Ameliorative potential of dietary supplements, ZnO-K, citrus essential oil, and pumpkin seed oil, on sperm quality in Nile tilapia: Insights from CASA, DNA integrity, antioxidant enzymes, and gene expressions

Improving sperm quality is essential for enhancing the reproductive efficiency of Oreochromis niloticus . This study evaluated the effect of three dietary antioxidant supplements, kaolinite-doped zinc oxide (ZnO-K), citrus essential oil (CEO), and pumpkin seed oil (PSO), on sperm quality parameters. A suite of integrative assessments, including Computer-Assisted Semen Analysis (CASA) parameters, spermatozoa DNA integrity, antioxidant enzyme assays, and gene expression profiling, was applied to validate sperm quality. A total of 192 adult males (mean weight 421.31 g ± 30.41 SD) were assigned to four dietary groups, each with three replicates. The control group received a basal diet without supplementation. The experimental groups were fed diets supplemented with ZnO-K (0.06 g kg⁻ ¹ ), CEO (10 g kg⁻ ¹ ), or PSO (15 g kg⁻ ¹ ). ZnO-K supplementation significantly ( P  < 0.05) increased milt volume (1.40 ± 0.10 ml) and sperm concentration (5.676 × 10 ⁹ sperm ml ⁻¹ ), along with marked improvements in CASA parameters (sperm motility and velocity) and DNA integrity. Antioxidant enzymes, catalase, CAT, glutathione peroxidase, GPX, and superoxide dismutase, SOD, were also significantly elevated in the ZnO-K group, recording activity values of 47.333 ± 1.452 U ml ⁻¹ milt, 65.667 ± 5.547 mU ml ⁻¹ milt and 60.667 ± 3.382 U ml ⁻¹ milt, respectively. Gene expression analysis revealed that CEO induced the highest upregulation of HSP70 , while both CEO and ZnO-K significantly upregulated CC chemokine expression compared to control. In contrast, PSO consistently showed the lowest performance across all measured parameters. Collectively, ZnO-K supplementation most effectively enhanced sperm physiological and biochemical quality, whereas CEO predominantly supported cellular stress protection through gene expression modulation. These findings offered practical implications for optimizing artificial reproduction and sperm cryopreservation strategies in tilapia aquaculture.