Effect of Tris-Citric-Fructose-Asolectin extender on mitochondrial activity and intracellular reactive oxygen species in cryopreserved dog semen

The aim of this study was to evaluate the cryoprotective capacity of Tris-Citrate-Fructose (TCF) extenders supplemented with soy lecithin (asolectin) at concentrations of 0.05% (Asol 0.05%) and 0.5% (Asol 0.5%), in comparison with the commonly used egg yolk-based extender (TCF-EY). Ten ejaculates, each obtained from a different dog, were included in the experiment. Both fresh and cryopreserved semen were assessed for total motility (TM) and progressive motility (PM) using computer-assisted sperm analysis (CASA). Plasma membrane integrity (LIVE), DNA fragmentation index (DFI), mitochondrial activity (MT-/PI+, MT+/PI+, MT+/PI-), and oxidative stress (CellROX+/DRD+, CellROX-/DRD-, CellROX+/DRD-) were measured by flow cytometry. Significant differences (p<0.05) were found in TM (%) between the TCF-EY and Asol 0.5% groups (51.16±5.80 vs. 22.33±9.62). However, no significant differences were observed in LIVE sperm. The DFI remained below 5% in all examined groups. Compared with the control TCF-EY extender, both soy-supplemented groups showed a significant reduction in the population of viable sperm without oxidative stress. The use of asolectin-based extenders also significantly reduced the percentage of viable sperm with active mitochondria, compared to the TCF-EY extender. There were no significant differences between the tested two soy lecithin concentrations in any of the evaluated parameters. In conclusion, the TCF-EY extender demonstrated superior efficacy in preserving semen quality after cryopreservation. Further research is needed to explore alternative phospholipid sources, including different types and concentrations of soy lecithin, and their effects on sperm fertilizing capacity.