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.