultracentrifugation at 100,000–120,000×g for 120 minutes, and the resulting pellet, containing EVs, was resuspended in 500 μL of phosphate-buffered saline (PBS). The isolated EVs were characterized according to the criteria established by the International Society for Extracellular Vesicles (ISEV). Size and Concentration Analysis of Particles The overall characterization of the nanoparticle populations isolated from each blastocyst's culture medium was performed using Nanoparticle Tracking Analysis (NTA). This technique enables the determination of both particle concentration (particles/mL) and size distribution (in nanometers). For this analysis, the isolated nanoparticle pellet was diluted 1:20 in PBS (30 μL of pellet in 570 μL PBS). Measurements were conducted using a NanoSight NS300 instrument (Malvern Instruments, UK) equipped with a 488 nm laser, following the manufacturer’s protocol (Malvern Instruments, 2015) and the guidelines described by Bachurski, Schuldner (37). Analysis of Molecular Markers Specific EV markers (CD9, CD63, CD81, and CD40L), as defined by the International Society for Extracellular Vesicles (19), were evaluated by flow cytometry, following the protocol described by (25). Nanoparticles were incubated with 4 μm aldehyde/sulfate latex beads (1.25 × 10⁵ particles/mL; ThermoFisher Scientific, Waltham, MA, USA). The nanoparticle-bead complexes were incubated with primary antibodies against CD63 (FITC-conjugated; catalog no. 18235, Abcam), CD9 (FITC-conjugated; catalog no. 34162, Abcam), CD81 (PE-conjugated; catalog no. 81436, Abcam), and CD40L (PE/Cy5®-conjugated; catalog no. 25044, Abcam) for 2 hours at 4°C. Negative controls were prepared by incubating latex beads alone with each antibody under identical conditions. The labeled complexes were then resuspended in focusing fluid and analyzed on an Attune™ NxT Flow Cytometer (ThermoFisher Scientific, Waltham, MA, USA). Transmission Electron Microscopy (TEM) Nanoparticles were deposited onto formvar-carbon-coated copper grids for whole-mount preparations and examined by TEM, following the protocol outlined by (36, 38, 39) with slight modifications. Briefly, 10 μL of nanoparticle suspension was thawed and mixed with an equal volume of 4% paraformaldehyde. For each sample, two grids were prepared, washed, and fixed with 1% glutaraldehyde. They were then contrasted with uranyl-oxalate (pH 7.0) and 4% uranyl acetate. Grids were observed using a Talos F200C G2 transmission electron microscope (ThermoFisher Scientific, Chile) at the Advanced Microscopy Facility of the Pontificia Universidad Católica, Santiago de Chile. The instrument, equipped with a Ceta 16M CMOS camera, has a resolution of 0.3 nm and was operated at 80 kV. At least five images per sample were captured and processed using ImageJ software (V1.47t, NIH, USA). EVs RNA Isolation and Quantification For each experimental group, three biological replicates were prepared, each consisting of a pool of 10 conditioned culture media samples. The experimental groups included IVV 5–7, IVV 7–9, IVP 5–7, and IVP 7–9. Total RNA was extracted using the Exosomal RNA Isolation Kit (Norgen Biotek, Thorold, Canada) according to the manufacturer's instructions and following the protocols described by Lässer, Eldh (39). RNA integrity was assessed with the RNA ScreenTape Assay (Agilent Technologies, Santa Clara, CA, USA). Next-generation sequencing (NGS) was outsourced to an external facility (Norgen Biotek, Thorold, Canada). Small RNA Sequencing and Data Analysis Small RNA libraries were prepared using the Small RNA Library Prep Kit (Norgen Biotek, Thorold, Canada), according to the manufacturer’s instructions. Sequencing was performed on an Illumina NextSeq 500 platform with the NextSeq 500/550 High Output Kit v2 (Illumina, San Diego, CA, USA). Library quality was evaluated using FastQC (Babraham Bioinformatics, Cambridge, UK). Adapter sequences were trimmed, reads aligned, and quantified using the SRNA bench pipeline (40). For miRNA analysis, reads with a Phred score above 20 and lengths between 18 and 30 base pairs were accepted. Reads were mapped against the reference genome ARS-UCD 1.2 and miRBase version 21 using Bowtie2 and miRDeep2 mapper. Gene counts were calculated with HTSeq, applying a cutoff of counts per million (CPM) ≥ 5 to filter low-abundance features. Statistical and Bioinformatic Analyses Embryonic morphological characteristics and measurable EV variables (mean size and concentration) for each experimental group were compared using the Mann-Whitney U test. The correlation between embryo quality scores and developmental competence was assessed via Pearson’s and Spearman’s correlation tests. Differential expression analysis of the miRNA profiles was performed using the EdgeR package. The analysis included normalization of read counts using the trimmed mean of M-values (TMM) method. Statistical significance was determined using the exact test implemented in EdgeR, and p-values were adjusted for multiple testing using the Benjamini–Hochberg false discovery rate (FDR) correction. MiRNAs were considered differentially expressed based on an adjusted FDR 1.2 (or <- 1.2). The results were represented using volcano plots, heat maps, and principal component analysis (PCA). The set of differentially expressed miRNAs was further analyzed for target gene prediction using miRinGO. Gene Ontology (GO) enrichment analysis was performed via the DAVID platform to identify biological processes and pathways associated with the miRNA targets. Venn 2.1 https://bioinfogp.cnb.csic.es/tools/venny/. RESULTS Embryo Development Bovine pre-implantation embryos were produced through both in vivo and in vitro methods and were classified into two groups based on the developmental stage to be analyzed: blastulation and hatching (windows 5-7 and 7-9, respectively) (Table 1). At the 5-7 developmental stage, 63.9% of IVV embryos reached the blastocyst stage, whereas only 24.7% (p<0.05) of those produced by IVP reached this stage. Blastocyst development up to day 11 was also significantly lower in embryos (10.5%) compared with IVV embryos (52.5%). A similar pattern was observed in the 7-9 window, with 71.2% of IVV and 26.7% of IVP embryos developing to the blastocyst stage, and 61.6% versus 13.1% reaching the hatched stage (p<0.05). In all cases, IVP embryos showed significantly reduced developmental progression compared with IVV counterparts (Table 1). The analysis of embryonic growth dynamics revealed significant differences across production methods and developmental windows (Figure 2). In general, embryos from the 5-7 windows exhibited smaller diameters throughout the culture period than those from the 7-9 windows (p<0.05). By day 11, embryos from the IVV 7-9 group reached the largest diameter, whereas those from the IVP 5-7 group showed the smallest size (p<0.05). In all cases, embryos derived from IVV exhibited greater overall growth compared with their IVP counterparts . Based on developmental competence, 133 culture media samples from the respective experimental groups (IVP 5-7, IVV 5-7, IVP 7-9, and IVV 7-9) were selected for further analysis. EVs characterization NTA analysis revealed that EVs isolated from all study groups ranged in size from 100 to 200 nm, consistent with exosomes and small microvesicles (Figure 3). The data analysis revealed significant variations in both the concentration and mean size of secreted EVs, influenced by embryonic origin and developmental stage (Table 2). In a general analysis, IVP embryos showed higher nanoparticle concentrations than IVV embryos, especially at the hatching stage (IVP 7-9), with a marked predominance of particles smaller than 200 nm. In contrast, IVV embryos displayed a decrease in nanoparticle concentration from blastulation (5-7 days) to hatching (7-9 days), while maintaining a uniform size distribution. During the blastulation window (Days 5-7), EV concentrations were similar between in vitro and in vivo-produced embryos. However, during the hatching window (Days 7-9), IVP embryos secreted a significantly higher number of EVs ( p <0.05) than IVV embryos. Regarding EV size, at the blastulation stage (D5-7), EVs obtained from IVV embryos were significantly larger on average ( p <0.05) than those secreted by IVP embryos. Conversely, at the hatching stage (D7-9), no significant differences in average EV size were observed between IVP embryos and IVV embryos. Flow cytometry analysis confirmed the classification of nanoparticles as EVs based on the expression of their specific surface markers CD9, CD63, CD81, and CD40 in all experimental groups, with no signal detected in the negative control (Table 3). TEM revealed a typical EV morphology, with diameters ranging from approximately 100 to 500 nm. A rounded, cup-shaped appearance and a clearly defined bilayer membrane were evident. No morphological differences were detected between embryo-derived EVs from the different experimental groups (Figure 4). Analysis of miRNA Cargo in EVs Principal component analysis (PCA) was performed to evaluate the variability in miRNA profiles between and within the experimental groups (Figure 5). The PCA demonstrated a clear separation between groups based on embryonic origin. Specifically, EVs derived from IVV embryos formed a distinct cluster separate from those produced by IVP during both blastulation (5-7) and hatching (7-9) stages. The first two principal components explained 51% of the total variance (36% for Dim1 and 15% for Dim2). Dim1 mainly accounted for differences associated with embryo origin (IVV versus IVP), whereas Dim2 described variation related to developmental stage. During the blastulation period (5-7), miRNA expression patterns, as defined by principal component analysis, differed between EVs from IVV and IVP embryos. Despite some intragroup variability, the distinct separation between the IVV and IVP clusters was maintained. Similarly, during the hatching period (7-9), the intergroup variability between IVV and IVP miRNA profiles persisted, although there was a slight increase in intragroup dispersion. A comparative analysis of the global miRNA representation in EVs (Figure 6A) allowed the identification of the most variable miRNAs between the IVP and IVV groups for each developmental window. Marked differences in miRNA expression were observed in EVs from IVP 5-7 compared with the other experimental groups. A group of miRNAs, including miR-3432a-2 and miR-1468, showed significant overrepresentation (red hues) in EVs from 7-9 windows (both IVP and IVV), contrasting with their low representation at earlier stages (5-7). Conversely, miRNAs such as miR-16b, miR-140, and miR-155 generally demonstrated higher representation in EVs from 5-7 windows (IVP and IVV), with a trend towards lower representation at 7-9 days. Distinct profiles of miRNA content in EVs from four groups of preimplantation bovine embryos were revealed by analysis (Figure 6B). Out of the 122 identified miRNAs, 65 (48.1%) were shared among all groups, representing a conserved EV miRNA core signature across embryonic origins and stages. Ten miRNAs (7.4%) were uniquely detected in EVs secreted by in vitro-produced blastocysts (IVP 5-7), among which miR-Let7c, miR-181a-1, miR-181a-2, and miR-23b-2 were predominant. In contrast, no exclusive miRNAs were detected in EVs from IVV 7-9. For IVV 5-7 produced blastocysts, 4 unique miRNAs were identified, including miR-1271, miR-485, and miR-409a. Finally, 3 exclusive miRNAs —miR-Let7d, miR-12a-2, and miR-16b —were detected in EVs from IVP 7-9. Additional file 1 provides a detailed summary of detected miRNAs, their normalized expression levels (log₂ CPM), and their distribution across experimental groups, including in vitro- and in vivo-derived embryos at the blastulation (D 5-7) and hatching (D 7-9) stages. Prediction of gene functions Gene ontology analysis demonstrated that specific biological processes regulated by miRNAs in EVs are associated with developmental stage and embryo origin. During the blastulation stage (5-7), both IVV and IVP embryos showed enrichment in glycerol and water transport, along with significant regulation of T-cell differentiation, purinergic nucleotide signaling, and glucocorticoid response in the in vivo group (Figures 7A and B). At the hatching stage (7-9), IVV embryos continued to exhibit processes such as cytolysis, glycerol transport, phospholipase C-activating G protein-coupled receptor signaling, and glucocorticoid response, along with additional responses to light, sodium excretion, and homeostasis. IVP embryos, however, showed responses to glucocorticoids and unfolded proteins, as well as cell proliferation, muscle development, calcium regulation, and sperm–egg recognition, indicating functional adaptations in EVs to different developmental conditions (Figures 7C-D). In the shared biological processes (Figure 8A), a unique process was identified for IVV 5-7 involving the regulation of synaptic and glutamatergic transmission. Additionally, seven processes (glycerol transport, sperm and egg recognition, GMP-mediated signaling, response to unfolded proteins, cell proliferation, skeletal muscle development, and positive regulation of calcium) were exclusive to the IVP 7-9 group. Water transport and cytolysis were common to all four groups. The gene distribution (Figure 8B) showed a more differentiated pattern at the hatching stage, IVV containing seven exclusive genes (KCNC2, GZMM, C6, LTB4R, GNAT1, CALCR, CYP4F12) and IVP showed the highest number, total 13 (KCNU1, NPPA, DNAJA1, FOXN1, ACTA1, CATSPER4, HTR2B, HSPA13, HSPA4, PAX1, FAM83A, PDS5B, LPAR3). In contrast, no exclusive genes were identified during IVP 5-7, and only one gene (TPRG1L) was identified in IVV 5-7. Four genes (AQP3, PRF1, GZMB, and AQP7) were shared across all four groups, indicating a progressive diversification of EV-associated gene cargo toward the hatching stage, particularly in IVP embryos. DISCUSSION This study explored the influence of developmental stage (blastulation/hatching) and embryonic origin (IVV/IVP) on the characteristics of EVs released by bovine embryos, with particular attention to their molecular composition, size, and concentration. The impact of this work lies in the growing recognition of EVs as a crucial mechanism of communication between the embryo and the maternal environment, particularly during the pre-implantation stage. Here, special attention is given to the effect of embryo origin and quality on EVs' microRNA cargo. IVV embryos showed improved developmental capacity, as evidenced by higher blastocyst rates and post-hatching survival. Furthermore, IVV embryos collected at the blastocyst stage (IVV 5–7) exhibited linear growth until day 11 of in vitro culture and a larger diameter. However, IVV embryos collected on day 5 had reduced growth compared to the other groups, which is indicative of the stress generated due to the change from the maternal environment to in vitro culture. The results align with the literature supporting the increased competence of IVV embryos and the detrimental effect of embryo manipulation and in vitro conditions (3, 41, 42). Once the differential competence of embryos from different groups was confirmed, the impact on the released EV population was assessed. The characterization of nanoparticles isolated from the embryo culture media of each experimental group confirmed the presence of EVs with a classical morphology and the presence of protein markers defined in the ISEV guidelines (19) and previous characterizations of bovine embryonic EVs (24, 43, 44). EV size decreased as embryonic development progressed, regardless of origin, but was more pronounced in in vivo-derived embryos. An opposite pattern has been observed in pigs and humans, with a progressive increase in particle size throughout pre-implantation development (25, 45, 46). The largest EVs size was observed in IVV embryos collected on day 5, which also showed slower growth and a smaller diameter on day 11 of development. Therefore, it appears that the higher stress associated with early collection of IVV embryos is affecting not only the quality of the embryos but also the population of released EVs. The works by Mellisho et al. (2019) and Dissanayake et al. (2020) showed an inverse pattern, with the lowest mean size of released EVs observed for lower-quality embryos. However, in both reports, all embryos analyzed were produced in vitro, and EV collection was performed at different developmental windows. On the other hand, EV concentration in IVV embryos was consistent across both developmental stages studied (blastulation and hatching). In contrast, IVP embryos showed an increase in EV concentration as development progressed. In previous studies, variations in EV concentration were associated with embryonic quality rather than with developmental stage (44, 47). Here, in agreement with the literature, IVP embryos, which are considered less competent, released a higher concentration of EVs compared to vivo-derived embryos. It is known that in vitro conditions alter embryo metabolism, developmental kinetics, and gene expression, leading to a stress profile during culture (48-50). In other cell types, stress promotes the release of more EVs to remove unwanted molecules (51, 52). Higher EV release by embryos with compromised competence may be a compensatory mechanism that mitigates molecular stress and allows continued development. The analysis of miRNA content in EVs has gained significance, as these molecules influence essential processes post-transcriptionally, including pluripotency, cellular differentiation, and endometrial preparation for implantation (53, 54). Several studies have demonstrated that the miRNA profile in EVs varies with the embryo's characteristics [42] and can influence gene expression and target cell function (17). PCA analysis revealed that miRNA composition varied by embryo origin and stage. The miRNA clusters across developmental windows showed distinct grouping, confirming dynamic reorganization of their content as development progresses. IVV embryos had more stable profiles, whereas IVP embryos displayed heterogeneous profiles, consistent with their lower competence. These findings align with previous studies showing that miRNAs in EVs vary across stages, including compaction, blastulation, and later development, potentially acting as key regulators of pluripotency, cell differentiation, and endometrial signaling (44, 55). Global expression profiling identifies 25 highly variable miRNAs that predominantly regulate pathways essential for implantation and early embryonic development. Numerous studies in mammalian embryos have demonstrated the presence of miR-1468-5p, miR-7, miR-124, and miR-29a in embryonic EVs, with regulatory functions associated with cell proliferation, apoptosis, and differentiation (56-59). Mechanistically, miR-1468-5p targets RRM1 to regulate cell-cycle progression and caspase-3 activation, while miR-7 modulates the TGF-β/Smad pathway controlling epithelial–mesenchymal transition (60, 61). Similarly, miR-124 represses SOX9 and PAX6 , facilitating lineage specification, and miR-29a promotes endometrial decidualization by negatively regulating inhibitors of the TGF-β/Smad signaling pathway, enabling stromal differentiation (62, 63). On the other hand, miR-9 was associated with the regulation of E-cadherin and VEGF , processes essential for vasculogenesis and maintenance of pluripotency. At the same time, miR-127 promotes mesendoderm differentiation during early embryonic development by suppressing Lefty2 , thereby enhancing Nodal/Smad2 signaling (64, 65). miR-155 contributed to osmotic regulation (AQP3/AQP7) and immune tolerance through Treg modulation (66). The exclusive expression of miR-Let-7g in in vivo -derived embryos supports its role in synchronizing embryonic growth with uterine receptivity by suppressing c-Myc/mTOR (67, 68). Overall, the literature confirms that these miRNAs cluster around three fundamental functional axes: pluripotency and differentiation, embryonic competence, and immune modulation related to embryonic viability. These findings support the notion that EV content provides a dynamic representation of embryonic development and of molecular communication with the endometrium. A conserved core of 65 miRNAs was identified, suggesting a foundational set of signals packaged in EVs that remain consistent across origins and developmental stages. Simultaneously, another subset of miRNAs was observed to be exclusively packaged in EVs, with their presence depending on both origin and stage, reflecting environmental adaptability and embryonic competence (54). The unique miRNAs in the blastulation window (D5-7) are let-7c, miR-181a, miR-23b, miR-92a, and miR-409, reflecting their importance during this period. Let-7c is associated with regulating cell proliferation and differentiation, particularly during the transition to pluripotency (69, 70). MiR-181a regulates proliferation and apoptosis (71). miR-23b is involved in energy metabolism and differentiation, serving a compensatory function against oxidative stress in culture (72, 73). Lastly, miR-409-3p modulates Wnt/β-catenin and MAPK/p38 signaling pathways involved in cell differentiation, which influence embryo-maternal communication, and its exclusive presence in early IVP embryos suggests activation of pathways not observed in IVV embryos (74). During the transition to hatching (D7-9), IVP embryos did not exhibit exclusive miRNAs, suggesting that the EV profile homogenizes as development progresses. This observation supports the idea that, although differences between IVV and IVP persist, miRNA profiles in EVs tend to converge during hatching (53). Conversely, EVs from IVV (D7-9) contained three exclusive miRNAs that could be associated with the activation of regulatory mechanisms promoting embryo-maternal communication during peri-implantation. The miR-Let7i, a member of the Let-7 family, acts as a modulator of uterine receptivity by suppressing the Wnt/β-catenin pathway under ovarian hormone regulation, facilitating cellular adhesion and embryonic attachment, and linking its expression to greater implantation success (68, 75). Its reduced expression in in vitro embryos, reported during blastulation, indicates loss of molecular equilibrium that may compromise trophoblast differentiation and maternal receptivity (76). Overexpression of miR-16-1 disrupts epigenetic regulation and promotes apoptosis by repressing Bcl-2 ( Suz12 , Kmt2a ), leading to impaired development and early embryonic death (77). Variation in miRNA profiles across embryonic origin and developmental stage is directly reflected in the biological processes they regulate. During blastulation (D5-7), biological processes exhibit a relatively conserved functional pattern, with transport and osmoregulation (e.g., glycerol and water transport) conserved across origins. This core function has expanded to include pathways related to cellular communication and immune modulation, signaling the preparation for interaction with the endometrium. Analysis of the biological processes and proteins common to each stage and embryo origin confirmed the existence of a conserved nucleus, with water transport and cytolysis standing out. Water transport, supported by AQP3 and AQP7, is an essential and widely conserved mechanism for the formation and maintenance of the blastocoel, as demonstrated in bovine embryos produced both in vivo and in vitro (78, 79). Cytolysis is the second most prevalent process, facilitated by the genes PRF1 and GZMB. Its functions are associated with cytotoxicity facilitated by T and NK cells (80, 81). In the context of embryonic EVs, their presence primarily indicates embryo-maternal communication and initial immunological regulation, as evidenced by bovine embryo-derived signals that elicit immunomodulatory responses in the endometrium (80, 82). Exclusive biological processes and genes originate from this conserved core, differentiated by their origin and stage. In IVP embryos during blastulation (D5-7), the glutamatergic synaptic transmission regulation was identified as an exclusive process, an uncommon observation in this context (83). Experimental evidence demonstrates that NMDA receptor subunits are expressed and functionally regulated during cellular differentiation in various tissues (83, 84). In line with these observations, the detection of glutamatergic pathway activation in embryos suggests that NMDA receptor–related signaling contributes to cellular differentiation processes, consistent with mechanisms described in other developmental systems. During IVP embryo hatching, the highest functional diversification was observed, involving processes related to glycerol transport, GMP-mediated signaling, and the unfolded protein response (UPR), as supported by the overexpression of DNAJA1, HSPA13, and HSPA4, molecular chaperones associated with cell survival under stress conditions (85, 86). In addition, Processes associated with cell proliferation and muscle fiber development were linked to the expression of structural and regulatory genes such as ACTA1 , FAM83A , and PDS5B , which play essential roles in sarcomeric organization, growth signaling (PI3K/AKT), and embryonic differentiation (87-89). Positive regulation of Ca²⁺ signaling was also detected, involving channels such as CATSPER4, which modulates intracellular calcium levels, influencing cellular activation and motility (90, 91). These exclusive processes in IVP embryos demonstrate an adaptive metabolic response characteristic of embryos developed under in vitro conditions. The activation of AQP7 and KCNU1 indicates osmotic regulation mechanisms that support blastocoel maintenance in suboptimal environments (92-94). The increased expression of molecular chaperones (HSPA4, HSPA13, DNAJA1) confirms the activation of the UPR, a process associated with endoplasmic reticulum stress and protein homeostasis (95). The concurrent enrichment of GMP-mediated signaling and Ca²⁺ regulation reflects coordinated second messenger activity that sustains cell viability and energy balance during late pre-implantation development (96, 97). In contrast, during the hatching stage, IVV embryos showed enriched biological processes primarily related to sodium homeostasis and T cell-mediated cytotoxicity, as evidenced by the expression of KCNC2, CALCR, GZMM, and C6. These findings highlight the role of ionic regulation in maintaining fluid balance and blastocoel integrity, which is essential for sustained embryonic expansion and viability (Fujishima et al., 2025). Simultaneously, the involvement of immune-related genes such as GZMM and C6 suggests early activation of immunomodulatory mechanisms, possibly contributing to the establishment of maternal tolerance and effective embryo-endometrial communication (98, 99). This coordinated activation of ion homeostasis and immune signaling may reflect the superior implantation potential of IVV embryos. This more limited and specialized profile in IVV reflects a functional repertoire aimed at interacting with