BACKGROUND 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. 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. RESULTS Twenty 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. CONCLUSIONS These 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.

中文翻译：

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批次效应对大鼠尿液代谢组和肠道微生物群的影响比尿毒症更大：一个警示故事。

背景啮齿动物模型对于在整个生物体的背景下研究生物过程是无价的。此类研究的可重复性基于实验动物之间代谢相似性的假设，由尽量减少遗传和环境变异的育种和饲养策略控制。在这里，我们着手证明实验性尿毒症对大鼠尿液代谢组和肠道微生物组的影响，但发现供应商发货批次在这两个方面的影响都大于尿毒症。结果 从同一商业供应商处分两个不同批次获得的 24 只 Wistar 大鼠接受了肾次全切除术或假手术。所有接受次全肾切除术的动物都出现了预期的尿毒症表型。使用 1H-NMR 光谱研究尿液代谢组，发现不同批次的动物之间存在显着差异，包括乳酸盐、乙酸盐、葡萄糖、氨基酸、胺和苯甲酸盐衍生物在内的多种物质的浓度存在显着差异。在一个批次的动物中，完全没有与微生物组相关的尿液代谢物马尿酸盐，而在另一批次的动物中以显着浓度存在。这些差异如此显着，以至于根据我们使用的动物批次，我们会得出关于尿毒症对尿液表型影响的完全不同的结论。当通过 16S rRNA 基因扩增子的测序评估时，不同批次动物之间的肠道微生物群存在相应差异，与第一批相比，第二批具有更高的 alpha 多样性和不同分布的 Proteobacteria subtaxa 和短链脂肪酸产生细菌。虽然我们还证明了与尿毒症相关的尿液代谢组和肠道微生物群的差异，但这些影响的规模小于与装运批次相关的影响。结论 这些结果挑战了从同一供应商获得的实验动物在代谢方面具有可比性的假设，并提供了代谢组学证据表明实验动物微生物组的批次间差异是实验研究中的重要混杂因素。我们讨论了减少这种可变性的策略，以及在有关实验动物供应的研究出版物中提高透明度的必要性。