Effect of olive, flaxseed, and grape seed nano-emulsion essential oils on semen buffalo freezability

The existing treatise targeted to compare the effects of adding different nano-emulsions essential oils (olive, flaxseed, and grapeseed oils) in freezing extender on semen quality and freezability in buffalo. Nano-emulsions were prepared from olive, flaxseed, and grapeseed oils and characterized for their sizes and shapes. Semen extended in four tubes were supplemented with 0 (control) and 3.5% nanoemulsion oils, including olive (NEO), flaxseed (NEFO) and grape seed oils (NEGSO) respectively. NEGSO resulted in the highest (p < 0.05) membrane integrity, vitality, progressive motility (P-motility) of sperm compared to the other groups in post-thawed buffalo bull semen (at 37 °C for 30 s). The addition of NEGSO had the best results for membrane integrity, progressive motility, and vitality of sperm after incubation (at 37 °C and 5% CO₂ for 2 h). A superior (p < 0.05) value of total antioxidant capacity in frozen-thawed spermatozoa was monitored in all supplemented groups as relative to the control. The values of malondialdehyde (MDA) and nitric oxide (NO) were lower (p < 0.05) in NEGSO group compared with other groups. Both NEO and NEFO exhibited the same results for MDA, and NO levels (p > 0.05). All supplemented groups exhibited lower hydrogen peroxide levels (p < 0.05) as relative to the un-treated group. The lowest (p < 0.05) caspase 3 levels were verified in NEGSO treatment, followed by NEFO and NEO treatments. Post-thawed sperm showed ultrastructural damages in the control group, and theses damages were attenuated or resorted by the NEGSO, NEFO and NEO supplemented to freezing extender. In consequences with in vitro results regarding the sperm attribute, a greater pregnancy rate (92%) was observed in NEGSO group as compared with NEFO (88%), NEO (76%) and CON (68%) groups. Our findings demonstrate that NEGSO (3.5%) could be used as a new strategy in enhancing sperm functionality, potential fertility and reducing the oxidative damage and apoptosis markers. This could be significantly applicable for sperm physiology cryopreservation in the milieu of assisted reproduction systems.