Dose-dependent effects of ursolic acid supplementation on post-thaw motility and mitochondrial activity of goat sperm.

Ursolic acid (UA) is a natural pentacyclic triterpene present in many fruits, vegetables and medicinal plants. We tested whether adding UA to the freezing extender improves the quality of frozen-thawed goat sperm. Ejaculates from six bucks were pooled at each collection, and the pooled sample was split into six equal aliquots diluted in Andromed® extender either without UA (UA-0, control) or with 0.5 µM (UA-1), 2 µM (UA-2), 5 µM (UA-3), 15 µM (UA-4) or 30 µM (UA-5). Samples were frozen using a standard protocol and thawed at 37 °C for 30 s. Post-thaw sperm quality was assessed by computer-assisted sperm analysis (CASA) for motility, the hypo-osmotic swelling test for membrane integrity, and flow cytometry for acrosome status (FITC-PSA/PI), phosphatidylserine externalization (Annexin V-FITC), mitochondrial membrane potential (Rhodamine 123) and intracellular reactive oxygen species (H₂DCFDA). Adding UA changed progressive motility (PR) and mitochondrial activity (MA), and both peaked at 0.5 µM. PR at 0.5 µM was significantly higher than at 15-30 µM, and MA at 0.5-2 µM was significantly higher than at 30 µM; however, no UA concentration differed significantly from the control. Total motility, membrane integrity (HOST), acrosome integrity, phosphatidylserine externalization and intracellular ROS did not differ among the treatments. In short, the response was dose-dependent: sperm frozen with 0.5 µM UA retained the best PR and MA, the high concentrations were harmful, and an improvement over the control itself could not be demonstrated. Confirming a fertility benefit will require in vitro fertilization and in vivo insemination trials.