Histopathological Examination of Thyroid and Gonad

For thyroid follicle examination, the fixed fish heads were placed in 10% EDTA (PH 7.4) solution for 1 week for decalcification. Then, fixed gonad and EDTA-treated head samples were dehydrated through arising grades of ethanol and then were cleared and embedded in paraffin wax. For thyroid follicle sections, serial frontal sections were cut at 7 μm from the ventral side of the head. The embedded gonad samples were sectioned at 5 µm thickness; the samples were stained with H&E stain and then observed microscopically (Hamed et al., 2021 (link); Sayed E.-D. H. et al., 2022 (link); Sayed et al., 2023b (link)). For scoring each histopathological parameter, six sections of four fish from each treatment were randomly selected and labeled as follows: control, unchanged (0–2); mild, + (>2–10%) area of section; moderate, ++ (>10–40%) partition area; and severe, +++ (>40% partition area) (Hamed et al., 2021 (link)). The sections were examined under an Olympus microscope (model BX50F4, Olympus Optical Co., Ltd., Tokyo, Japan).