Curcumin-loaded niosomal nanocarriers offer a promising approach to improve quality characteristics, apoptotic gene expression, and flow cytometry assessments of stallion spermatozoa after thawing.

The optimization of cryopreservation media to reduce oxidative damage on post-thaw spermatozoa is crucial. This research aimed to assess the antioxidant properties of curcumin-loaded niosomal nanocarriers (CurLNN) on the functional characteristics, the relative expression of apoptotic genes, and flow cytometry assessments of apoptotic-like changes, reactive oxygen species production (ROS), mitochondrial membrane potential, and chromatin integrity in stallion spermatozoa following thawing. Twenty-five ejaculates were diluted in INRA96 freezing media supplemented with 20 μM of either curcumin (Cur) or CurLNN and then cryopreserved. Results demonstrated that spermatozoa treated with Cur, particularly CurLNN, exhibited higher percentages of total and progressive motility, as well as VAP, VSL, and STR kinematics. Additionally, the functionality of the plasma membrane was enhanced, and there was a decrease in spermatozoa abnormality (P < 0.05). The incorporation of cryo-diluent medium with Cur and CurLNN led to increased viability (P < 0.05), while simultaneously reducing the levels of MDA. Flow cytometry analysis revealed a significant enhancement in mitochondrial potential activity, a reduction (P < 0.05) in ROS production, and an increase (P < 0.05) in the proportion of live and a decrease in late apoptotic stallion post-thawed spermatozoa treated with both Cur and CurLNN. Moreover, the relative expression of the Bcl₂ anti-apoptotic gene increased (P < 0.05) by the addition of cur and CurLNN in cryo-diluent extender, while inclusion of CurLNN in cryo-diluent medium resulted in a significant reduction (P < 0.05) in the relative expression of the Bax pro-apoptotic gene in stallion post-thawed spermatozoa. In summary, the findings of this study demonstrated that CurLNN exhibits enhanced antioxidant properties, which contribute to the improved functional quality of spermatozoa by alleviating oxidative stress during the cryopreservation process.