the endometrium and modulating the local immune system, aligning with the greater effectiveness of EVs derived from in vivo sources in inducing coordinated responses in the uterine epithelium (27, 53). Overall, IVP embryos tend to activate adaptive stress pathways. In contrast, their IVV counterparts emphasize ionic balance and controlled immunomodulation, both of which contribute to establishing a more effective dialogue with the endometrium. This interpretation is consistent with previous studies showing that in vitro embryos exhibit greater molecular heterogeneity and greater dependence on compensatory mechanisms, whereas in vivo embryos maintain more stable, implantation-oriented molecular profiles (24, 27, 44, 53, 55). Subsequently, the functional validation of anticipated pathways and miRNA targets will be crucial to ascertaining their roles. Optimizing culture conditions to replicate vivo signaling may also improve IVP embryo competence. CONCLUSION This study confirms that both the origin (IVV vs. IVP) and developmental stage (blastulation vs. hatching) of bovine embryos critically influence the characteristics and molecular composition of EVs, particularly their miRNA cargo. IVV embryos exhibit superior developmental competence, more stable EV profiles, and miRNA signatures linked to ionic balance and controlled immunomodulation. In contrast, IVP embryos exhibit increased EV release, greater heterogeneity in miRNA profiles, and activation of stress-related pathways, suggesting adaptive mechanisms in response to suboptimal culture conditions. The observed divergence in EV cargo and associated biological processes reveals that EVs play a dynamic role in pre-implantation embryo–maternal communication. These differences have profound implications for reproductive biotechnology, as they provide a potential non-invasive biomarker platform to assess embryo quality and competence. Future research should focus on functionally validating the predicted targets of EV-associated miRNAs and on evaluating their impact on maternal endometrial responses. Such insights will contribute to the development of novel diagnostic and therapeutic strategies to enhance implantation success and pregnancy rates in assisted reproduction programs. Abbreviations Ca²⁺: calcium ion; CPM: counts per million; EMT: epithelial–mesenchymal transition; EVs: extracellular vesicles; GO: Gene Ontology; GMP: cyclic guanosine monophosphate; IVP: in vitro-produced; IVV: in vivo-derived; Log FC: log₂ fold change; miRNA/miRNAs: microRNA(s); NTA: nanoparticle tracking analysis; PCA: principal component analysis; SEM: standard error of the mean; TEM: transmission electron microscopy; TGF-β: transforming growth factor beta; UPR: unfolded protein response. Declarations ETHICS APPROVAL The study was approved by the Ethics Committee of the Faculty of Veterinary Sciences, University of Concepción, for the execution of research project No. 1210334. CONSENT FOR PUBLICATION Not applicable. DATA AVAILABILITY The datasets generated and analyzed during the current study, including miRNA profiles and EV characterization data, are available from the corresponding author upon reasonable request. CONFLICT OF INTEREST The authors declare that they have no conflict of interests. FUNDING This research was supported by FONDEF Project ID18I10082; FONDECYT Projects 1170310 and 1210334; ANID Scholarship 21201280 and 21220042 (National Agency for Research and Development) AUTHOR CONTRIBUTIONS Co-first authorship: M.A.G.-R. and I.M.-H contributed equally to this work and shared first authorship. Conceptualization: L.R.-A.; F.O.C. Methodology: L.R.-A.; M.A.G.-R. Investigation: M.A.G.-R; I.M.-H.; Y.S.W.; F.N.; B.M.-B.; C.A. Data curation: M.A.G.-R; I.M.-H.; L.R.-A.; Y.S.W.; F.N.; C.A. Formal analysis: M.A.G.-R; I.M.-H.; Y.S.W; L.R.-A. Resources: L.R.-A.; F.O.C. Supervision: L.R.-A.; F.O.C. Writing–original draft: M.A.G.-R; I.M.-H; L.R.-A. Writing–review and editing: I.M.-H.; L.R.-A.; F.O.C. All authors read and approved of the final manuscript. 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Development and progression of bovine embryos produced in vitro (IVP) and in vivo (IVV) during the blastulation (Days 5–7) and hatching (Days 7–9) stages. Groups N° presumptive zygotes N° collected embryos % morulae (n) % blastocyst (n) % hatched blastocyst (n) IVP 5-7 612 44.9 (275) a 24.7 (151) a 10.5 (64) a IVV 5-7 61 78.7 (48) b 63.9 (39) b 52.5 (32) b IVP 7-9 701 42.2 (289) a 26.7 (187) a 13.1 (92) a IVV 7-9 73 83.6 (61) b 71.2 (52) b 61.6 (45) b Different superscript letters (a, b) within the same column indicate statistically significant differences between groups ( p < 0.05). TABLE 2. Concentration and size parameters of extracellular vesicles (EVs) secreted by bovine embryos during the blastulation (Days 5–7) and hatching (Days 7–9) stages under in vitro (IVP) and in vivo (IVV) conditions. EV Parameters Blastulation Hatching IVP 5-7 IVV 5-7 IVP 7-9 IVV 7-9 Concentration (particles x ml) 2,9 x 10 9 ± 0,3 a(§) 2,6 x 10 9 ± 0,8 a 3,8 x 10 9 ± 0,3 b(ţ) 2,2 x 10 9 ± 0,3 a Size (nm) 124,7±3,1 a(§) 148,5±4,1 b(§) 118,1±2,1 a(ţ) 115,4±1,8 a(ţ) Superscript letters (a, b) indicate statistically significant differences between embryo origins within the same stage ( p < 0.05). Symbols (§, ţ) indicate statistically significant differences between developmental stages within the same embryo origin ( p < 0.05). TABLE 3. Characterization of surface markers on extracellular vesicles (EVs) secreted by bovine embryos produced in vitro (IVP) and in vivo (IVV) during the blastulation and hatching stages. Groups SURFACE MARKERS (POSITIVE PERCENTAGE) CD9 (%) CD63 (%) CD81 (%) CD40 (%) IVP 5-7 10,5 24,4 36,6 9,3 IVV 5-7 3,5 7,6 3,8 20,8 IVP 7-9 17,9 3,9 15,7 9,2 IVV 7-9 7,4 13,0 7,8 18,5 Bovine blood serum (positive control) 14,5 6,5 9,6 12,0 Beads with antibodies (Negative control) 0 0 0 0 Values represent the percentage of positivity for surface markers CD9 , CD63 , CD81 , and CD40 on EVs, as determined by flow cytometry. Additional Declarations No competing interests reported. Supplementary Files ADDITIONALFILE.docx Additional file 1. miRNA expression profiles detected in extracellular vesicles (EVs) secreted by bovine embryos produced in vivo (IVV) and in vitro (IVP) during the blastulation (Days 5–7) and hatching (Days 7–9) stages. CPM: normalized readings in log₂ counts per million Cite Share Download PDF Status: Published Journal Publication published 13 Apr, 2026 Read the published version in Journal of Animal Science and Biotechnology → Version 1 posted Editorial decision: Revision requested 15 Dec, 2025 Reviews received at journal 13 Dec, 2025 Reviews received at journal 11 Dec, 2025 Reviewers agreed at journal 08 Dec, 2025 Reviewers agreed at journal 08 Dec, 2025 Reviewers invited by journal 08 Dec, 2025 Editor assigned by journal 03 Dec, 2025 Submission checks completed at journal 03 Dec, 2025 First submitted to journal 01 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8248877","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":557289689,"identity":"a282c3ac-03a9-444b-95f4-8763cd0c0fca","order_by":0,"name":"Miguel A. 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Values represent the mean ± SEM of three independent replicates. Statistical differences between groups are indicated by * (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"FIGUIRE2.png","url":"https://assets-eu.researchsquare.com/files/rs-8248877/v1/473a88412762b4984ddc7a3a.png"},{"id":97808444,"identity":"b2966413-d11f-4e2c-b826-0862ac977ceb","added_by":"auto","created_at":"2025-12-09 15:20:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4196876,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtracellular vesicle (EV) concentration and size distribution determined by nanoparticle tracking analysis (NTA, NanoSight©).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEVs were isolated from embryo-conditioned media collected from bovine embryos produced in vivo (IVV) or in vitro (IVP) during the blastulation (Days 5–7) and hatching (Days 7–9) stages. Each curve represents the average particle size and concentration from three replicates per group. Negative controls (EV-depleted medium) showed no detectable signal.\u003c/p\u003e","description":"","filename":"FIGUIRE3.png","url":"https://assets-eu.researchsquare.com/files/rs-8248877/v1/a1bd0ee659b102ea3ddae559.png"},{"id":97808439,"identity":"111227e1-f43e-4b88-a0d9-989880beaa89","added_by":"auto","created_at":"2025-12-09 15:20:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1663902,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphology of extracellular vesicles (EVs) secreted by bovine embryos under different experimental conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative transmission electron microscopy (TEM) images of EVs isolated from embryo-conditioned media of bovine embryos produced in vitro (IVP; A, C) or in vivo (IVV; B, D) during blastulation (Days 5–7) and hatching (Days 7–9) stages. Panels (A–B): blastulation stages. Panels (C–D): hatching stages. Scale bars represent 100 nm.\u003c/p\u003e","description":"","filename":"FIGURE4.png","url":"https://assets-eu.researchsquare.com/files/rs-8248877/v1/ad0c4cbdbb4f5112b2615bf1.png"},{"id":97808442,"identity":"b2c106cc-fc8d-4c13-8027-93bc606fa6f5","added_by":"auto","created_at":"2025-12-09 15:20:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":230359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrincipal component analysis (PCA) of miRNA profiles in extracellular vesicles (EVs) secreted by bovine embryos.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe plot represents the dimensional distribution of EV-associated miRNA expression profiles according to embryo origin and developmental stage. Colors denote experimental groups: Green, IVP 5–7; Black, IVV 5–7; Red, IVP 7–9; and Blue, IVV 7–9. Axes represent the principal components calculated from log₂ fold-change (Log FC) values. Each point corresponds to an independent experimental replicate.\u003c/p\u003e","description":"","filename":"FIGURE5.png","url":"https://assets-eu.researchsquare.com/files/rs-8248877/v1/285bb1330b76941dcae5bc63.png"},{"id":97898074,"identity":"53e5ff09-e690-4e23-ad07-349dc29899e1","added_by":"auto","created_at":"2025-12-10 15:38:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":10538996,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution and expression profiles of miRNAs in extracellular vesicles (EVs) from bovine embryos produced in vitro (IVP) and in vivo (IVV) during blastulation (5–7) and hatching (7–9).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eHeatmap representing the hierarchical clustering of miRNA expression levels identified in EVs secreted by bovine embryos. Each row corresponds to a specific miRNA, and each column to an experimental replicate (IVP 5–7, IVV 5–7, IVP 7–9, IVV 7–9). The color scale indicates relative expression levels; Red: upregulated miRNAs, Green: downregulated miRNAs. Dendrograms depict clustering based on the similarity of expression profiles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Venn diagram showing the distribution and overlap of miRNAs detected in EVs according to embryonic origin and developmental stage. Numbers within each area represent the number and percentage of miRNAs shared or uniquely expressed among the groups IVP 5–7, IVV 5–7, IVP 7–9, IVV 7–9.\u003c/p\u003e","description":"","filename":"FIGURE6.png","url":"https://assets-eu.researchsquare.com/files/rs-8248877/v1/86f2e3517f7fa58a0558ae66.png"},{"id":97808441,"identity":"43a650c7-1469-43bf-be89-8b504aa4e481","added_by":"auto","created_at":"2025-12-09 15:20:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1432928,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiological processes regulated by differentially expressed miRNAs in extracellular vesicles (EVs) from bovine embryos during blastulation and hatching under in vivo (IVV) and in vitro (IVP) conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePie charts illustrate the distribution of genes associated with significantly enriched biological processes (Gene Ontology analysis). The number within each segment indicates the count of genes involved in each process, while \u003cem\u003ep\u003c/em\u003e-values (shown in parentheses). (A) IVV 5–7 (blastulation), (B) IVP 5–7 (blastulation), (C) IVV 7–9 (hatching), and (D) IVP 7–9 (hatching).\u003c/p\u003e","description":"","filename":"FIGURE7.png","url":"https://assets-eu.researchsquare.com/files/rs-8248877/v1/2fa68ae50f38a6f3e4130b9b.png"},{"id":97808443,"identity":"e11e6cf0-3c62-4bad-9831-ef715c945036","added_by":"auto","created_at":"2025-12-09 15:20:04","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":854754,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution and interaction of biological processes and genes regulated by overexpressed miRNAs in extracellular vesicles (EVs) secreted during bovine embryonic development under in vivo (IVV) and in vitro (IVP) conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eVenn diagram showing the distribution of biological processes regulated by overexpressed miRNAs according to embryo origin and developmental stage. Shared and exclusive biological processes are indicated, with the number and percentage of processes represented within each region.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eA\u003cstrong\u003e \u003c/strong\u003eVenn diagram illustrates the distribution of genes targeted by overexpressed miRNAs. Shared and exclusive genes are shown for each condition. Specific biological processes and genes associated with each experimental group are listed adjacent to the diagrams: Yellow, IVV 5–7; Blue, IVP 5–7; Green, IVV 7–9; and Red, IVP 7–9.