Nigella sativa-Based Extender Enhances Nilem (Osteochilus vittatus) Sperm Quality: Implications for Sustainable Aquaculture Practices

<ns7:p> Abstract* Background and Aim Preservation of Nilem ( <ns7:italic>Osteochilus vittatus</ns7:italic> ) spermatozoa is essential for sustainable aquaculture and the development of reproductive biotechnology. This study evaluated the effectiveness of a commercial Habbatussauda ( <ns7:italic>Nigella sativa</ns7:italic> ) extract as a natural antioxidant-based extender for maintaining sperm quality during 48 h cold storage at 4 °C. Materials and Methods Sperm samples were supplemented with commercial habbatussauda extract at concentrations of 1% (P <ns7:sub>1</ns7:sub> ), 2% (P <ns7:sub>2</ns7:sub> ), 3% (P <ns7:sub>3</ns7:sub> ), and 4% (P <ns7:sub>4</ns7:sub> ). Untreated sperm served as a negative control (P <ns7:sub>0</ns7:sub> ), while sperm treated with 0.005% pure thymoquinone acted as a positive control (P <ns7:sub>TQ</ns7:sub> ). All samples were stored at 4 °C for 48 hours with six replicates per treatment. Post-storage assessments included sperm viability, motility, and morphological abnormalities. Ultrastructural integrity of spermatozoa was qualitatively evaluated using Cryogenic Scanning Electron Microscopy (CRYO-SEM). Results The 2% habbatussauda extract (P <ns7:sub>2</ns7:sub> ) yielded the highest sperm viability (84.80 ± 5.54%) and motility (73.25 ± 10.82%) and significantly reduced abnormality rates compared to the untreated control. CRYO-SEM analysis revealed severe ultrastructural damage in untreated spermatozoa, including plasma membrane disruption, head deformation, and flagellar breakage. In contrast, spermatozoa preserved with 1–2% extract, particularly P <ns7:sub>2</ns7:sub> , maintained intact plasma membranes, normal head morphology, and well-preserved flagella. Higher extract concentrations (3–4%) induced mild ultrastructural alterations. Conclusion A 2% commercial habbatussauda ( <ns7:italic>Nigella sativa</ns7:italic> ) extract effectively preserves Nilem sperm quality and ultrastructure during short-term cold storage, supporting its application as a natural antioxidant protectant in aquaculture. </ns7:p>