Conditioned Medium from Canine Amniotic Membrane-Derived Mesenchymal Stem Cells Improved Dog Sperm Post-Thaw Quality-Related Parameters

SIMPLE SUMMARY: Mesenchymal stem cells and their derivatives are used in clinical studies for their anti-apoptotic, anti-oxidant, immunomodulatory, and regenerative properties. Their use in reproductive medicine is increasing as they have been proved to be beneficial for infertility treatment. Mesenchymal stem cells can secrete factors that influence biological processes in target tissues or cells; these factors are either directly secreted by the cells or mediated through their derivatives. Although the amniotic membrane is easy to obtain and is a good source of stem cells, clinical trials using amniotic membrane-derived mesenchymal stem cells are still uncommon, especially in reproductive medicine or artificial reproductive technologies. The objective of the present study was to demonstrate the effects of conditioned medium prepared from amniotic membrane-derived stem cells on dog sperm cryopreservation. Our results showed that 10% of the conditioned medium enhanced the quality-related parameters of frozen–thawed sperm cells because of the presence of antioxidants and growth factors in the medium, which probably protected spermatozoa during the freeze–thaw process. These results suggest that conditioned media prepared from amniotic membrane-derived mesenchymal stem cells might have clinical applications in assisted reproductive technologies. ABSTRACT: This study investigated the effects of conditioned medium (CM) from canine amniotic membrane-derived MSCs (cAMSCs) on dog sperm cryopreservation. For this purpose, flow cytometry analysis was performed to characterize cAMSCs. The CM prepared from cAMSCs was subjected to proteomic analysis for the identification of proteins present in the medium. Sperm samples were treated with freezing medium supplemented with 0%, 5%, 10%, and 15% of the CM, and kinetic parameters were evaluated after 4–6 h of chilling at 4 °C to select the best concentration before proceeding to cryopreservation. Quality-related parameters of frozen–thawed sperm were investigated, including motility; kinetic parameters; viability; integrity of the plasma membrane, chromatin, and acrosome; and mitochondrial activity. The results showed that 10% of the CM significantly enhanced motility, viability, mitochondrial activity, and membrane integrity (p < 0.05); however, the analysis of chromatin and acrosome integrity showed no significant differences between the treatment and control groups. Therefore, we concluded that the addition of 10% CM derived from cAMSC in the freezing medium protected dog sperm during the cryopreservation process.