Abstract* Background and Aim Preservation of Nilem ( Osteochilus vittatus ) spermatozoa is essential for sustainable aquaculture and the development of reproductive biotechnology. This study evaluated the effectiveness of a commercial Habbatussauda ( Nigella sativa ) 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 1 ), 2% (P 2 ), 3% (P 3 ), and 4% (P 4 ). Untreated sperm served as a negative control (P 0 ), while sperm treated with 0.005% pure thymoquinone acted as a positive control (P TQ ). 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 2 ) 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 2 , 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 ( Nigella sativa ) extract effectively preserves Nilem sperm quality and ultrastructure during short-term cold storage, supporting its application as a natural antioxidant protectant in aquaculture.
Astuti et al. (Fri,) studied this question.