Seminal plasma-derived extracellular vesicles improve post-thaw function of cryopreserved ram spermatozoa and in vitro embryo development

Seminal plasma-derived extracellular vesicles (SP-EVs) are emerging as bioactive regulators of sperm function; however, their application during semen cryopreservation remains poorly defined. This study investigated the dose-dependent effects of SP-EV supplementation on post-thaw sperm function and subsequent in vitro embryo development in sheep. SP-EVs were isolated from ram seminal plasma, characterized, and supplemented at 0× (Control), 1×, 4×, and 16× concentrations during ram semen cryopreservation. Post-thaw sperm quality and functional parameters, in vitro embryo development, and embryonic gene expression were evaluated. SP-EV supplementation significantly improved total and progressive motility, viability, and mitochondrial membrane potential while reducing intracellular oxidative stress and DNA fragmentation. Quadratic regression revealed significant non-linear, curvilinear dose–response relationships for several sperm parameters; however, because only four dose levels were tested, these models characterize the response pattern rather than identify a specific optimal concentration. In contrast, the 1× treatment yielded the highest cleavage and blastocyst development rates and was associated with distinct expression patterns of NANOG , GLUT , and IFNT . Overall, SP-EV supplementation improved several post-thaw sperm functional parameters and was associated with higher subsequent cleavage and blastocyst development rates. Importantly, the supplementation level associated with the most favorable post-thaw sperm parameters differed from that associated with the highest cleavage and blastocyst development rates, indicating distinct concentration-dependent responses across reproductive outcomes.