Histological Assessment of Fish Tissues

For hepatic, muscle and intestine histological evaluation, three fish in each treatment were randomly euthanized with AQUI-S at 175 mg/L and dissected to excise liver. Liver and muscle tissues were immediately fixed in 10% buffered formalin prior to dehydrate in series of ethanol and equilibrate in xylene. Then the tissues were embedded in paraffin wax, sectioned at approximately 5 μm using a rotary microtome machine and stained with hematoxylin and eosin (H&E) according to standard histological procedures. Photographs of histological slides were scanned under a light microscope (BX40F4, Olympus, Tokyo, Japan). Intestinal sections were stained with Alcian Blue (AB) pH 2.5 to visualize acidic mucins which were counted from ten highest intact villi under a light microscope (BX40F4, Olympus, Tokyo, Japan), following the methods of Sewaka, Trullas [21 (link)] and Chaklader, Siddik [22 (link)].