A randomized controlled field trial using a 2 × 2 factorial design was conducted at the Kansas State University (KSU) Beef Cattle Research Center and approved by the KSU Institutional Animal Care and Use Committee (Animal Use Protocol #3334, 2 September 2014). In total, 80 steers were placed in individual pens, stratified by weight and then randomly assigned by weight block to a treatment group. Treatments were: supra-nutritional zinc fed at elevated feeding concentrations (300 ppm; n = 20 steers), menthol (fed as 0.3% of dry matter; n = 20 steers), a combination of supra-nutritional zinc and menthol (n = 20 steers) and a control group fed neither zinc nor menthol (n = 20 steers). Animals were allowed to acclimate to their pens for two weeks to ensure proper equilibration of enteric bacterial flora with neighboring cattle and the environment prior to the onset of the trial.

Fecal samples were collected per rectum from each steer starting with Day 0 prior to initiating the experimental regimens and then again at Day 21. Animals were fed their respective treatment diet for three weeks, with the peak of treatment effect expected at Day 21 [33]. Samples were processed in the laboratory into a tube with 50% sterile glycerol at a 1:1 ratio of glycerol to feces. Tubes were then stored at −80 °C until later use.

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