Oxidative stress impairs sperm function and fertility in rams, most notably during cryopreservation, and is characterized by compromised sperm function and motility. Mitochondria contribute immensely towards sperm function, such as mitochondrial membrane potential (MMP) which is crucial for ATP production. The purpose of this study was to assess the role of MitoPBN supplementation on cryopreserved sperm quality in mature Ghezel ram sperm. Semen samples were treated with 0 (control), 100, 150, 200, and 250 µmol/L MitoPBN and then analyzed for key sperm factors. Results indicated that supplementation with 100 and 150 µmol/L MitoPBN significantly enhanced total motility, increasing to 52.36 ± 4.26% and 54.16 ± 3.19%, respectively. Progressive motility similarly improved to 24.82 ± 3.27% and 26.77 ± 3.46%, respectively. Additionally, sperm membrane integrity was markedly increased to 50.01 ± 4.22% and 52.54 ± 2.24%, while mitochondrial activity was significantly augmented from 35.14 ± 4.09% to 46.16 ± 4.02% and 50.26 ± 6.69%. Sperm viability also improved, rising to 48.99 ± 3.98% and 52.20 ± 3.17%, respectively. Notably, reactive oxygen species (ROS) levels were reduced to 2.95 ± 0.16% and 2.80 ± 0.11%, respectively, paralleled by enhancements in total antioxidant capacity (TAC; 1.85 ± 0.21% and 1.93 ± 0.16%, respectively) and glutathione peroxidase (GPx) activity (61.16 ± 4.77% and 63.36 ± 4.95%, respectively). Moreover, ATP content reached a peak of 116.29 ± 5.83 in the 150 µmol/L group. In conclusion, our results evidence that oxidative stress during ram sperm cryopreservation is effectively countered by MitoPBN, improving sperm quality.
