Cryoprotective effects of mesenchymal stem cell and seminal plasma-derived extracellular vesicles on canine sperm.

In this study, the lack of standardized freezing protocols for sperm cryopreservation in dogs and the limited research on using exosomes in cryopreservation were considered. Additionally, unlike previous studies on sperm cryopreservation, we introduced an innovative approach using the Aqueous Two-Phase System (ATPS) method for exosome isolation. This study aimed to evaluate the sperm-protective effects of adipose tissue-derived mesenchymal stem cell exosomes (MSC-exo) and seminal plasma exosomes (SP-exo) in dog sperm cryopreservation. Ejaculates from six dogs were processed with Tris-based diluents and divided into four groups: MSC-exo, 1.5 % SP-exo, 2 % SP-exo, and control, and frozen. After thawing, sperm motility, viability, membrane integrity, chromatin integrity, morphological integrity, and gene expression levels were analyzed. The results showed that the MSC-exo group had significantly higher total motility (%60.31 ± 6.12), progressive motility (%22.09 ± 3.34), plasma membrane integrity (%66.94 ± 2.24), and viability (%70.88 ± 1.95) compared to the other groups (P < 0.05). Additionally, the normal chromatin packaging rate was highest in the MSC-exo group (%91.33 ± 0.61, P < 0.05). While some improvements were observed in the SP-exo groups, they were not as pronounced as in the MSC-exo group. No significant differences were found in gene expression levels, although an improvement trend was observed in the MSC-exo group. In conclusion, MSC-exo reduced cryopreservation-induced sperm damage and provided overall protection in sperm parameters. These findings suggest that MSC-exo could be a potential biological additive in dog sperm freezing protocols.