Thymus vulgaris extract as a potent cryoprotectant enhancing antioxidant defense and sperm quality in roosters

Cryopreservation is a key method for conservation of poultry genetic resources but cryopreserved spermatozoa are highly sensitive to oxidative stress and cell damage induced by the freeze-thaw cycle. In this study, the effects of Thymus vulgaris extract addition in the cryopreservation extender on rooster sperm post-thaw quality were evaluated. Semen samples were cryopreserved in the presence of Thymus vulgaris extract at 0, 30, 60, 90, and 120 μg/mL, and post-thaw analysis involved motility, membrane integrity, mitochondrial activity, viability, apoptosis, and oxidative stress markers. The results showed that the 30 μg/mL concentration significantly improved total motility (60.13 ± 0.70 %) and progressive motility (24.76 ± 0.75 %) when compared with the control group (42.78 ± 1.97 % and 18.15 ± 0.71 %, respectively; P < 0.05). Integrity of the plasma membrane (57.23 ± 1.49 %) and mitochondrial function (110.35 ± 3.24 ATP content) were also improved, while apoptotic sperm rates (14.40 ± 1.68 %) and reactive oxygen species levels (2.50 ± 0.06) were significantly lower. In contrast, higher concentrations (90 and 120 μg/mL) exerted a negative effect, leading to compromised motility, elevated apoptosis, and mitochondrial dysfunction. The findings suggest that Thymus vulgaris extract at an optimal concentration improves sperm cryotolerance through the alleviation of oxidative stress and the stabilization of membrane and mitochondrial integrity. The results provide strong evidence for the application of natural antioxidants in avian sperm cryopreservation.