Fitc 4000 fd 4

Immediately after dissection, segments of recto-sigmoid were sampled and the antemesenteric region (corresponding to the hypoganglionic region in the BAC group) was mounted in Ussing chamber (Physiologic instruments, San Diego, CA) exposing 0.5 cm 2 of tissue area to 2.5 mL circulating oxygenated Krebs solution maintained at 39°C.
Conductance (G) and FITC-4000 (FD4, Sigma Aldrich) and horseradish peroxidase (HRP, Sigma Aldrich) fluxes were measured as already described [30] (link).

At necropsy, pigs were sedated with tiletamine HCl and zolazepam HCl (Telazol ® ; Zoetis Inc.) before blood draw, and then euthanised with sodium pentobarbital (Fatal Plus ® ; Vortech Pharmaceuticals Ltd). Pancreas, liver and intestine were removed and weighed immediately after euthanasia. Small intestine length was determined and used to calculate the midpoint of the small intestine to standardise a location for collecting jejunum samples. Tissue samples from the duodenum (10 cm distal to the pyloric sphincter), jejunum (mid-point) and ileum (20 cm proximal to the ileocaecal junction) were collected and rinsed with PBS to remove intestinal contents before fixing in 4 % buffered formalin solution. Samples were processed, stained with haematoxylin and eosin, imaged and villi length and width, crypt depth and lamina propria thickness measured for five well-oriented crypt-villi units per sample as previously described (19) . Barrier function was assessed ex vivo by preparing and mounting jejunum tissue in Ussing chambers (Physiological Instruments) with electrical conductance and the flux of FITC-4000 (FD-4; Sigma-Aldrich) used to measure paracellular permeability (flow between cells) and the flux of horseradish peroxidase (HRP Type II; Sigma-Aldrich) used to measure transcellular transport (flow within cells), as previously described (11) .