Microbial Profiling of Outbred CD-1 Mice

Six CD1GM1 and eight CD1GM4 (MU Mutant Mouse Resource and Research Center (MMRRC), Columbia, MO, USA) mice were obtained at 9 weeks of age and set up in same GM profile breeding pairs. These mice were produced by existing colonies of mice generated several years ago and maintained as genetically similar outbred colonies of mice, differing in that each colony harbors a distinct supplier-origin microbiome, with GM1 originating from C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME, USA) and GM4 originating from C57BL/6NHsd mice (Envigo, Indianapolis, IN, USA). In summary, these colonies were generated via the embryo transfer of CD-1 germplasm into pseudopregnant C57BL/6J and C57BL/6NHsd surrogate dams. Pups born to those dams acquired their respective supplier-origin microbiomes and served as founders for outbred colonies that have been maintained at the MU MMRRC since their initial description [22 (link),24 (link)]. Outbred status was maintained via careful rotational breeding and the annual introduction of new genetic stock from the CD-1 vendor via the embryo transfer of a newly acquired CD-1 germplasm into surrogate dams from the existing colonies. Additionally, the microbiome of these colonies was monitored on a quarterly basis via random sampling of 10 cages of adult breeding trios per colony and 16S rRNA amplicon sequencing of fecal DNA. Three litters per microbiome were culled down to eight mice at birth (with a goal of four male and four female pups) to reduce the possible effects of litter size and differential maternal care on preweaning growth. Due to difficulties in accurately sexing neonatal pups, a total of 20 GM1 offspring (10 cages, 5 male, and 5 female) and 22 GM4 offspring (11 cages, 5 male, and 6 female) were available for weaning into same-sex pairs at three weeks of age. Coprophagy leads to a shared cage-level microbiome and the single housing of mice is nonstandard husbandry that is considered stressful to mice. For these reasons, all adult experimental outcomes were based on the cage (i.e., mouse pair) as the experimental unit in an effort to eliminate cage effects and allow for normal activity and feeding behaviors. All mice were housed under barrier conditions in microisolator cages on ventilated racks with pelleted paper bedding and nestlets as enrichment. All mice had ad libitum access to an irradiated diet (LabDiet 5058 for breeder animals and LabDiet 5053 for feed study animals, LabDiet, St. Louis, MO, USA) and autoclaved tap water under a 12:12 light/dark cycle. The mice were determined to be free from opportunistic bacterial pathogens including Bordetella bronchiseptica; cilia-associated respiratory (CAR) bacillus; Citrobacter rodentium; Clostridium piliforme; Corynebacterium bovis; Corynebacterium kutscheri; Helicobacter spp.; Mycoplasma spp.; Pasteurella pneumotropica; Pneumocystis carinii; Salmonella spp.; Streptobacillus moniliformis; Streptococcus pneumoniae; adventitious viruses including H1, Hantaan, KRV, LCMV, MAD1, MNV, PVM, RCV/SDAV, REO3, RMV, RPV, RTV, and Sendai viruses; intestinal protozoa including Spironucleus muris, Giardia muris, Entamoeba muris, trichomonads, and other large intestinal flagellates and amoebae; intestinal parasites including pinworms and tapeworms; and external parasites including all species of lice and mites via quarterly sentinel testing performed by IDEXX BioAnalytics (Columbia, MO, USA).