\u003c/p\u003e","description":"","filename":"FIGURE8.png","url":"https://assets-eu.researchsquare.com/files/rs-8248877/v1/99055537ab6a931e0ff6c7c8.png"},{"id":107352799,"identity":"f5e4e9f2-fe0f-4fc4-a50a-ff7e7f5b7ccd","added_by":"auto","created_at":"2026-04-20 16:15:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":35255383,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8248877/v1/3028bf39-7ac4-4b40-b705-2308bff41089.pdf"},{"id":97808438,"identity":"0462f010-a8ca-41d5-97c2-cfa5ebc72201","added_by":"auto","created_at":"2025-12-09 15:20:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":45337,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1. \u003c/strong\u003emiRNA expression profiles detected in extracellular vesicles (EVs) secreted by bovine embryos produced in vivo (IVV) and in vitro (IVP) during the blastulation (Days 5–7) and hatching (Days 7–9) stages. \u003cstrong\u003eCPM:\u003c/strong\u003e normalized readings in log₂ counts per million\u003c/p\u003e","description":"","filename":"ADDITIONALFILE.docx","url":"https://assets-eu.researchsquare.com/files/rs-8248877/v1/8405232c3848ec6b5afe2de8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative profiling of extracellular vesicles and miRNA cargo from in vivo- and in vitro-derived bovine embryos during blastulation and hatching","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAssisted reproductive technologies (ART) are crucial to the bovine industry because they accelerate the propagation of genetically superior livestock. Recently, a shift has occurred from in vivo-produced (IVV) to in vitro-produced (IVP) embryos as the preferred strategy for maximizing genetic gains in cattle (1). Despite notable advances in IVP protocols and the optimization of culture media to enhance gamete and embryo competence, the cryotolerance and post-transfer pregnancy rates of IVP embryos remain lower than those of IVV embryos (2, 3).\u003c/p\u003e\n\u003cp\u003eThe in vitro environment exerts a profound influence on early embryonic development, rendering them less competent than their in vivo counterparts. Morphological selection of blastocysts often fails to predict implantation potential, despite its widespread adoption for its simplicity and non-invasiveness. Even blastocysts considered of the highest quality (Grade 1) according to IETS grading (4) may exhibit compromised implantation capacity (5). Most pregnancy losses occur during the peri-implantation period (6-8). The in vitro environment impacts epigenetic and gene expression patterns, effects that may not be evident at the blastocyst stage but manifest later during embryo-maternal recognition processes (9-11), due to the compromised signaling required for successful maternal recognition (12, 13).\u003c/p\u003e\n\u003cp\u003eIn bovines, interferon tau (IFN-\u0026tau;) secreted by the embryo is the primary signal for maternal recognition of pregnancy. Despite the critical role of IFN-\u0026tau; in pregnancy recognition, there is evidence that earlier signals secreted by the embryo may also prepare the maternal side for implantation (14, 15). Pre-implantation embryos release extracellular vesicles (EVs), which are internalized by endometrial cells and participate in early embryo-maternal communication (16, 17).\u003c/p\u003e\n\u003cp\u003eEVs are nanometer-sized particles surrounded by a lipid bilayer and are naturally secreted into the extracellular environment by various cell types, including embryonic cells (18, 19). During their biogenesis, EVs are loaded with diverse molecules, including proteins, lipids, RNAs, and DNA, initially described as mechanisms for cellular waste disposal (20). However, EVs are now recognized as key mediators of intercellular communication, mediating the transfer of bioactive molecules to recipient cells through their molecular cargo (21, 22).\u003c/p\u003e\n\u003cp\u003eEVs have been linked to several physiological processes, including gamete and embryo development. Pre-implantation embryos from various species, including humans, pigs, mice, and cattle, secrete EVs (23-25). The characteristics (size, concentration, and molecular cargo) of EV populations vary according to the origin, developmental stage, and quality of the embryo (26). The differential molecular composition of embryonic EVs reflects the embryo\u0026apos;s developmental competence and may modulate embryo-maternal communication. Although the number of studies is still limited, emerging evidence suggests that embryo-maternal communication mediated by EVs may differ depending on whether the embryo was produced in vitro or in vivo (17, 27-29). However, our understanding of how EVs derived from IVV or IVP produced embryos influence the establishment of a receptive maternal environment and proper implantation remains scarce.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study aimed to characterize EV populations derived from in vivo- and in vitro-produced bovine embryos at distinct developmental stages to elucidate their role in embryo\u0026ndash;maternal communication. It was hypothesized that both the origin and developmental stage exert significant influences on population characteristics and miRNA content, potentially affecting molecular pathways involved in embryo-maternal communication and implantation. Understanding these differences was anticipated to offer novel insights into the mechanisms underlying the diminished success rates observed in in vitro-produced embryos and to inform the development of enhanced strategies for embryo selection and culture.\u0026nbsp;\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eAll experiments were conducted at the University of Concepci\u0026oacute;n, Campus Chill\u0026aacute;n, except for transmission electron microscopy, which was carried out at the Advanced Microscopy Facility of the Pontificia Universidad Cat\u0026oacute;lica de Chile.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo experiments were conducted to characterize EV populations during specific windows of early embryonic development to elucidate how the characteristics of EVs secreted by bovine embryos are influenced by their origin (IVV or IVP) and developmental stage (blastulation and hatching) (Figure 1). For both experiments, embryos were produced through \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e procedures. In Experiment 1, corresponding to the blastulation stage (Days 5\u0026ndash;7), a total of 30 in vivo (IVV) and 60 in vitro (IVP) embryos were obtained. In Experiment 2, which focused on the hatching stage (Days 7\u0026ndash;9), 30 IVV and 30 IVP embryos were produced.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eExperiment 1:\u003c/u\u003e\u003c/strong\u003e Evaluation of Characteristics and miRNA Profiles of EVs Secreted During Blastulation in In Vitro and In Vivo Bovine Embryos.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eIVV 5-7 Group: Morulae obtained via superovulation and artificial insemination.\u003c/li\u003e\n \u003cli\u003eIVP 5-7 Group: Morulae generated through in vitro fertilization.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eExperiment 2:\u003c/u\u003e\u003c/strong\u003e Evaluation of Characteristics and miRNA Profiles of EVs Secreted During Hatching in In Vitro and In Vivo Bovine Embryos.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eIVV 7-9 group: Blastocysts obtained via superovulation and artificial insemination.\u003c/li\u003e\n \u003cli\u003eIVP 7-9 group: Blastocysts generated through in vitro fertilization.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eIn both experiments, the culture media of the blastocysts on Day-7 and Day-9 were collected and stored at -80\u0026deg;C for subsequent EV and content analysis.\u003c/p\u003e\n\u003cp\u003eEV characterization included size and concentration assessment via nanoparticle tracking analysis (NTA), as well as surface marker detection (CD9, CD40, CD63, CD81) using cytometry and transmission electron microscopy (TEM). Additionally, miRNA profiles within the EVs were evaluated through sequencing. All blastocysts were transferred to a new drop of EV-depleted SOF medium and maintained in individual culture until day 11 to assess their post-hatching developmental capacity. Only conditioned media from these competent embryos were used for the analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe IETS classification system was applied at each stage of embryo development. Only embryos graded 1-2 on days 5 and 9 of development were considered for further study. Morphological evaluation was performed under blind conditions, in accordance with IETS guidelines. After classification, embryos were randomly and blindly assigned to the corresponding experimental groups. Subsequently, embryos that showed linear growth and a diameter greater than 270 \u0026micro;m on day 11 were considered competent (24).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBovine Embryo \u003cem\u003eIn Vitro\u003c/em\u003e Production\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCumulus-oocyte complexes (COCs) were obtained from ovaries collected from slaughtered beef cattle at a local abattoir (Frigosur, Chill\u0026aacute;n, Chile). The collection, maturation, fertilization, and culture procedures followed protocols described by (25, 30). COCs were aspirated from antral follicles (3\u0026ndash;6 mm diameter) using a 19-G needle. COCs were first transferred to manipulation medium (TCM199 supplemented with 4 mM bicarbonate, 18 mM HEPES, 10% fetal bovine serum (FBS), and 50 mg/mL gentamicin), pre-warmed to 38\u0026deg;C, for washing and morphological assessment. Selected COCs were matured in groups in 500 \u0026mu;L of TCM199-based medium supplemented with 0.6 mM glutamine, 0.2 mM pyruvate, 0.01 IU/mL FSH and LH, 1 \u0026mu;g/mL estradiol, 50 \u0026mu;g/mL gentamicin, 10 ng/mL epidermal growth factor (EGF), and 10% FBS. Cultures were incubated at 38\u0026deg;C in a humidified atmosphere of 5% CO₂\u0026nbsp;for 20-22 hours.\u003c/p\u003e\n\u003cp\u003eSpermatozoa were obtained from commercial semen (Semex, Madison, WI, USA) and separated using a Percoll gradient method. Each matured oocyte was inseminated with 10,000 motile spermatozoa in 500 \u0026mu;L of fertilization medium (TALP-FIV) supplemented with 0.01 mg/mL heparin, 2 mM pyruvate, 50 mg/mL gentamicin, and 6 mg/mL fatty acid-free serum albumin (BSA). Fertilization was carried out at 39\u0026deg;C under 5% CO₂.\u003c/p\u003e\n\u003cp\u003eEighteen hours after fertilization, presumptive zygotes were mechanically denuded of cumulus cells by vortexing for 3 minutes in TCM199 medium containing HEPES and 0.3 mg/mL hyaluronidase. Zygotes were then cultured in groups of 25\u0026ndash;30 in 500 \u0026mu;L of synthetic oviduct fluid (SOF) medium supplemented with 0.37 mM trisodium citrate, 2.77 mM myo-inositol, 10 ng/mL EGF, 2% FBS, and 3 mg/mL fatty acid-free BSA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor Experiment 1, on day 5 of development, Grade 1 morula-stage embryos were selected and cultured individually in 96-well plates containing 80 \u0026micro;l of EV-depleted SOF culture medium. Incubation was maintained until day 7 of development, after which the embryos were evaluated and classified based on morphological quality, and the culture medium was collected. In Experiment 2, on day 7, the embryos were classified. Grade I blastocysts were then selected and cultured individually in 96-well plates with 80 \u0026micro;l of EV-depleted SOF culture medium until day 9 of development. On day 9, the embryos were re-evaluated, classified by morphological quality, and the culture medium was collected for further analysis. In both cases, the blastocysts were transferred to fresh SOF medium for continued monitoring until day 11 of development. Incubation was carried out at 38.5\u0026deg;C under a gas mixture of 5% CO₂, 5% O₂, and 90% N₂, with a humidity of 85%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBovine \u003cem\u003eIn Vivo\u003c/em\u003e Embryo Production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe donor females underwent follicular population assessment via ultrasonography (Sonoscape X3V with a 5 MHz sector transducer). To synchronize the emergence of the follicular wave among the donors, a 1.38 g intravaginal progesterone device (CIDR\u0026reg;; Zoetis) combined with 2.5 mg of estradiol benzoate (Zoetis) administered intramuscularly and 50 mg of progesterone administered intramuscularly (31, 32).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHormonal stimulation for superovulation (SOV) began on Day 4 and continued until Day 7. Each donor in the control group received a total of 250 mg of NIH-FSH-P1 (Folltropin-V; Bioniche Animal Health, Belleville, Ontario, Canada), divided into eight intramuscular injections with decreasing doses from 50 mg to 10 mg, administered every 12 hours over 4 days.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn Day 6, two doses of prostaglandin F2\u0026alpha; (cloprostenol, Ciclase\u0026reg;, Zoetis), each 500 \u0026micro;g, were administered intramuscularly in the morning and evening, with a 12-hour interval. The CIDR devices were removed 12 hours before the last NIH-FSH-P1 injection on Day 7 (33). Following the onset of estrus, artificial insemination was performed at 12 hours and again at 48 hours after estrus detection.