Epididymosome-Supplemented Extender Induces Changes in Morpho-Functional Traits and microRNA Levels of Post-Thaw...

While molecular signature contributes to sperm quality and fertility potential, sperm may lost key molecules during cryopreservation. Since sperm cells are transcriptionally inert and rely on interactions with epididymosomes (epEVs) to acquire molecules, herein, we investigated the effects of the addition of epEVs to the cryopreservation extender on post-thaw sperm quality and fertility potential. Epididymal sperm were obtained from six bulls (n = 6) and a pool of epEVs was formed from the epididymal fluid of 15 bulls. After, a total of 30 × 10⁶ sperm was cryopreserved in the presence (epEVs) or not (Control) of PKH67 green-labeled epEVs. Post-thaw, sperm were evaluated for interaction with epEVs by flow cytometry (FC) and fluorescence microscopy (FM); motility, vigor, and morphology by microscopy; membrane integrity by FC and FM; microRNA levels by qPCR; and fertility potential by in vitro embryo production (IVEP). EpEVs-sperm showed interaction with green-epEVs, and higher motility and plasma membrane integrity compared to Control-sperm. Levels of 28 microRNAs were higher or uniquely detected in epEVs-sperm. IVEP with epEVs-sperm resulted in a higher rate of hatched blastocyst. Thus, epEVs-supplemented to the sperm cryopreservation extender improved post-thaw sperm quality and embryo developmental potential. These findings provide new insights into the molecular modulation of sperm fertility.