2.3 | Intestinal morphology analysis
Three fish tissues from midgut for each feeding habit was collected and
prepared for intestinal morphology analysis as described previously
(Limbu et al., 2018). The tissues were fixed with 4% paraformaldehyde
for 24 h, then dehydrated with 75% absolute ethanol. The tissues were
then transferred into xylene twice for transparent, immersed into
paraffin wax three times, embedded and cooled. The intestine tissues
were sliced transversely into pieces with approximately 5 to 6 µm, dried
and stained by using hematoxylin and eosin (H & E). The slides were
finally examined by using electronic biological microscope KOPPACE at
40x to 1600x (Kopace Technology Co., LTD, Shenzhen, China). Villi height
(VH), villi width (VW) and muscularis thickness (MT) were measured from
at least 30 segments for each feeding habit by using Case Viewer
software. Villi height index (VHI), villi width index (VWI) and
muscularis thickness index (MTI) were calculated as VH, VW and MT
divided by individual fish body weight1/3.