Gaspak ez anaerobic pouch system

We used the AN MicroPlate (Biolog) containing 95 carbon sources to determine metabolic activity of F. necrophorum. The assay was carried out according to the manufacturer’s instructions. Briefly, F. necrophorum KG34 (GenBank accession no. SRX5402669) was grown on Wilkins-Chalgren agar medium (Sigma-Aldrich) and suspended in 14 mL of AN inoculating fluid (Biolog) to make bacterial suspension with a transmittance level of about 65%. A 100 μL of bacterial suspension was quickly plated into each well of the AN MicroPlate and incubated at 37 °C in the GasPak EZ anaerobic pouch system (BD). Utilization of the carbon sources by F. necrophorum was indicated by tetrazolium violet forming a blue color, which was measured at 3, 6, 9, 12, 18, and 24 h post inoculation at OD590 using SmartSpec 3000 spectrophotometer (Bio-Rad).

In July 2018, fresh individual faecal samples from 5 subjects, 3 (2 adults and 1 young) housed under semi-natural conditions in Parco Natura Viva-Garda Zoological Park (Bussolengo, Verona, Italy) and 2 (adults) in Bioparco (Roma, Italy) were collected from the ground using a sterile spoon, put into a sterile plastic tube, and stored under anaerobic conditions in an anaerobic jar (Merck) at 4 °C. The anaerobic atmosphere was obtained using the GasPak EZ Anaerobic Pouch system (BD).
Samples of faeces were collected by the animal-care staff (keepers) during their routine cleaning of the enclosure and were taken promptly to the laboratory (within 2 h). Animals were free from intestinal infections and did not receive antibiotics or probiotics for two months before samples were collected.
The two diets consisted mostly of grass, fruits and vegetables, and protein-based feed. (For details, please see Supplementary Table S1).