Jejunal Histomorphometry and Microbial Counts of Digesta

A distal jejunum segment of 5 cm (close to Meckel's diverticulum) was excised, flushed with sterile PBS, and fixed by immersion in a 3.7 to 4.0% neutral buffered formaldehyde solution (PanReac AppliChem, Castellar del Vallès, Spain). Once fixed, 6 cross sections were obtained from each jejunum, embedded in paraffin, and prepared and stained with hematoxylin/eosin for further observations in a light microscope (BHS-Olympus, Tokyo, Japan). The histomorphometric indexes evaluated were villus height (VH: from the villus tip to the crypt junction), crypt depth (CD: from the villus bottom to the crypt), and the ratio between VH and CD (VH:CD). The analysis was performed on 10 well-oriented and intact villi and 10 crypts, according to methodology described by Schiavone et al. (2018). Measures were taken using ImageJ (1.8.0; NIH; Bethesda, MD) via software analysis.

Jejunal digesta samples were collected in sterile conditions and were stored at 4°C until further analysis. Samples were 10-fold serial diluted in Lactated Ringer's Solution (Sigma-Aldrich Química, Madrid, Spain) to perform microbiota counts. Diluted digesta samples were inoculated on azide glucose, MacConkey, and MRS (de Man, Rogosa and Sharpe) agar (Oxoid Limited, Hampshire, United Kingdom) for microbial counts of Enterococcus faecium, Enterobacteria, and Lactobacillus spp., respectively. Counts were manually read after 24-h incubation at 37°C.