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

Abstract

The freeze-thaw process in sperm cryopreservation generates reactive oxygen species (ROS) that impair motility, viability, and fertilizing capacity in fish, including the Javaen barb (Systomus rubripinnis), an endemic Indonesian cyprinid of conservation concern. Cryopreservation underpins gene bank development for aquatic genetic resources and supports biodiversity conservation alongside hatchery-based stock enhancement. 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). Each antioxidant was supplemented into the Kurokura's cryopreservation extender across a graded concentration series (BLE 1-4%, NFE and MLE 0.5-1.5%, SLE 6-9%, and HEY 1-2% with a fixed 5% egg yolk) and compared with an antioxidant-free control. 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. The most effective doses tested were doses of 1% for BLE, NFE, and MLE, 1.25% for HEY, and 7% for SLE. BLE at 1% gave the best overall profile (motility 85.00 ± 0.88%; duration 7.14 ± 0.38 min; viability 82.00 ± 1.86%; abnormality 18.26 ± 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 locally available, plant-derived antioxidants offer a low-cost route to germplasm conservation of Javaen barb, strengthening conservation hatchery and broodstock management, sustainable aquaculture production, and food security for inland fisheries communities.