Natural Antioxidants Enhance Post-Thaw Sperm Quality and Fertilization Performance in Javan Barb (Systomus rubripinnis)

The freeze-thaw process in sperm cryopreservation generates reactive oxygen species (ROS) that impair motility, viability, and fertilizing capacity in fish, including the Javan barb (Systomus rubripinnis), an endemic Indonesian cyprinid of conservation concern. This study evaluated five natural antioxidants: basil leaf extract (BLE), noni fruit extract (NFE), moringa leaf extract (MLE), sambiloto leaf extract (SLE), and honey-egg yolk (HEY). Radical-scavenging capacity was measured by DPPH assay (IC₅₀: BLE 134.06, MLE 297.04, NFE 422.86, SLE 3010.46 ppm), and post-thaw motility, duration of motility, viability, abnormality, fertilization rate, and ATP bioluminescence were assessed. Optimum doses were 1% for BLE, NFE, and MLE, 1.25% for HEY, and 7% for SLE. BLE at 1% gave the best overall profile (motility 85.15 ± 0.88%; duration 7.14 ± 0.38 min; viability 82.21 ± 1.86%; abnormality 18.29 ± 0.16%), significantly exceeding the control (65.00 ± 2.31%; 4.56 ± 0.33 min; 68.00 ± 2.42%; 23.54 ± 0.28%; p < 0.05), whereas SLE sustained the longest motility. All extracts supported fertilization rates above 82% versus 70.08 ± 3.82% in the control, and 1% BLE preserved post-thaw luminescence equivalent to fresh sperm (165,217 vs. 164,087 RLU), interpreted comparatively. These plant-derived antioxidants are promising supplements for Javan barb germplasm conservation.