4.8.1. Zebrafish Maintenance

Zebrafish in their adult stage were purchased from Seoul Aquarium, Korea. The fish were maintained in acrylic tanks under controlled conditions (28.5 ◦C, with a 14/10 h light/dark cycle). Fish were fed with tetramin flake, including live brine shrimp, three times per day in equal intervals for 6 days of the week. Natural spawning was stimulated with lights on conditions to obtain the embryos and completed collection within 30 min.

## 4.8.2. Polysaccharide Application to Zebrafish Embryos

The embryos were transferred to 12 well plates after 7–9 h post-fertilization (hpf). The embryos were maintained in an embryo medium. Samples were treated and incubated for 1 h and stimulated with H2O2 (5 mM) and continued incubation for 24 hpf. The live embryos were counted after 3 days of post-fertilization (dpf) to obtain the survival rate.

#### 4.8.3. Intracellular ROS, Lipid Peroxidation, and Viability Analysis

At 2 days of post fertilization (dpf), the heartbeat rate was evaluated. Both the atrium and ventricle heartbeat rate was assessed under microscope for 1 min. The zebrafish were initially treated with sample and was induced with H2O2 [57].

DCF-DA was used to detect the intracellular ROS levels in the zebrafish embryos while lipid peroxidation was assessed via DPPP. Moreover, cell death was evaluated with acridine orange staining. Following each staining method, the embryos, zebrafish larvae were rinsed with embryo media and anaesthetized with 2-phenox ethanol. A microscope equipped with CoolSNAP-Pro color digital camera (Olympus, Tokyo, Japan) was assisted in observation and photography. The intensity quantification was completed with the ImageJ program [58].