\u003c/p\u003e\n\u003cp\u003eEmbryos were collected from donor females on days 5 and 7 after the onset of estrus using a closed transrectal uterine lavage system (34). Uterine catheters of the Foley silicon type with two channels were used, along with commercial collection media ABT360 Complete Flush (35).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the IVV cultures, embryos were collected on day 5 (Experiment 1) and day 7 (Experiment 2), using a procedure similar to the in vitro method. The embryos were cultured under the same conditions, and the media and EVs were collected on the respective days for subsequent analysis. In both experiments, the blastocysts were transferred to fresh SOF medium for continued monitoring until day 11 of development. Incubation was maintained under the same conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEV Isolation and Characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIsolation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCulture media from each group were individually processed for EV purification using the protocol described by Th\u0026eacute;ry, Amigorena (36) and validated in our laboratory by Mellisho, Vel\u0026aacute;squez (25). The procedure involves sequential centrifugation designed to remove cells and debris. Initially, samples were centrifuged at 200\u0026ndash;700 \u0026times; g for 10\u0026ndash;20 minutes to remove cellular debris. The supernatant was then subjected to ultracentrifugation at 100,000\u0026ndash;120,000\u0026times;g for 120 minutes, and the resulting pellet, containing EVs, was resuspended in 500 \u0026mu;L of phosphate-buffered saline (PBS). The isolated EVs were characterized according to the criteria established by the International Society for Extracellular Vesicles (ISEV).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSize and Concentration Analysis of Particles\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe overall characterization of the nanoparticle populations isolated from each blastocyst\u0026apos;s culture medium was performed using Nanoparticle Tracking Analysis (NTA). This technique enables the determination of both particle concentration (particles/mL) and size distribution (in nanometers). For this analysis, the isolated nanoparticle pellet was diluted 1:20 in PBS (30 \u0026mu;L of pellet in 570 \u0026mu;L PBS). Measurements were conducted using a NanoSight NS300 instrument (Malvern Instruments, UK) equipped with a 488 nm laser, following the manufacturer\u0026rsquo;s protocol (Malvern Instruments, 2015) and the guidelines described by Bachurski, Schuldner (37).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnalysis of Molecular Markers\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecific EV markers (CD9, CD63, CD81, and CD40L), as defined by the International Society for Extracellular Vesicles (19), were evaluated by flow cytometry, following the protocol described by (25). Nanoparticles were incubated with 4 \u0026mu;m aldehyde/sulfate latex beads (1.25 \u0026times; 10⁵ particles/mL; ThermoFisher Scientific, Waltham, MA, USA). The nanoparticle-bead complexes were incubated with primary antibodies against CD63 (FITC-conjugated; catalog no. 18235, Abcam), CD9 (FITC-conjugated; catalog no. 34162, Abcam), CD81 (PE-conjugated; catalog no. 81436, Abcam), and CD40L (PE/Cy5\u0026reg;-conjugated; catalog no. 25044, Abcam) for 2 hours at 4\u0026deg;C. Negative controls were prepared by incubating latex beads alone with each antibody under identical conditions. The labeled complexes were then resuspended in focusing fluid and analyzed on an Attune\u0026trade; NxT Flow Cytometer (ThermoFisher Scientific, Waltham, MA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTransmission Electron Microscopy (TEM)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNanoparticles were deposited onto formvar-carbon-coated copper grids for whole-mount preparations and examined by TEM, following the protocol outlined by (36, 38, 39) with slight modifications. Briefly, 10 \u0026mu;L of nanoparticle suspension was thawed and mixed with an equal volume of 4% paraformaldehyde. For each sample, two grids were prepared, washed, and fixed with 1% glutaraldehyde. They were then contrasted with uranyl-oxalate (pH 7.0) and 4% uranyl acetate. Grids were observed using a Talos F200C G2 transmission electron microscope (ThermoFisher Scientific, Chile) at the Advanced Microscopy Facility of the Pontificia Universidad Cat\u0026oacute;lica, Santiago de Chile. The instrument, equipped with a Ceta 16M CMOS camera, has a resolution of 0.3 nm and was operated at 80 kV. At least five images per sample were captured and processed using ImageJ software (V1.47t, NIH, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEVs RNA Isolation and Quantification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor each experimental group, three biological replicates were prepared, each consisting of a pool of 10 conditioned culture media samples. The experimental groups included IVV 5\u0026ndash;7, IVV 7\u0026ndash;9, IVP 5\u0026ndash;7, and IVP 7\u0026ndash;9. Total RNA was extracted using the Exosomal RNA Isolation Kit (Norgen Biotek, Thorold, Canada) according to the manufacturer\u0026apos;s instructions and following the protocols described by L\u0026auml;sser, Eldh (39). RNA integrity was assessed with the RNA ScreenTape Assay (Agilent Technologies, Santa Clara, CA, USA). Next-generation sequencing (NGS) was outsourced to an external facility (Norgen Biotek, Thorold, Canada).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSmall RNA Sequencing and Data Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSmall RNA libraries were prepared using the Small RNA Library Prep Kit (Norgen Biotek, Thorold, Canada), according to the manufacturer\u0026rsquo;s instructions. Sequencing was performed on an Illumina NextSeq 500 platform with the NextSeq 500/550 High Output Kit v2 (Illumina, San Diego, CA, USA). Library quality was evaluated using FastQC (Babraham Bioinformatics, Cambridge, UK). Adapter sequences were trimmed, reads aligned, and quantified using the SRNA bench pipeline (40).\u003c/p\u003e\n\u003cp\u003eFor miRNA analysis, reads with a Phred score above 20 and lengths between 18 and 30 base pairs were accepted. Reads were mapped against the reference genome ARS-UCD 1.2 and miRBase version 21 using Bowtie2 and miRDeep2 mapper. Gene counts were calculated with HTSeq, applying a cutoff of counts per million (CPM) \u0026ge; 5 to filter low-abundance features.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical and Bioinformatic Analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEmbryonic morphological characteristics and measurable EV variables (mean size and concentration) for each experimental group were compared using the Mann-Whitney U test. The correlation between embryo quality scores and developmental competence was assessed via Pearson\u0026rsquo;s and Spearman\u0026rsquo;s correlation tests. Differential expression analysis of the miRNA profiles was performed using the EdgeR package. The analysis included normalization of read counts using the trimmed mean of M-values (TMM) method. Statistical significance was determined using the exact test implemented in EdgeR, and p-values were adjusted for multiple testing using the Benjamini\u0026ndash;Hochberg false discovery rate (FDR) correction. MiRNAs were considered differentially expressed based on an adjusted FDR \u0026lt; 0.05 and an absolute log₂\u0026nbsp;fold change \u0026gt; 1.2 (or \u0026lt;- 1.2). The results were represented using volcano plots, heat maps, and principal component analysis (PCA).\u003c/p\u003e\n\u003cp\u003eThe set of differentially expressed miRNAs was further analyzed for target gene prediction using miRinGO. Gene Ontology (GO) enrichment analysis was performed via the DAVID platform to identify biological processes and pathways associated with the miRNA targets. Venn 2.1 https://bioinfogp.cnb.csic.es/tools/venny/.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eEmbryo Development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBovine pre-implantation embryos were produced through both in vivo and in\u003cem\u003e\u0026nbsp;\u003c/em\u003evitro methods and were classified into two groups based on the developmental stage to be analyzed: blastulation and hatching (windows 5-7 and 7-9, respectively) (Table 1).\u003c/p\u003e\n\u003cp\u003eAt the 5-7 developmental stage, 63.9% of IVV embryos reached the blastocyst stage, whereas only 24.7% (p\u0026lt;0.05) of those produced by IVP reached this stage. Blastocyst development up to day 11 was also significantly lower in embryos (10.5%) compared with IVV embryos (52.5%). A similar pattern was observed in the 7-9 window, with 71.2% of IVV and 26.7% of IVP embryos developing to the blastocyst stage, and 61.6% versus 13.1% reaching the hatched stage (p\u0026lt;0.05). In all cases, IVP embryos showed significantly reduced developmental progression compared with IVV counterparts\u003cem\u003e\u0026nbsp;\u003c/em\u003e(Table 1).\u003c/p\u003e\n\u003cp\u003eThe analysis of embryonic growth dynamics revealed significant differences across production methods and developmental windows (Figure 2). In general, embryos from the 5-7 windows exhibited smaller diameters throughout the culture period than those from the 7-9 windows (p\u0026lt;0.05). By day 11, embryos from the IVV 7-9 group reached the largest diameter, whereas those from the IVP 5-7 group showed the smallest size (p\u0026lt;0.05). In all cases, embryos derived from IVV exhibited greater overall growth compared with their IVP counterparts\u003cem\u003e.\u0026nbsp;\u003c/em\u003eBased on developmental competence, 133 culture media samples from the respective experimental groups (IVP 5-7, IVV 5-7, IVP 7-9, and IVV 7-9) were selected for further analysis.\u003c/p\u003e\n\u003ch2\u003eEVs characterization\u003c/h2\u003e\n\u003cp\u003eNTA analysis revealed that EVs isolated from all study groups ranged in size from 100 to 200 nm, consistent with exosomes and small microvesicles (Figure 3). The data analysis revealed significant variations in both the concentration and mean size of secreted EVs, influenced by embryonic origin and developmental stage (Table 2). In a general analysis, IVP embryos showed higher nanoparticle concentrations than IVV embryos, especially at the hatching stage (IVP 7-9), with a marked predominance of particles smaller than 200 nm. In contrast, IVV embryos displayed a decrease in nanoparticle concentration from blastulation (5-7 days) to hatching (7-9 days), while maintaining a uniform size distribution.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDuring the blastulation window (Days 5-7), EV concentrations were similar between in vitro and in vivo-produced embryos. However, during the hatching window (Days 7-9), IVP embryos secreted a significantly higher number of EVs (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) than IVV embryos. Regarding EV size, at the blastulation stage (D5-7), EVs obtained from IVV embryos were significantly larger on average (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) than those secreted by IVP embryos. Conversely, at the hatching stage (D7-9), no significant differences in average EV size were observed between IVP embryos and IVV embryos.\u003c/p\u003e\n\u003cp\u003eFlow cytometry analysis confirmed the classification of nanoparticles as EVs based on the expression of their specific surface markers CD9, CD63, CD81, and CD40 in all experimental groups, with no signal detected in the negative control (Table 3). TEM revealed a typical EV morphology, with diameters ranging from approximately 100 to 500 nm. A rounded, cup-shaped appearance and a clearly defined bilayer membrane were evident. No morphological differences were detected between embryo-derived EVs from the different experimental groups (Figure 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of miRNA Cargo in EVs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrincipal component analysis (PCA) was performed to evaluate the variability in miRNA profiles between and within the experimental groups (Figure 5). The PCA demonstrated a clear separation between groups based on embryonic origin. Specifically, EVs derived from IVV embryos formed a distinct cluster separate from those produced by IVP during both blastulation (5-7) and hatching (7-9) stages. The first two principal components explained 51% of the total variance (36% for Dim1 and 15% for Dim2). Dim1 mainly accounted for differences associated with embryo origin (IVV versus IVP), whereas Dim2 described variation related to developmental stage. During the blastulation period (5-7), miRNA expression patterns, as defined by principal component analysis, differed between EVs from IVV and IVP embryos. Despite some intragroup variability, the distinct separation between the IVV and IVP clusters was maintained. Similarly, during the hatching period (7-9), the intergroup variability between IVV and IVP miRNA profiles persisted, although there was a slight increase in intragroup dispersion.