Intestinal Microbiota Contribute to the Endogenous Formation of Thiouracil in Livestock

Abstract

In recent years, the frequent detection of the banned substance thiouracil (TU) in livestock urine has been related to its endogenous formation following consumption of glucosinolate-rich Brassicaceae crops. Besides, TU residues have been recovered in these crops upon plant-derived myrosinase hydrolysis. Through in vitro bovine and porcine static digestive simulations, the influence of gastrointestinal digestion of Brassicaceae-derived matrixes on TU formation was assessed. Following derivatization and LC-MS(2) analysis, TU was detected in colonic suspensions with traditional rapeseed, coarse colza "00" meal, cauliflower, and broccoli ranging from 3.47 to 30.96 μg kg(-1) (bovine) and from 3.55 to 26.34 μg kg(-1) (porcine). In stomach and small intestinal fluids, TU remained unfound, whereas upon rumen simulation TU was detected for coarse colza "00" meal (4.43 μg kg(-1)) and grounded traditional rapeseed (0.35 μg kg(-1)). The origin of this detection was investigated through filter-sterilizing and autoclaving the fecal inoculum causing a significant decrease in TU concentration, thereby reinforcing the possibility of an active bacterial involvement, which however was characterized with a high interanimal variation. In conclusion, these results support the previously proven endogenous origin of TU and acknowledge the active role of the gastrointestinal bacteria in TU formation, through production of an extracellular component.

Thiouracil-Forming Bacteria Identified and Characterized upon Porcine In Vitro Digestion of Brassicaceae Feed

“…Autoclaved rapeseed and control incubations with pure strains did not differ significantly in TU concentrations. Addition of rapeseed as a whole did, however, significantly increase TU formation upon incubation with pure strains, reaffirming its importance as a source of TU precursors, in line with previous in vitro (28) and in vivo (1) reports. During in vitro digestion with the porcine fecal inoculum producing TU at a high level, levels of TU formation did not significantly differ between autoclaved and nonautoclaved rapeseed administrations.…”

“…For general preculturing and cultivation of porcine microorganisms, brain heart infusion broth (BHI) (CM1135) (Oxoid Ltd., Hampshire, England) was used (28). Specialized media were used for the cultivation of specific microbial strains: Sabouraud dextrose liquid medium (SDx) (CM0147) for fungi and yeasts, Wilkins-Chalgren anaerobe broth (WCh) (CM0643) for anaerobes, MacConkey broth (MacC) (CM0005) for coliforms, and de Man Rogosa Sharpe broth (MRS) (CM0359) for lactic acid bacteria (Oxoid).…”

“…Consequently, the focus of the present study was to further investigate the potential and identity of the intestinal microbiota as well as the mechanisms involved in TU formation upon Brassicaceae digestion in livestock by conducting static in vitro colonic-digestion simulations in porcine livestock, because of their high TU-producing capacity (28). Institute for Agriculture and Fisheries Research [ILVO], Melle, Belgium) was used for the in vitro digestion experiments as a substrate for high TU production upon bacterial fermentation.…”

“…The protocol applied for the static in vitro colonic-digestion simulations in the present study was adapted from the scientific literature on simulations of the gastrointestinal tract of humans and porcine livestock (28,34,35).…”

In vitro simulation of the equine hindgut as a tool to study the influence of phytosterol consumption on the excretion of anabolic–androgenic steroids in horses

“…As no standardized horse gut simulation protocol was available [29], the in vitro digestive simulation protocol applied during this study was adapted from previously reported in vitro models, simulating the upper and lower gastrointestinal tract of bovines, porcines and humans [30][31][32][33][34]. A recent study however confirmed the valuable use of in vitro assays to study the horse hindgut, using an in vitro digestion assay to identify lactate-utilizing bacteria that differentially respond to starch induction, in the light of laminitis in horses [23].…”