This study was designed to develop a protocol for repeated intramuscular indomethacin injections to replicate leaky-gut-like symptoms in male Holstein calves to model and study the detrimental effects of leaky gut on gut tissue function and inflammatory response. A generalized randomized block design was used to evaluate how repeated indomethacin intramuscular injections affected the development of leaky gut in 18 male Holstein calves. Animals were enrolled at 3 ± 1 d of life, and after 21 d of adaptation, they were randomly assigned to 1 of 3 treatments consisting of intramuscular saline or indomethacin injections every 12 h for 48 h: (1) control (CTL), saline injection, (2) low intramuscular indomethacin (INDO-L) dosed at 1.2 mg/kg of body weight (BW), and (3) high intramuscular indomethacin (INDO-H) dosed at 2.4 mg/kg of BW. During the challenge, milk intake, starter intake, fecal scores, and rectal temperature were measured daily, and BW was measured at the beginning and at the end of the challenge. Plasma samples were used to measure the recovery of markers of intestinal permeability before and after the challenge by dosing lactulose, d-mannitol, and chromium-EDTA. In addition, several cytokines were measured in plasma during the challenge. Calves were dissected at the end of the challenge to obtain tissue and digesta samples from the gastrointestinal tract and liver. No treatment differences were observed for starter and milk intakes, fecal scores, BW, and rectal temperature. The difference in marker concentrations between pre and post challenges was higher for INDO calves compared with CTL calves in the case of lactulose and chromium-EDTA. In addition, chemokine ligand 2 and 4 and IL-6 were higher for INDO-H calves compared with CTL. Both doses of indomethacin resulted in re-ductions in villus length and surface area in the distal jejunum and ileum and reductions in crypt depth and width in the colon. We showed that repeated indomethacin injections over a 48-h period induced leaky-gutlike symptoms in a region-specific manner, affecting mainly the distal section of the intestine. This outcome was characterized by histomorphological changes in the distal jejunum, ileum, and colon and by increased gut permeability. Interestingly, changes in liver morphology and immune function also occurred, possibly due to the increased translocation of foreign antigens breaching the epithelial cell wall. The leaky gut challenge model described here could be used to improve understanding of the pathogenesis of intestinal disorders in cattle and provide a reliable alternative for testing feed additives with intestinal health benefits.

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