Improving the cryo-resistance of Egyptian buffalo semen via chlorogenic acid and rutin supplementation: molecular docking, antioxidant, and anti-apoptotic pathways

Abstract This study compared the antioxidant effects of chlorogenic acid (CGA) and rutin (RU) supplementation in freezing extenders on the cryo-resistance of buffalo spermatozoa. Over eight weeks, 56 ejaculates were collected from seven fertile buffalo ( Bubalus bubalis ) bulls. To eliminate individual animal variability, high-quality ejaculates were pooled and extended in a Tris–egg yolk medium. The pooled semen was divided into three treatment groups: control (no additive), 0.6 mM RU, and 150 µM CGA. All groups were cryopreserved using a standard protocol and stored in liquid nitrogen for one month prior to post-thaw analysis. The findings revealed that the addition of either RU or CGA substantially enhanced post-thaw sperm viability, progressive motility, plasma membrane integrity, and acrosomal integrity compared with the control group ( P < 0.05). Furthermore, both treatments improved sperm kinematics and antioxidant profiles by significantly increasing total antioxidant capacity and catalase levels ( P < 0.05). This was accompanied by a significant decline in oxidative markers (MDA, ROS, and H 2 O 2 ) ( P < 0.05), indicating robust antioxidant activity. Supplementation with RU or CGA significantly increased sperm viability and notably reduced apoptotic percentages ( P < 0.05). Furthermore, CGA treatment resulted in the lowest total bacterial and coliform counts among all experimental groups ( P < 0.05). Molecular docking predictions suggest a potential interaction between CGA and RU against target proteins antioxidant status (HSP70) and mitochondrial function (mitochondrial uncoupling protein 1 and mitochondrial cytochrome c) in sperm. Overall, supplementing with rutin and chlorogenic acid offers robust protection against cryodamage. By mitigating oxidative stress and preserving membrane stability, these antioxidants substantially enhance post-thawed quality, and physiological function of buffalo spermatozoa.