\u003c/p\u003e\n\u003cp\u003eA comparative analysis of the global miRNA representation in EVs (Figure 6A) allowed the identification of the most variable miRNAs between the IVP and IVV groups for each developmental window. Marked differences in miRNA expression were observed in EVs from IVP 5-7 compared with the other experimental groups. A group of miRNAs, including miR-3432a-2 and miR-1468, showed significant overrepresentation (red hues) in EVs from 7-9 windows (both IVP and IVV), contrasting with their low representation at earlier stages (5-7). Conversely, miRNAs such as miR-16b, miR-140, and miR-155 generally demonstrated higher representation in EVs from 5-7 windows (IVP and IVV), with a trend towards lower representation at 7-9 days. Distinct profiles of miRNA content in EVs from four groups of preimplantation bovine embryos were revealed by analysis (Figure 6B).\u003c/p\u003e\n\u003cp\u003eOut of the 122 identified miRNAs, 65 (48.1%) were shared among all groups, representing a conserved EV miRNA core signature across embryonic origins and stages. Ten miRNAs (7.4%) were uniquely detected in EVs secreted by in vitro-produced blastocysts (IVP 5-7), among which miR-Let7c, miR-181a-1, miR-181a-2, and miR-23b-2 were predominant. In contrast, no exclusive miRNAs were detected in EVs from IVV 7-9. For IVV 5-7 produced blastocysts, 4 unique miRNAs were identified, including miR-1271, miR-485, and miR-409a. Finally, 3 exclusive miRNAs \u0026mdash;miR-Let7d, miR-12a-2, and miR-16b \u0026mdash;were detected in EVs from IVP 7-9. \u0026nbsp;Additional file 1 provides a detailed summary of detected miRNAs, their normalized expression levels (log₂\u0026nbsp;CPM), and their distribution across experimental groups, including in vitro- and in vivo-derived embryos at the blastulation (D 5-7) and hatching (D 7-9) stages.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrediction of gene functions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene ontology analysis demonstrated that specific biological processes regulated by miRNAs in EVs are associated with developmental stage and embryo origin. During the blastulation stage (5-7), both IVV and IVP embryos showed enrichment in glycerol and water transport, along with significant regulation of T-cell differentiation, purinergic nucleotide signaling, and glucocorticoid response in the in vivo group (Figures 7A and B).\u003c/p\u003e\n\u003cp\u003eAt the hatching stage (7-9), IVV embryos continued to exhibit processes such as cytolysis, glycerol transport, phospholipase C-activating G protein-coupled receptor signaling, and glucocorticoid response, along with additional responses to light, sodium excretion, and homeostasis. IVP embryos, however, showed responses to glucocorticoids and unfolded proteins, as well as cell proliferation, muscle development, calcium regulation, and sperm\u0026ndash;egg recognition, indicating functional adaptations in EVs to different developmental conditions (Figures 7C-D).\u003c/p\u003e\n\u003cp\u003eIn the shared biological processes (Figure 8A), a unique process was identified for IVV 5-7 involving the regulation of synaptic and glutamatergic transmission. Additionally, seven processes (glycerol transport, sperm and egg recognition, GMP-mediated signaling, response to unfolded proteins, cell proliferation, skeletal muscle development, and positive regulation of calcium) were exclusive to the IVP 7-9 group. Water transport and cytolysis were common to all four groups.\u003c/p\u003e\n\u003cp\u003eThe gene distribution (Figure 8B) showed a more differentiated pattern at the hatching stage, IVV containing seven exclusive genes (KCNC2, GZMM, C6, LTB4R, GNAT1, CALCR, CYP4F12) and IVP showed the highest number, total 13 (KCNU1, NPPA, DNAJA1, FOXN1, ACTA1, CATSPER4, HTR2B, HSPA13, HSPA4, PAX1, FAM83A, PDS5B, LPAR3). In contrast, no exclusive genes were identified during IVP 5-7, and only one gene (TPRG1L) was identified in IVV 5-7. Four genes (AQP3, PRF1, GZMB, and AQP7) were shared across all four groups, indicating a progressive diversification of EV-associated gene cargo toward the hatching stage, particularly in IVP embryos.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study explored the influence of developmental stage (blastulation/hatching) and embryonic origin (IVV/IVP) on the characteristics of EVs released by bovine embryos, with particular attention to their molecular composition, size, and concentration. The impact of this work lies in the growing recognition of EVs as a crucial mechanism of communication between the embryo and the maternal environment, particularly during the pre-implantation stage. Here, special attention is given to the effect of embryo origin and quality on EVs\u0026apos; microRNA cargo.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIVV embryos showed improved developmental capacity, as evidenced by higher blastocyst rates and post-hatching survival. Furthermore, IVV embryos collected at the blastocyst stage (IVV 5\u0026ndash;7) exhibited linear growth until day 11 of in vitro culture and a larger diameter. However, IVV embryos collected on day 5 had reduced growth compared to the other groups, which is indicative of the stress generated due to the change from the maternal environment to in vitro culture. The results align with the literature supporting the increased competence of IVV embryos and the detrimental effect of embryo manipulation and in vitro conditions (3, 41, 42). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOnce the differential competence of embryos from different groups was confirmed, the impact on the released EV population was assessed. The characterization of nanoparticles isolated from the embryo culture media of each experimental group confirmed the presence of EVs with a classical morphology and the presence of protein markers defined in the ISEV guidelines (19) and previous characterizations of bovine embryonic EVs (24, 43, 44).\u003c/p\u003e\n\u003cp\u003eEV size decreased as embryonic development progressed, regardless of origin, but was more pronounced in in vivo-derived embryos. An opposite pattern has been observed in pigs and humans, with a progressive increase in particle size throughout pre-implantation development (25, 45, 46). The largest EVs size was observed in IVV embryos collected on day 5, which also showed slower growth and a smaller diameter on day 11 of development. Therefore, it appears that the higher stress associated with early collection of IVV embryos is affecting not only the quality of the embryos but also the population of released EVs. The works by Mellisho et al. (2019) and Dissanayake et al. (2020) showed an inverse pattern, with the lowest mean size of released EVs observed for lower-quality embryos. However, in both reports, all embryos analyzed were produced in vitro, and EV collection was performed at different developmental windows. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the other hand, EV concentration in IVV embryos was consistent across both developmental stages studied (blastulation and hatching). In contrast, IVP embryos showed an increase in EV concentration as development progressed. In previous studies, variations in EV concentration were associated with embryonic quality rather than with developmental stage (44, 47). Here, in agreement with the literature, IVP embryos, which are considered less competent, released a higher concentration of EVs compared to vivo-derived embryos. It is known that in vitro conditions alter embryo metabolism, developmental kinetics, and gene expression, leading to a stress profile during culture (48-50). In other cell types, stress promotes the release of more EVs to remove unwanted molecules (51, 52). Higher EV release by embryos with compromised competence may be a compensatory mechanism that mitigates molecular stress and allows continued development.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe analysis of miRNA content in EVs has gained significance, as these molecules influence essential processes post-transcriptionally, including pluripotency, cellular differentiation, and endometrial preparation for implantation (53, 54). Several studies have demonstrated that the miRNA profile in EVs varies with the embryo\u0026apos;s characteristics [42] and can influence gene expression and target cell function (17).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePCA analysis revealed that miRNA composition varied by embryo origin and stage. The miRNA clusters across developmental windows showed distinct grouping, confirming dynamic reorganization of their content as development progresses. IVV embryos had more stable profiles, whereas IVP embryos displayed heterogeneous profiles, consistent with their lower competence. These findings align with previous studies showing that miRNAs in EVs vary across stages, including compaction, blastulation, and later development, potentially acting as key regulators of pluripotency, cell differentiation, and endometrial signaling (44, 55).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGlobal expression profiling identifies 25 highly variable miRNAs that predominantly regulate pathways essential for implantation and early embryonic development. Numerous studies in mammalian embryos have demonstrated the presence of miR-1468-5p, miR-7, miR-124, and miR-29a in embryonic EVs, with regulatory functions associated with cell proliferation, apoptosis, and differentiation (56-59). Mechanistically, miR-1468-5p targets \u003cem\u003eRRM1\u003c/em\u003e to regulate cell-cycle progression and caspase-3 activation, while miR-7 modulates the \u003cem\u003eTGF-\u0026beta;/Smad\u003c/em\u003e pathway controlling epithelial\u0026ndash;mesenchymal transition (60, 61). Similarly, miR-124 represses \u003cem\u003eSOX9\u003c/em\u003e and \u003cem\u003ePAX6\u003c/em\u003e, facilitating lineage specification, and miR-29a promotes endometrial decidualization by negatively regulating inhibitors of the TGF-\u0026beta;/Smad signaling pathway, enabling stromal differentiation (62, 63).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the other hand, miR-9 was associated with \u003cem\u003ethe regulation of E-cadherin and VEGF\u003c/em\u003e, processes essential for vasculogenesis and maintenance of pluripotency. At the same time, miR-127 promotes mesendoderm differentiation during early embryonic development by suppressing \u003cem\u003eLefty2\u003c/em\u003e, thereby enhancing Nodal/Smad2 signaling (64, 65). miR-155 contributed to osmotic regulation (AQP3/AQP7) and immune tolerance through Treg modulation (66). The exclusive expression of miR-Let-7g in \u003cem\u003ein vivo\u003c/em\u003e-derived embryos supports its role in synchronizing embryonic growth with uterine receptivity by suppressing c-Myc/mTOR (67, 68).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOverall, the literature confirms that these miRNAs cluster around three fundamental functional axes: pluripotency and differentiation, embryonic competence, and immune modulation related to embryonic viability. These findings support the notion that EV content provides a dynamic representation of embryonic development and of molecular communication with the endometrium. A conserved core of 65 miRNAs was identified, suggesting a foundational set of signals packaged in EVs that remain consistent across origins and developmental stages. Simultaneously, another subset of miRNAs was observed to be exclusively packaged in EVs, with their presence depending on both origin and stage, reflecting environmental adaptability and embryonic competence (54).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe unique miRNAs in the blastulation window (D5-7) are let-7c, miR-181a, miR-23b, miR-92a, and miR-409, reflecting their importance during this period. Let-7c is associated with regulating cell proliferation and differentiation, particularly during the transition to pluripotency (69, 70). MiR-181a regulates proliferation and apoptosis (71). miR-23b is involved in energy metabolism and differentiation, serving a compensatory function against oxidative stress in culture (72, 73). Lastly, miR-409-3p modulates Wnt/\u0026beta;-catenin and MAPK/p38 signaling pathways involved in cell differentiation, which influence embryo-maternal communication, and its exclusive presence in early IVP embryos suggests activation of pathways not observed in IVV embryos (74).\u003c/p\u003e\n\u003cp\u003eDuring the transition to hatching (D7-9), IVP embryos did not exhibit exclusive miRNAs, suggesting that the EV profile homogenizes as development progresses. This observation supports the idea that, although differences between IVV and IVP persist, miRNA profiles in EVs tend to converge during hatching (53). Conversely, EVs from IVV (D7-9) contained three exclusive miRNAs that could be associated with the activation of regulatory mechanisms promoting embryo-maternal communication during peri-implantation. The miR-Let7i, a member of the Let-7 family, acts as a modulator of uterine receptivity by suppressing the Wnt/\u0026beta;-catenin pathway under ovarian hormone regulation, facilitating cellular adhesion and embryonic attachment, and linking its expression to greater implantation success (68, 75). Its reduced expression in in vitro embryos, reported during blastulation, indicates loss of molecular equilibrium that may compromise trophoblast differentiation and maternal receptivity (76). Overexpression of miR-16-1 disrupts epigenetic regulation and promotes apoptosis by repressing \u003cem\u003eBcl-2\u003c/em\u003e (\u003cem\u003eSuz12\u003c/em\u003e, \u003cem\u003eKmt2a\u003c/em\u003e), leading to impaired development and early embryonic death (77).