title: Handling stress may confound murine gut microbiota studies.
abstract: 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, includingBifidobacterium longumsubsp.infantis (B. infantis), a species sensitive to host stress (Bailey & Coe, 2004). Exposure to gut bacteria likeB. 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, particularlyB. infantis, we predicted the pups receiving a prebiotic solution in a less-invasive manner would have the highest amount ofBifidobacteriain 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 ofBifidobacterium,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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Human Evolutionary Biology, Harvard University , Cambridge , MA , United States.
authors.forename: Cary R
authors.initials: CR
authors.lastname: Allen-Blevins
authors.affiliation: Department of Food Science, University of Massachusetts , Amherst , MA , United States.
authors.forename: Xiaomeng
authors.initials: X
authors.lastname: You
authors.affiliation: Center for Evolution and Medicine, Arizona State University, Tempe, AZ, United States; School of Human Evolution and Social Change, Arizona State University, Tempe, AZ, United States.
authors.forename: Katie
authors.initials: K
authors.lastname: Hinde
authors.affiliation: Department of Food Science, University of Massachusetts, Amherst, MA, United States; Department of Microbiology, University of Massachusetts, Amherst, MA, United States; Center for Microbiome Research, University of Massachusetts Medical School, Worcester, MA, United States.
authors.forename: David A
authors.initials: DA
authors.lastname: Sela
keywords: Bifidobacteria; Methods; Microbiota; Milk oligosaccharides; Mouse study