Animal procedures were approved by Cornell University’s Institutional Animal Care and Use Committee. Mice from the C57BL/6J inbred strain and the B6.129S1-Tlr5tm1Flv/J (TLR5KO) congenic strain were acquired from the Jackson Laboratory (Bar Harbor, ME) and each bred separately in conventional housing in our animal facility. C57BL/6J is the recommended control strain for TLR5KO (28 (link),30 (link)). Animals were housed in plastic cages filled with 1/4-inch corn cob bedding (The Andersons’ Lab Bedding, Ohio), fed with standard laboratory chow (Teklad LM-485 Mouse/Rat Sterilizable Diet) and water ad libitum, and provided a cardboard refuge environmental enrichment hut (Ketchum Manufacturing; Brockville, Ontario). Male mice were divided into four groups: two groups treated to disrupt the gut microbiota (C57BL/6J: n=7, TLR5KO: n=8) and two untreated groups (C57BL/6J: n=12, TLR5KO: n=16). Mice with disrupted microbiota are referred to as “ΔMicrobiota.” Mice were housed in cages with other animals from the same genetic background/treatment group. Treated groups received broad-spectrum antibiotics (1.0 g/L ampicillin, 0.5 g/L neomycin) in their drinking water from weaning at 4 weeks of age until skeletal maturity (16 weeks of age) (28 (link)). Chronic antibiotics used in this manner causes consistent disruptions to the gut microbiota over a prolonged time period (31 (link)). Ampicillin and neomycin have poor bioavailability, thereby limiting extra-intestinal effects of treatment (28 (link),32 (link)). Additionally, neomycin and ampicillin have never been associated with impaired bone growth. Animals were euthanized at 16 weeks of age. Femora, tibiae, epididymal fat pads, and spleen were collected immediately after euthanasia. Fecal pellets were collected one day prior to euthanasia to allow analysis of the microbiota.