Metabolomic Analysis of Feces vs. Cecum Content in Animals: A Comparative Study Investigated by 1H-NMR

<b>Background</b><b>:</b> Feces and cecum content are commonly involved in metabolomic analysis to understand the gut metabolic profile of the host, while, in fact, they are different. Feces represent the terminal excretory product after extensive host enzymatic digestion, absorption, and significant modification by the distal gut microbiota. In contrast, cecum content reflects the localized, in situ metabolic microenvironment at that specific site. However, it is worth noting that feces are the most accessible sample type for non-invasive studies, which could be considered proxies for cecum content in some specific cases. Unfortunately, the validity of fecal samples as an alternative to cecum content has rarely been assessed. <b>Methods</b>: The current study attempted to illustrate the distinct metabolomic and microbiota features of feces and cecum content in eight animals (mouse, pig, chicken, duck, rabbit, Gansu yak, Sichuan yak, and sheep) by means of ¹H-NMR and 16S rRNA, respectively. <b>Results</b>: A total of 116 molecules were characterized in feces and cecum content samples. Among them, 22 molecules were shared in all groups. Taking advantage of the univariate analysis, twenty-seven of the quantified molecules were significantly different between feces and cecum content, mainly pertaining to amino acids and organic acids. Moreover, in terms of mammals and non-mammals, short-chain fatty acids could be considered the main factor discriminating the metabolomic profiles between feces and cecum content. Furthermore, to better understand the mechanism of their metabolomic differences, 16S rRNA sequencing analysis was performed on feces and cecum content samples of mice, which is the most widely used animal model. The result showed that the Ace, Shannon, and Sobs indexes in feces were significantly higher than those of cecum content (<i>p</i> < 0.05). At the phylum and genus levels, the microbiota structures of feces and cecum content were similar, while the relative abundances of their microbiota exhibited distinct features. <b>Conclusions</b>: The present study could reduce this gap in information by characterizing, for the first time, the metabolomic differences between feces and cecum content using ¹H-NMR. Moreover, this study is meant as a reference guide for researchers wishing to apply a metabolomics approach to the gut of the host.