*4.15. Animal Grouping and Administration*

Transgenic Tg (flk1a: EGFP) zebrafish embryos (24 hpf) were stripped of their egg membranes in Petri dishes containing Pronase E (1 mg/mL) and randomly distributed in 24-well plates with 10 strips/well, using 3 replicates set up for each concentration group. The plates were then treated with different concentrations of DGS, the specific concentrations administered are illustrated in the corresponding results. After 72 h of treatment at 28.5 ◦C, the development and mortality of each group of zebrafish were enumerated.

Transgenic Tg (flk1a: EGFP) zebrafish embryos (24 hpf) were stripped of their egg membranes with Pronase E (1 mg/mL) in Petri dishes, which were randomly distributed in 6-well plates and then treated with different concentrations of DGS as indicated in the corresponding results. After treatment at 28.5 ◦C for 24 h, the zebrafish larvae were rinsed several times with water and then transferred to a 96-well plate. The total length of the intersegment vessels (ISVs) of zebrafish larvae was observed and imaged under a fluorescent microscope. The total ISV length was quantified for each zebrafish using the Image Pro Plus and the average ISVs length was calculated for each group of zebrafish larvae.

Transgenic Tg (flk1a: EGFP) zebrafish larvae (72 hpf) were randomly distributed in 6-well plates and then treated with different concentrations of DGS, as indicated in the corresponding results. After treatment at 28.5 ◦C for 24 h, the zebrafish larvae were rinsed several times with water and then transferred into a 96-well plate with the appropriate amount of anesthetic. The total length of the main subintestinal vein (MSIV) of zebrafish larvae was determined under a fluorescent microscope. The total MSIV length of each zebrafish was quantified using Image Pro Plus, and the mean MSIV length was calculated for each group of zebrafish larvae.
