Incorporating olive (Olea europaea L) fruit extracts in a tris-based extender improves buffalo semen cryotolerance by reducing oxidative stress

This work aimed to evaluate whether supplementing the freezing extender with olive fruit extract (OFE) would improve the antioxidant defense of buffalo sperm, resulting in improved post-thaw semen quality. Ejaculates (two per 16 Murrah buffalo bulls) were split into four aliquots that were diluted in an extender supplemented with different doses of OFE (0, D50, D100, and D150, based on mu M concentrations of hydroxytyrosol, the most represented polyphenol) and frozen according to standard procedures. At thawing, sperm motility, kinetics, viability, acrosome integrity, and membrane functionality were evaluated. Based on the dose-response results, biological antioxidant potential (BAP) and reactive oxygen metabolites (ROMs) were assessed after thawing in D50 and control groups. The pre-freezing supplementation of the extender with D50 OFE showed higher (P < 0.05) total and progressive sperm motility, as well as straight-line velocity compared to the control. Treatment with D50 OFE of buffalo semen also improved (P < 0.01) post-thaw sperm viability, membrane functionality, and acrosome integrity compared to the control. The enrichment of the extender with D50 OFE increased (P < 0.01) the post-thaw BAP and reduced (P < 0.05) the ROMs levels. The highest concentration tested (D150 OFE) negatively affected (P < 0.05) total and progressive motility, and the percentage of sperm with functional membranes and intact acrosomes, compared to the control. In conclusion, low doses of OFE added to the extender significantly improved post-thawing buffalo semen quality by protecting the spermatozoa from cryopreservation-induced oxidative stress. Further studies should investigate its effectiveness on in vivo and in vitro fertility, for potential commercial applications.