Synergistic effect of extracellular adenosine triphosphate and quercetin on post-thaw quality and fertilization potential of Lohi ram sperm

This study determined the individual and combined effects of extracellular adenosine triphosphate (ATP) and quercetin (QUE) on the quality of post-thawed sperm and the fertilization potential of Lohi rams. In experiment 1, semen samples from four Lohi rams were pooled and extended with different concentrations of ATP or QUE (control; no ATP or QUE, 1 or 2 mM ATP and 10 or 20 μM QUE). In experiment 2, pooled semen samples were extended with various combinations of ATP and QUE (control; no ATP and QUE, 1 mM ATP + 10 μM QUE, 1 mM ATP + 20 μM QUE, 2 mM ATP + 10 μM QUE and 2 mM ATP + 20 μM QUE). All samples in both experiments were cryopreserved and analyzed for post-thawed sperm quality. In experiment 3, the best combination of ATP and QUE from experiment 2 was to extend semen, which was then used for laparoscopic insemination in estrus-synchronized ewes (n = 83). The results of experiment 1 showed that 1 mM ATP and 20 μM QUE treatments resulted in higher total motility, progressive motility, viability, plasma membrane intactness (PMI), and motion kinetics (VCL, VSL, VAP, LIN, and STR) compared to other treatments (p < 0.05). In experiment 2, the 1 mM ATP +10 μM QUE-treated group exhibited significantly higher total and progressive motility, PMI, and motion kinetics (VSL, VCL, VAP, STR, and BCF) compared to the control group (p < 0.05). In experiment 3, the fertilizing potential of sperms treated with 1 mM ATP +10 μM QUE was greater than that of untreated controls (58.1% vs. 27.5%, respectively, p-value = 0.012). In conclusion, the quality of post-thawed ram semen is enhanced when the extender is supplemented with extracellular 1 mM ATP and 20 μM QUE, whether used separately or in combination with 1 mM ATP and 10 μM QUE. Furthermore, the inclusion of 1 mM ATP and 10 μM QUE together in the extender significantly improves in vivo fertility in Lohi ram.