Abstract
Cryopreservation of fish sperm is a vital tool for advancing aquaculture, genetic studies, and biodiversity preservation. Nevertheless, the freeze-thaw cycle subjects spermatozoa to considerable cryodamage, primarily driven by oxidative stress and resulting cellular injury, which compromises post-thaw viability and fertility. In this study, pomegranate peel extract (PPE) was investigated as a possible natural antioxidant supplement for the cryopreservation medium of Nile tilapia (Oreochromis niloticus) sperm. 0%, 0.5%, 1.0%, 1.5%, or 2.0% PPE were added to extenders. The 1.0% PPE treatment yielded the highest outcomes, significantly boosting post-thaw total sperm motility to 72.4% and progressive motility to 58.9%, compared to control values of 45.2% and 32.1%, respectively. Sperm viability was 78.5% (control: 51.3%), and membrane integrity was 65.8% (control: 40.2%). Malondialdehyde levels, an indicator of lipid peroxidation, were lowest at 1.0% PPE, whereas antioxidant enzyme activity (SOD, CAT, and GPx) were highest. The DNA fragmentation index significantly decreased from 25.7% to 8.4% in comparison to the control. Gene expression study revealed elevated antioxidant genes (SOD1, CAT, and GPX1) and a positive anti-apoptotic shift. These results indicate that 1.0% PPE mitigates cryodamage by bolstering antioxidant defenses and inhibiting apoptosis, thereby improving post-thaw sperm quality and presenting a sustainable, eco-friendly option for tilapia germplasm banking.