Nutrient digestibility was calculated as described by Wang et al. [22 (link)]. For the digestive enzyme activity of duodenum and jejunum determination, the mucosa of the duodenum and jejunum were collected and homogenized by adding sterile 4% saline solution to prepare 10% (W:V) homogenates. The homogenate was centrifuged at 1000× g for 10 min at 4 °C, then each digestive enzyme activity (protease, amylase, and lipase) in the supernatant was determined by spectrophotometry using a commercial kit according to the manufacturer’s instruction (Sigma-Aldrich Co, Saint Louis, MO, USA). The middle duodenum was collected and fixed in 10% PBS-buffered formalin and embedded in paraffin. The 0.2 μm thick paraffin sections were cut and stained with hematoxylin and eosin (H&E). Images of the H&E-stained sections were acquired through a Leica DM3000 Microsystem (Leica, Germany). Then, the morphology of the duodenum was evaluated by scanning electron microscopy (SEM, TM-1000, Hitachi, Japan) and transmission electron microscopy (TEM, H-7650, Hitachi, Japan) as previously described [23 (link)]. SEM and TEM images were acquired and analyzed using a Hamamatsu ORCA-HR digital camera system operated with AMT software (Advanced Microscopy Techniques Corp., Danvers, MA, USA) and Semicaps 2000 software, respectively.
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