Handling Stress May Confound Murine Gut Microbiota Studies

Background: Accumulating evidence indicates interactions between human milk composition, particularly sugars (human milk oligosaccharides or HMO), the gut microbiota of human infants, and behavioral effects. Some HMO secreted in human milk are unable to be endogenously digested by the human infant but are able to be metabolized by certain species of gut microbiota, including Bifidobacterium longum subsp. infantis (B. infantis), a species sensitive to host stress (Bailey & Coe, 2004). Exposure to gut bacteria like B. infantisduring critical neurodevelopment windows in early life appears to have behavioral consequences; however, environmental, physical, and social stress during this period can also have behavioral and microbial consequences. While rodent models are a useful method for determining causal relationships between HMO, gut microbiota, and behavior, murine studies of gut microbiota usually employ oral gavage, a technique stressful to the mouse. Our aim was to develop a less-invasive technique for HMO administration to remove the potential confound of gavage stress. Under the hypothesis that stress affects gut microbiota, particularly B. infantis, we predicted the pups receiving a prebiotic solution in a less-invasive manner would have the highest amount of Bifidobacteria in their gut.

Methods: This study was designed to test two methods, active and passive, of solution administration to mice and the effects on their gut microbiome. Neonatal C57BL/6J mice housed in a specific-pathogen free facility received increasing doses of fructooligosaccharide (FOS) solution or deionized, distilled water. Gastrointestinal (GI) tracts were collected from five dams, six sires, and 41 pups over four time points. Seven fecal pellets from unhandled pups and two pellets from unhandled dams were also collected. Qualitative real-time polymerase chain reaction (qRT-PCR) was used to quantify and compare the amount of Bifidobacterium, Bacteroides, Bacteroidetes, and Firmicutes.

Results: Our results demonstrate a significant difference between the amount of Firmicutes in pups receiving water passively and those receiving FOS actively (p-value = 0.009). Additionally, we found significant differences between the fecal microbiota from handled and non-handled mouse pups.

Discussion: From our results, we conclude even handling pups for experimental purposes, without gavage, may induce enough stress to alter the murine gut microbiota profile. We suggest further studies to examine potential stress effects on gut microbiota caused by experimental techniques. Stress from experimental techniques may need to be accounted for in future gut microbiota studies.