Nanozyme conjugated chitosan nanoparticles improve Buffalo Bull Sperm Cryopreservation by Enhancing Antioxidant Defense and Mitochondrial Function

Ramya Ahmad Sindi¹Mohammed A. Alfattah²Mahmoud A. E. Hassan³Ehab El-Haroun⁴Ahmed E. Noredlin⁵Sameh A. Abdelnour^6*

• ¹Umm Al-Qura University College of Applied Medical Sciences, Makkah, Saudi Arabia
• ²Jazan University, Jazan, Saudi Arabia
• ³Agriculture Research Centre, Ministry of Agriculture, Giza, Egypt
• ⁴United Arab Emirates University College of Agriculture and Veterinary Medicine, Al Ain, United Arab Emirates
• ⁵Damanhour University Faculty of Veterinary Medicine, Damanhour, Egypt
• ⁶Faculty of Agriculture, Zagazig University, Zagazig, Egypt

Introduction: Nanozymes, a cutting-edge development in nanotechnology, offer a versatile and promising tool for mitigating oxidative stress caused by cryo-injury. This study targeted to assess the protective effects of selenium conjugated chitosan nanoparticles (SeCN; as a nanozyme) when added to semen freezing extenders in buffalo bulls. Methods: Semen samples were extended with 0 (SeCN0), 0.5 (SeCN0.5), 1 (SeCN1), or 2 (SeCN2) μg/mL SeCN, frozen at −196 °C, and assessed post-thaw for sperm quality, antioxidant status, mitochondrial activity and ultrastructural changes. Results: The SeCN supplementation significantly improved all post-thawed sperm parameters in a dose-dependent manner (P < 0.001). A significant linear increase (P<0.01) was monitored in viability, sperm progressive motility, and plasma membrane integrity with increasing concentrations of SeCN supplementation. Sperm abnormalities decreased linearly with SeCN supplementation, reaching the lowest rate in the SeCN2 group (P<0.01 compared to other groups). Chromatin damage decreased significantly in a cubic manner in the SeCN1 and SeCN2 groups compared to the other groups (P<0.01). A clear linear increase in both SOD levels and TAC levels (P<0.01). The SeCN2 group exhibited the highest SOD activity, while the SeCN1 and SeCN2 groups showed the highest TAC values (P<0.01) compared to other groups. Additionally, GPx activity in all SeCN-supplemented groups demonstrated a cubic increase (P<0.01) compared to the control group. The SeCN2 group showed the most effective reduction in MDA levels, followed by the SeCN1 and SeCN0.5 groups (p <0.001). Nitric oxide was significantly decreased in a linear manner by the addition of SeCN (p < 0.001). A significant cubic increase in mitochondrial membrane potential (MMP) was observed, with the addition of 1 or 2 μg of SeCN/mL (p < 0.001). Ultrastructural analysis via transmission electron microscopy confirmed improved preservation of acrosomal, mitochondrial, and plasma membrane integrity of buffalo spermatozoa. Discussion: At a concentration of 1 or 2 μg/mL, SeCN demonstrates potent cryoprotective effects by enhancing sperm function, reducing oxidative stress, and preserving mitochondrial activity and ultrastructure changes of sperm. Incorporating SeCN into semen extenders may improve cryosurvival in buffalo and represents a promising strategy for optimizing artificial insemination outcomes in livestock breeding programs.