Abstract
BackgroundRodent models are invaluable for studying biological processes in the context of whole organisms. The reproducibility of such research is based on an assumption of metabolic similarity between experimental animals, controlled for by breeding and housing strategies that minimise genetic and environmental variation. Here, we set out to demonstrate the effect of experimental uraemia on the rat urinary metabolome and gut microbiome but found instead that the effect of vendor shipment batch was larger in both areas than that of uraemia.ResultsTwenty four Wistar rats obtained from the same commercial supplier in two separate shipment batches underwent either subtotal nephrectomy or sham procedures. All animals undergoing subtotal nephrectomy developed an expected uraemic phenotype. The urinary metabolome was studied using 1H-NMR spectroscopy and found to vary significantly between animals from different batches, with substantial differences in concentrations of a broad range of substances including lactate, acetate, glucose, amino acids, amines and benzoate derivatives. In animals from one batch, there was a complete absence of the microbiome-associated urinary metabolite hippurate, which was present in significant concentrations in animals from the other batch. These differences were so prominent that we would have drawn quite different conclusions about the effect of uraemia on urinary phenotype depending on which batch of animals we had used. Corresponding differences were seen in the gut microbiota between animals in different batches when assessed by the sequencing of 16S rRNA gene amplicons, with higher alpha diversity and different distributions of Proteobacteria subtaxa and short-chain fatty acid producing bacteria in the second batch compared to the first. Whilst we also demonstrated differences in both the urinary metabolome and gut microbiota associated with uraemia, these effects were smaller in size than those associated with shipment batch.ConclusionsThese results challenge the assumption that experimental animals obtained from the same supplier are metabolically comparable, and provide metabolomic evidence that batch-to-batch variations in the microbiome of experimental animals are significant confounders in an experimental study. We discuss strategies for reducing such variability and the need for transparency in research publications about the supply of experimental animals.
Highlights
Rodent models are invaluable for studying biological processes in the context of whole organisms
Animal research has been based on the assumption that whilst experimental animals in different facilities may have differences at species level between their gut microbiota [3], at a population level, in healthy laboratory animals on identical diets, these diverse collections of microorganisms achieve a shared set of basic metabolic functions—an assumption supported by evidence of significant functional redundancy within gut microbial communities [4]
We found that the effect of shipment batch had a larger effect in both areas than uraemia and that conclusions drawn about the effect of uraemia on gut-derived metabolites would have been radically different depending on the batch of animals used
Summary
Rodent models are invaluable for studying biological processes in the context of whole organisms. The reproducibility of such research is based on an assumption of metabolic similarity between experimental animals, controlled for by breeding and housing strategies that minimise genetic and environmental variation. We set out to demonstrate the effect of experimental uraemia on the rat urinary metabolome and gut microbiome but found instead that the effect of vendor shipment batch was larger in both areas than that of uraemia. Based on an assumption of metabolic similarity between experimental animals, we sought to investigate this ‘gut-kidney axis’ in a rodent model of uraemia, by demonstrating the effect of experimental uraemia on the urinary metabolome and gut microbiota of rats, purchased from the same supplier in two separate shipment batches for logistical reasons. We found that the effect of shipment batch had a larger effect in both areas than uraemia and that conclusions drawn about the effect of uraemia on gut-derived metabolites would have been radically different depending on the batch of animals used
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