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVariation in miRNA profiles across embryonic origin and developmental stage is directly reflected in the biological processes they regulate. During blastulation (D5-7), biological processes exhibit a relatively conserved functional pattern, with transport and osmoregulation (e.g., glycerol and water transport) conserved across origins. This core function has expanded to include pathways related to cellular communication and immune modulation, signaling the preparation for interaction with the endometrium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnalysis of the biological processes and proteins common to each stage and embryo origin confirmed the existence of a conserved nucleus, with water transport and cytolysis standing out. Water transport, supported by AQP3 and AQP7, is an essential and widely conserved mechanism for the formation and maintenance of the blastocoel, as demonstrated in bovine embryos produced both in vivo and in vitro (78, 79). Cytolysis is the second most prevalent process, facilitated by the genes PRF1 and GZMB. Its functions are associated with cytotoxicity facilitated by T and NK cells (80, 81). In the context of embryonic EVs, their presence primarily indicates embryo-maternal communication and initial immunological regulation, as evidenced by bovine embryo-derived signals that elicit immunomodulatory responses in the endometrium (80, 82).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExclusive biological processes and genes originate from this conserved core, differentiated by their origin and stage. In IVP embryos during blastulation (D5-7), the glutamatergic synaptic transmission regulation was identified as an exclusive process, an uncommon observation in this context (83). Experimental evidence demonstrates that NMDA receptor subunits are expressed and functionally regulated during cellular differentiation in various tissues (83, 84). In line with these observations, the detection of glutamatergic pathway activation in embryos suggests that NMDA receptor\u0026ndash;related signaling contributes to cellular differentiation processes, consistent with mechanisms described in other developmental systems.\u003c/p\u003e\n\u003cp\u003eDuring IVP embryo hatching, the highest functional diversification was observed, involving processes related to glycerol transport, GMP-mediated signaling, and the unfolded protein response (UPR), as supported by the overexpression of DNAJA1, HSPA13, and HSPA4, molecular chaperones associated with cell survival under stress conditions (85, 86). In addition, Processes associated with cell proliferation and muscle fiber development were linked to the expression of structural and regulatory genes such as \u003cem\u003eACTA1\u003c/em\u003e, \u003cem\u003eFAM83A\u003c/em\u003e, and \u003cem\u003ePDS5B\u003c/em\u003e, which play essential roles in sarcomeric organization, growth signaling (PI3K/AKT), and embryonic differentiation (87-89). Positive regulation of Ca\u0026sup2;⁺\u0026nbsp;signaling was also detected, involving channels such as CATSPER4, which modulates intracellular calcium levels, influencing cellular activation and motility\u0026nbsp;(90, 91).\u003c/p\u003e\n\u003cp\u003eThese exclusive processes in IVP embryos demonstrate an adaptive metabolic response characteristic of embryos developed under in vitro conditions. The activation of AQP7 and KCNU1 indicates osmotic regulation mechanisms that support blastocoel maintenance in suboptimal environments (92-94). The increased expression of molecular chaperones (HSPA4, HSPA13, DNAJA1) confirms the activation of the UPR, a process associated with endoplasmic reticulum stress and protein homeostasis (95). The concurrent enrichment of GMP-mediated signaling and Ca\u0026sup2;⁺\u0026nbsp;regulation reflects coordinated second messenger activity that sustains cell viability and energy balance during late pre-implantation development\u0026nbsp;(96, 97).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn contrast, during the hatching stage, IVV embryos showed enriched biological processes primarily related to sodium homeostasis and T cell-mediated cytotoxicity, as evidenced by the expression of KCNC2, CALCR, GZMM, and C6. These findings highlight the role of ionic regulation in maintaining fluid balance and blastocoel integrity, which is essential for sustained embryonic expansion and viability (Fujishima et al., 2025). Simultaneously, the involvement of immune-related genes such as GZMM and C6 suggests early activation of immunomodulatory mechanisms, possibly contributing to the establishment of maternal tolerance and effective embryo-endometrial communication (98, 99). This coordinated activation of ion homeostasis and immune signaling may reflect the superior implantation potential of IVV embryos.\u003c/p\u003e\n\u003cp\u003eThis more limited and specialized profile in IVV reflects a functional repertoire aimed at interacting with the endometrium and modulating the local immune system, aligning with the greater effectiveness of EVs derived from in vivo sources in inducing coordinated responses in the uterine epithelium (27, 53). Overall, IVP embryos tend to activate adaptive stress pathways. In contrast, their IVV counterparts emphasize ionic balance and controlled immunomodulation, both of which contribute to establishing a more effective dialogue with the endometrium. This interpretation is consistent with previous studies showing that in vitro embryos exhibit greater molecular heterogeneity and greater dependence on compensatory mechanisms, whereas in vivo embryos maintain more stable, implantation-oriented molecular profiles (24, 27, 44, 53, 55). Subsequently, the functional validation of anticipated pathways and miRNA targets will be crucial to ascertaining their roles. Optimizing culture conditions to replicate vivo signaling may also improve IVP embryo competence.\u0026nbsp;\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study confirms that both the origin (IVV vs. IVP) and developmental stage (blastulation vs. hatching) of bovine embryos critically influence the characteristics and molecular composition of EVs, particularly their miRNA cargo. IVV embryos exhibit superior developmental competence, more stable EV profiles, and miRNA signatures linked to ionic balance and controlled immunomodulation. In contrast, IVP embryos exhibit increased EV release, greater heterogeneity in miRNA profiles, and activation of stress-related pathways, suggesting adaptive mechanisms in response to suboptimal culture conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe observed divergence in EV cargo and associated biological processes reveals that EVs play a dynamic role in pre-implantation embryo\u0026ndash;maternal communication. These differences have profound implications for reproductive biotechnology, as they provide a potential non-invasive biomarker platform to assess embryo quality and competence. Future research should focus on functionally validating the predicted targets of EV-associated miRNAs and on evaluating their impact on maternal endometrial responses. Such insights will contribute to the development of novel diagnostic and therapeutic strategies to enhance implantation success and pregnancy rates in assisted reproduction programs.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCa\u0026sup2;⁺: calcium ion; CPM: counts per million; EMT: epithelial\u0026ndash;mesenchymal transition; EVs: extracellular vesicles; GO: Gene Ontology; GMP: cyclic guanosine monophosphate; IVP: in vitro-produced; IVV: in vivo-derived; Log FC: log₂ fold change; miRNA/miRNAs: microRNA(s); NTA: nanoparticle tracking analysis; PCA: principal component analysis; SEM: standard error of the mean; TEM: transmission electron microscopy; TGF-\u0026beta;: transforming growth factor beta; UPR: unfolded protein response.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethics Committee of the Faculty of Veterinary Sciences, University of Concepci\u0026oacute;n, for the execution of research project No. 1210334.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study, including miRNA profiles and EV characterization data, are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by FONDEF Project ID18I10082; FONDECYT Projects 1170310 and 1210334; ANID Scholarship 21201280 and 21220042\u0026nbsp;(National Agency for Research and Development)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-first authorship:\u003c/strong\u003e M.A.G.-R. and I.M.-H contributed equally to this work and shared first authorship. \u003cstrong\u003eConceptualization:\u003c/strong\u003e L.R.-A.; F.O.C. \u003cstrong\u003eMethodology:\u003c/strong\u003e L.R.-A.; M.A.G.-R. \u003cstrong\u003eInvestigation:\u003c/strong\u003e M.A.G.-R; I.M.-H.; Y.S.W.; F.N.; B.M.-B.; C.A. \u003cstrong\u003eData curation:\u003c/strong\u003e M.A.G.-R; I.M.-H.; L.R.-A.; Y.S.W.; F.N.; C.A. \u003cstrong\u003eFormal analysis:\u003c/strong\u003e M.A.G.-R; I.M.-H.; Y.S.W; L.R.-A. \u003cstrong\u003eResources:\u003c/strong\u003e L.R.-A.; F.O.C. \u003cstrong\u003eSupervision:\u003c/strong\u003e L.R.-A.; F.O.C. \u003cstrong\u003eWriting\u0026ndash;original draft:\u003c/strong\u003e M.A.G.-R; I.M.-H; L.R.-A. \u003cstrong\u003eWriting\u0026ndash;review and editing:\u003c/strong\u003e I.M.-H.; L.R.-A.; F.O.C.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved of the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the staff of the Animal Biotechnology Laboratory at the Universidad de Concepci\u0026oacute;n for technical support, and the local slaughterhouse for providing bovine reproductive material. We also acknowledge the assistance of the microscopy facility for TEM imaging.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eViana JHM. Development of the world farm animal embryo industry over the past 30 years. Theriogenology. 2024;230:151\u0026ndash;6. https://doi.org/10.1016/j.theriogenology.2024.09.012\u003c/li\u003e\n\u003cli\u003eNedambale TL, Dinny\u0026eacute;s A, Yang X, Tian XC. Bovine Blastocyst Development In Vitro: Timing, Sex, and Viability Following Vitrification1. Biology of Reproduction. 2004;71(5):1671\u0026ndash;6. https://doi.org/10.1095/biolreprod.104.027987\u003c/li\u003e\n\u003cli\u003eRizos D, Guti\u0026eacute;rrez-Ad\u0026aacute;n A, P\u0026eacute;rez-Garnelo S, de la Fuente J, Boland MP, Lonergan P. Bovine Embryo Culture in the Presence or Absence of Serum: Implications for Blastocyst Development, Cryotolerance, and Messenger RNA Expression1. Biology of Reproduction. 2003;68(1):236\u0026ndash;43. https://doi.org/10.1095/biolreprod.102.007799\u003c/li\u003e\n\u003cli\u003eStringfellow DA, Givens MD, International Embryo Transfer S. Manual of the International Embryo Transfer Society : a procedural guide and general information for the use of embryo transfer technology emphasizing sanitary procedures. 4th ed. Savory, Ill.: International Embryo Transfer Society; 2010 2010. \u003c/li\u003e\n\u003cli\u003eRodr\u0026iacute;guez-Alvarez L, Manriquez J, Velasquez A, Castro FO. Constitutive expression of the embryonic stem cell marker OCT4 in bovine somatic donor cells influences blastocysts rate and quality after nucleus transfer. In Vitro CellDevBiol-Animal. 2013;49(9):657\u0026ndash;67. https://doi.org/10.1007/s11626-013-9650-0\u003c/li\u003e\n\u003cli\u003eBerg DK, van Leeuwen J, Beaumont S, Berg M, Pfeffer PL. Embryo loss in cattle between Days 7 and 16 of pregnancy. 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JNCI: Journal of the National Cancer Institute. 2000;92(19):1564\u0026ndash;72. https://doi.org/10.1093/jnci/92.19.1564\u003c/li\u003e\n\u003cli\u003eFatima S, Gupta A, Priya S. Cellular DNAJBs are selectively associated with proteotoxic stress and underlying mechanisms in neurodegenerative conditions. 2025 \u003c/li\u003e\n\u003cli\u003eOchala J, Ravenscroft G, Laing N, Nowak K. Nemaline Myopathy-Related Skeletal Muscle \u0026alpha;-Actin (ACTA1) Mutation, Asp286Gly, Prevents Proper Strong Myosin Binding and Triggers Muscle Weakness. PLoS ONE. 2012;7 https://doi.org/10.1371/journal.pone.0045923\u003c/li\u003e\n\u003cli\u003e\u0026lt;Viana - 2019 Statistics of embryo production and transfer in domestic farm animals.pdf\u0026gt;. \u003c/li\u003e\n\u003cli\u003eGarg A, Jansen S, Greenberg L, Zhang R, Lavine K, Greenberg M. Dilated cardiomyopathy\u0026ndash;associated skeletal muscle actin (ACTA1) mutation R256H disrupts actin structure and function and causes cardiomyocyte hypocontractility. Proceedings of the National Academy of Sciences of the United States of America. 2024;121 https://doi.org/10.1073/pnas.2405020121\u003c/li\u003e\n\u003cli\u003eChung J, Shim S, Zhuang X, Clapham D. Catsper Channel Organizes and Regulates Calcium Signaling Molecules in Spermatozoa. Biophysical Journal. 2013;104 https://doi.org/10.1016/j.bpj.2012.11.3501\u003c/li\u003e\n\u003cli\u003eHuang X, Miyata H, Wang H, Mori G, Iida-Norita R, Ikawa M, et al. A CUG-initiated CATSPER\u0026theta; functions in the CatSper channel assembly and serves as a checkpoint for flagellar trafficking. Proceedings of the National Academy of Sciences of the United States of America. 2023;120 https://doi.org/10.1101/2023.03.17.532952\u003c/li\u003e\n\u003cli\u003eSha X-y, Xiong Z-f, Liu H-s, Di X-d, Ma T-h. Maternal-fetal fluid balance and aquaporins: from molecule to physiology. Acta Pharmacologica Sinica. 2011;32(6):716\u0026ndash;20. \u003c/li\u003e\n\u003cli\u003eWang F, Kooistra M, Lee M, Liu L, Baltz JM. Mouse Embryos Stressed by Physiological Levels of Osmolarity Become Arrested in the Late 2-Cell Stage Before Entry into M Phase1. Biology of Reproduction. 2011;85(4):702\u0026ndash;13. https://doi.org/10.1095/biolreprod.111.090910\u003c/li\u003e\n\u003cli\u003eSchliffka MF, Dumortier JG, Pelzer D, Mukherjee A, Ma\u0026icirc;tre J-L. Inverse blebs operate as hydraulic pumps during mouse blastocyst formation. Nature Cell Biology. 