C1022

wITT was determined in 10 single-housed male and 10 female, non-fasted animals on a total of 13 different experimental days (1 to 4 animals each experimental day) starting at 07:00 and ending at 17:00 by oral application of the non-absorbable marker carmine red and checking pellets for red coloring every 15 min. Each guinea pig was orally administered a 6% carmine red solution (Sigma-Aldrich C1022) dissolved in 0.5% methylcellulose (Sigma-Aldrich 274429, 0.5 mL/100 g bodyweight (BW)) a single time at 07:00. Preliminary experiments administering different amounts of the 6% carmine red solution revealed that the chosen solution and amount were sufficient for intensive coloring of fecal pellets (see Supplementary Materials Figure S1), without any side effects for the animal. wITT was defined as the time of appearance of the first red colored fecal pellet, and guinea pigs were further observed for their well-being until 17:00. Afterwards, they were returned to their home cage. In addition to wITT, FI and FPO were measured in these animals between 07:00 and 17:00 as described above.

Gastrointestinal transit time was assessed immediately following the fourth oral challenge with intragastric OVA, on day 24 after allergic sensitization at ZT3. Mice were gavaged with a 0.25 ml solution with 6% carmine red (C1022, Sigma-Aldrich), 0.5% methylcellulose (M0512, Sigma-Aldrich) and 40 mg grade III OVA. After oral gavage and for the duration of the assay, mice were individually placed in clean cages containing normal bedding. Mice had free access to food and water and were monitored for the occurrence of diarrhoea. The gastrointestinal transit time was measured as the time between oral gavage and the appearance of the first faecal pellet containing the red carmine dye. Mice were grouped together at the end of the assay.

Whole gastric time was quantified administering a 6% carmine (C1022, Sigma-Aldrich) solution in 0,5% methylcellulose (M0512, Sigma-Aldrich) dissolved in distilled water, via oral gavage and recording time of passage in the fecal stream. In detail, each mouse received a gavage with 200 μL of 6% carmine solution and time of administration was recorded for each individual mouse. Given that the whole mouse gut transit time typically > 2 hours, observation started 90 minutes after carmine solution administration. Each mouse was transferred in an individual cage until a red fecal pellet was produced. Time of expulsion of the red fecal pellet was recorded and the mouse was returned to its original cage immediately after the production of the red fecal pellet. Whole gastric time was calculated as the time interval between the gavage and the first observed red fecal pellet. Importantly we did not squeeze or apply pressure to the mouse abdomen to stimulate passage of fecal pellets, as stress also can influence gut transit.

Mice were gavaged with 300 µl of 6% carmine red (Sigma–Aldrich, C1022) suspended in 0.5% methylcellulose (Sigma–Aldrich, M0512) solution. Following administration of the solution, the mice were allowed free access to food and water ad libitum. Gastrointestinal transit time was measured as the time interval between the begin of gavage and the appearance of the first red fecal pellet.