Modern cattle reproduction is based on the implementation of in vitro technologies; however, the quality of such embryos often remains inferior to their in vivo counterparts due to the lack of paracrine signals from the maternal organism. This study evaluated the effect of exosomes isolated from an endometrial cell culture on embryo morphology, developmental dynamics, and viability. The study was based on oocyte–cumulus complexes obtained after the slaughter of cows and processed according to standard IVP protocols. Exosomes isolated by sequential ultrafiltration using a 100 kDa cut-off were added to the culture at a ratio of 1 cell equivalent per 5 embryos. To analyze efficiency, 2–4-cell embryos were randomly assigned to a control group and two experimental groups, in which exosomes were introduced at 48 hours (Exo-48h) and 96 hours (Exo-96h) of culture, respectively. This made it possible to determine the role of extracellular vesicles in compensating for the absence of the maternal environment. It was found that the addition of exosomes derived from bovine endometrium to the in vitro system exerted a pronounced stimulatory effect on the preimplantation development of embryos, ensuring a significant increase in the blastocyst rate regardless of the time of their supplementation. As early as on day 7 of culture (168 h), the use of exosomes improved blastulation efficiency: in the Exo-48h group, this parameter increased to 26.7 ± 0.0%, while in the Exo-96h group it reached its maximum of 27.8 ± 2.2%, which was 1.66-fold higher than in the control group (16.7 ± 1.9%). Analysis of morphological parameters revealed a significant intensification of developmental kinetics: whereas only early blastocysts (stage code 5) were present in the control group on day 7 of culture, the experimental groups showed a significant proportion of mature blastocysts (stage code 6), at 8.9–10.0% (P < 0.05). A specific receptive period for the paracrine effect of vesicles on embryo development was observed at the morula stage (96 h). Exosome supplementation during this period provided the highest viability parameters at the final stages of observation. On day 8 (192 h), the experimental groups showed a shift in the morphological profile towards later embryogenesis: the percentage of expanded blastocysts (stage code 7) was 2.33-fold higher than in the control group (15.6 ± 1.1% vs. 6.7 ± 1.9%). A decisive advantage of adding exosomes to the culture medium was the activation of the hatching process (stage code 8), which was completely absent under control conditions on day 8, whereas in the Exo-96h group it reached 11.1 ± 1.1%. On the final, day 9 (216 h) of observation, the prolonged effect of exosomes was confirmed by a substantial increase in implantation potential. The percentage of embryos that successfully overcame metabolic barriers and emerged beyond the zona pellucida in the Exo-96h group reached 35.5 ± 2.9%, which was twice the control value (17.8 ± 1.1%) and significantly higher than the results of early supplementation (Exo-48h — 26.7 ± 1.9%). Thus, endometrial exosomes act as effective mediators of cellular communic a tion, enabling simulation of the maternal microenvironment, accelerating cavitation and intensifying hatching, thereby su b stantially improving the quality of bovine embryos produced in an in vitro sy s tem.