2024;26(10):1669\u0026ndash;77. https://doi.org/10.1038/s41556-024-01501-z\u003c/li\u003e\n\u003cli\u003eHemagirri M, Chen Y, Gopinath SCB, Sahreen S, Adnan M, Sasidharan S. Crosstalk between protein misfolding and endoplasmic reticulum stress during ageing and their role in age-related disorders. Biochimie. 2024;221:159\u0026ndash;81. https://doi.org/https://doi.org/10.1016/j.biochi.2023.10.019\u003c/li\u003e\n\u003cli\u003eTiyyagura SR, Kazerounian S, Schulz S, Waldman SA, Pitari GM. Reciprocal regulation and integration of signaling by intracellular calcium and cyclic GMP. Vitam Horm. 2004;69:69\u0026ndash;94. https://doi.org/10.1016/s0083-6729(04)69003-0\u003c/li\u003e\n\u003cli\u003eKashir J, Lai FA, Nomikos M. Editorial: The role of calcium signaling in gametogenesis and early embryogenesis. Front Cell Dev Biol. 2022;10:1051435. https://doi.org/10.3389/fcell.2022.1051435\u003c/li\u003e\n\u003cli\u003eDai CL, Yang HX, Liu QP, Rahman K, Zhang H. CXCL6: A potential therapeutic target for inflammation and cancer. Clin Exp Med. 2023;23(8):4413\u0026ndash;27. https://doi.org/10.1007/s10238-023-01152-8\u003c/li\u003e\n\u003cli\u003eWang Y, Chang Y, Li Z. Extracellular Vesicles in Pregnancy: Functional Insights, Diagnostic Potential for Maternal\u0026ndash;Fetal Disorders, and Therapeutic Implications. Extracellular Vesicle: Biology and Translational Application2024. p. 261\u0026ndash;93. https://doi.org/10.1007/978-981-97-5536-3_13\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTABLE 1. Development and progression of bovine embryos produced in vitro (IVP) and in vivo (IVV) during the blastulation (Days 5\u0026ndash;7) and hatching (Days 7\u0026ndash;9) stages.\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"662\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroups\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN\u0026deg; presumptive zygotes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN\u0026deg; collected embryos\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% morulae\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% blastocyst (n)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% hatched blastocyst\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIVP 5-7\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e612\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e44.9 (275) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e24.7 (151) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e10.5 (64) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIVV\u003cem\u003e\u0026nbsp;\u003c/em\u003e5-7\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e78.7 (48) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e63.9 (39) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e52.5 (32) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIVP 7-9\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e42.2 (289) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e26.7 (187) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e13.1 (92) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIVV 7-9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e83.6 (61) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e71.2 (52) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e61.6 (45) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eDifferent superscript letters (a, b) within the same column indicate statistically significant differences between groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTABLE 2.\u003c/strong\u003e \u003cstrong\u003eConcentration and size parameters of extracellular vesicles (EVs) secreted by bovine embryos during the blastulation (Days 5\u0026ndash;7) and hatching (Days 7\u0026ndash;9) stages under in vitro (IVP) and in vivo (IVV) conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"695\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEV Parameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 248px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlastulation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 248px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHatching\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIVP\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;5-7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIVV\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e5-7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIVP\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;7-9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIVV\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e7-9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eConcentration (particles x ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e2,9 x 10\u003csup\u003e9\u003c/sup\u003e\u0026plusmn; 0,3\u003csup\u003ea(\u0026sect;)\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e2,6 x 10\u003csup\u003e9\u003c/sup\u003e\u0026plusmn; 0,8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e3,8 x 10\u003csup\u003e9\u003c/sup\u003e\u0026plusmn; 0,3\u003csup\u003eb(ţ)\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e2,2 x 10\u003csup\u003e9\u003c/sup\u003e\u0026plusmn; 0,3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eSize (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e124,7\u0026plusmn;3,1\u003csup\u003ea(\u0026sect;)\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e148,5\u0026plusmn;4,1\u003csup\u003eb(\u0026sect;)\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e118,1\u0026plusmn;2,1\u003csup\u003ea(ţ)\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e115,4\u0026plusmn;1,8\u003csup\u003ea(ţ)\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eSuperscript letters (a, b) indicate statistically significant differences between embryo origins within the same stage (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Symbols (\u0026sect;, ţ) indicate statistically significant differences between developmental stages within the same embryo origin (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTABLE 3.\u003c/strong\u003e \u003cstrong\u003eCharacterization of surface markers on extracellular vesicles (EVs) secreted by bovine embryos produced in vitro (IVP) and in vivo (IVV) during the blastulation and hatching stages.\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"614\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroups\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 349px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSURFACE MARKERS (POSITIVE PERCENTAGE)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD9 (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD63 (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD81 (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD40 (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIVP 5-7\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e10,5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e24,4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e36,6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e9,3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIVV 5-7\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e3,5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e7,6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e3,8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e20,8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIVP\u003cem\u003e\u0026nbsp;\u003c/em\u003e7-9\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e17,9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e3,9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e15,7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e9,2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIVV\u003cem\u003e\u0026nbsp;\u003c/em\u003e7-9\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e7,4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e13,0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e7,8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e18,5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBovine blood serum (positive control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e14,5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e6,5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e9,6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e12,0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBeads with antibodies (Negative control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eValues represent the percentage of positivity for surface markers \u003cstrong\u003eCD9\u003c/strong\u003e, \u003cstrong\u003eCD63\u003c/strong\u003e, \u003cstrong\u003eCD81\u003c/strong\u003e, and \u003cstrong\u003eCD40\u003c/strong\u003e on EVs, as determined by flow cytometry.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-animal-science-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jasb","sideBox":"Learn more about [Journal of Animal Science and Biotechnology](http://jasbsci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jasb/default.aspx","title":"Journal of Animal Science and Biotechnology","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"bovine embryo, extracellular vesicles, microRNA, in vitro fertilization, blastulation, hatching, embryo–maternal communication","lastPublishedDoi":"10.21203/rs.3.rs-8248877/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8248877/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eExtracellular vesicles (EVs) play a central role in embryo–maternal communication, transporting bioactive molecules that reflect the embryo's physiological state and developmental competence. This study examined how embryonic origin and developmental stage shape the characteristics and molecular content of EVs secreted by bovine embryos.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eIVV embryos exhibited higher developmental competence and secreted larger EVs whose concentrations remained stable across developmental windows. In contrast, IVP embryos released smaller and more abundant vesicles, particularly during hatching, indicating origin- and stage-specific regulation of EV output. Distinct miRNA profiles clearly separated both embryo types. EVs from IVV embryos were enriched in miRNAs associated with implantation and lineage specification (e.g., miR-124, miR-125, miR-181), whereas EVs from IVP embryos contained higher levels of miRNAs linked to stress response, apoptosis, and the unfolded protein response (e.g., miR-23b, miR-92a, miR-409). Consistently, functional enrichment analyses revealed that IVV-derived miRNAs targeted pathways related to immune modulation and purinergic signaling, while IVP-derived miRNAs were associated with calcium transport and endoplasmic reticulum stress pathways. Together, these differences point to divergent regulatory programs shaped simultaneously by embryonic origin and developmental progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eEmbryonic origin and developmental stage modulate both the biophysical properties and molecular signatures of embryo-secreted EVs. The distinct miRNA landscapes identified in IVV and IVP embryos reflect contrasting developmental trajectories and support the potential of EV-derived miRNAs as non-invasive biomarkers of embryo quality and developmental competence.\u003c/p\u003e","manuscriptTitle":"Comparative profiling of extracellular vesicles and miRNA cargo from in vivo- and in vitro-derived bovine embryos during blastulation and hatching","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-09 15:19:59","doi":"10.21203/rs.3.rs-8248877/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-15T06:10:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-13T10:52:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-11T18:49:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148437864306914167141593648783441646762","date":"2025-12-09T01:16:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"183901191234590773951958579425494068990","date":"2025-12-08T09:38:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-08T07:14:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-03T09:56:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-03T09:55:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Animal Science and Biotechnology","date":"2025-12-01T09:14:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-animal-science-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jasb","sideBox":"Learn more about [Journal of Animal Science and Biotechnology](http://jasbsci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jasb/default.aspx","title":"Journal of Animal Science and Biotechnology","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"eda2b63d-c5d1-42a3-891e-ab96b5bf7342","owner":[],"postedDate":"December 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T16:15:22+00:00","versionOfRecord":{"articleIdentity":"rs-8248877","link":"https://doi.org/10.1186/s40104-026-01378-y","journal":{"identity":"journal-of-animal-science-and-biotechnology","isVorOnly":false,"title":"Journal of Animal Science and Biotechnology"},"publishedOn":"2026-04-13 15:57:14","publishedOnDateReadable":"April 13th, 2026"},"versionCreatedAt":"2025-12-09 15:19:59","video":"","vorDoi":"10.1186/s40104-026-01378-y","vorDoiUrl":"https://doi.org/10.1186/s40104-026-01378-y","workflowStages":[]},"version":"v1","identity":"rs-8248877","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8248877","identity":"rs-8248877","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}