Limitations of Feces as Proxy for Gut Microbiota

A myriad of factors can influence the gut microbiota composition of laboratory rats and mice: diet, stress, circadian rhythm, vendor source, how they are housed, and more. Since different gut microbiota compositions can elicit different phenotypes under otherwise similar experimental conditions, understanding what shapes the rodent microbiome is becoming increasingly important for researchers.

Husbandry-Induced Microbiome Effects

The husbandry of laboratory animals is highly variable between facilities. Different practices may elicit effects on the microbiome, in turn contributing to lack of reproducibility between institutions. However, there are only a few studies on how different cage types, bedding materials, and chow diets affect the gut microbiota, such as this new study out of University of Missouri.

In the study, the authors purchased outbred mice in one single order and subjected them to different husbandry conditions, in the same animal room, in a full factorial study design. The husbandry variables were:

  • Cage ventilation:
    Static microisolator cages with filter top on open shelves or individually ventilated cages (IVCs) on an IVC-rack
  • Bedding material:
    Compressed paper or aspen chips
  • Chow diet:
    Purina LabDiet 5008, 5053 or 5058
Fecal pellets were collected one week after arrival, and the fecal gut microbiota composition was analyzed in all the groups. It was found that all mice shared a comparable baseline microbiota.

When the researchers analyzed the fecal microbiota 12 weeks later, they found that it was still largely comparable between all the groups, meaning that diet, cage and bedding type had very little influence on the fecal microbiota.

Limitations of Feces as Proxy for Gut Microbiota
Source: Ericsson AC et al. Sci Rep. 2018 Mar 6;8(1):4065.

Significant Changes in Cecal Microbiota

When the mice were euthanized, the authors sampled intestinal contents from different sections of the small intestine and from the cecum. When the microbiota composition from these different sections was analyzed, it was revealed that caging and bedding type had a highly significant effect on the cecal microbiota.

Specifically, mice housed in static cages with aspen bedding had a clearly different cecal microbiota composition compared to all other groups. This was not observed in the group with static cages and paper bedding, nor in the IVC group with aspen bedding. The authors concluded that the effect was a consequence of interaction between the cage ventilation and bedding variables.

It is not clear why this happened, but the authors hypothesize that unknown volatile or aromatic compounds, e.g. urinary ammonia, could play a role in shaping of the microbiota. Such compounds could be differently absorbed by the two types of bedding and would be removed to a larger extent in the IVCs versus the static cages. The combination of aspen bedding and static caging may therefore favor a cage microenvironment that influences the cecal microbiota by hitherto unknown mechanisms.

Limited Effect of Diet

Interestingly, the different diets had very little effect on the intestinal microbiota. Diet is usually perceived as one of the biggest influences on the gut microbiome. In this case, as is the case for many chow diets, the diets did not differ much in the sources of macronutrients and only relatively little in the proportions of crude protein and fat.

More dramatic diet regimes, such as Western diet and synthetic diets, are known to elicit significant changes in the gut microbiota. According to this study, however, the differences between more broadly similar chow diets may be of less concern when comparing studies.

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Limitations of Fecal Analysis

An important finding of this study is that these husbandry effects were not discovered when analyzing fecal pellets. When researchers are looking for microbiota effects in studies, there is a risk of false negative conclusions if they rely only on analyzing fecal microbiota.

The obvious advantage of fecal pellet sampling is ease of longitudinal gut microbiota surveys, but including groups of mice to be euthanized in intervals would also support analysis of the cecal microbiota.


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