2.2 Fish Sampling
The experimental period was 60 d. After being fasted for 24 h, and being
placed under anesthesia in buffered tricaine methanesulfonate (35–40
mg/L; MS-222, Sigma-Aldrich, St. Louis, MO), nine fish were randomly
selected from each experimental tank on days 0, 7, 14, 30, and 60.
Caudal vein blood, sampled with a syringe, was stored in lithium heparin
anticoagulant tubes at 4 °C, centrifuged within 1 h at 3000 rpm for 10
min, then stored at −80 °C. The liver, kidney, and spleen samples were
stored in liquid nitrogen for genetic analysis.
Histological samples were stored overnight in 4% paraformaldehyde, then
in 70% ethanol for long-term storage. For histological analysis, the
livers were dehydrated using an increasing alcohol gradient, and
embedded in paraffin. Embedded samples were sectioned to 5 µm using a
microtome (RM 2235, Leica, Wetzlar, Germany) and stained with
hematoxylin and eosin (Suvarna 2012).