Optimisation of inoculum concentration and incubation duration for an in vitro hindgut dry matter digestibility assay

The aim was to optimise inoculum concentration and incubation duration for a published in vitro hindgut digestibility assay using ileal digesta (sampled from the chicken or rat) pertaining to a mixed human diet as the substrate. The study also sought to investigate the digestibility of the inoculum itself and the importance of correcting for this in the in vitro hindgut digestion assay. For two assays, hindgut dry matter digestibility (DMD) generally increased with inoculum concentration. A sharp increase in DMD observed at high inoculum concentrations may have been related to problems with filtering the inoculum. An inoculum concentration of 160 g/L was considered optimal based on close agreement of observed values with previously published in vivo hindgut dry matter digestibility for similar diets. One of the methods was chosen for optimisation of the duration of incubation. Ileal substrate organic matter digestibility (OMD) increased with increasing time of incubation for all diets. An incubation duration of 18 h using a mean inoculum digestibility value for calculation purposes was considered optimal based on observed in vivo hindgut DMD values in humans, but there was little difference in estimated in vitro hindgut DMD between 18 and 24h incubation durations. Although considerably lower than the OM digestibility of the substrate (no less than 51% after 48 h), the OM digestibility of the inoculum (13% after 48 h) itself was of significance in calculating estimated digestibility. The optimised assay gave realistic hindgut OMD values ranging from 55% to 79% (Wheat Bran Diet and Pectin Diet, respectively) using an 18-h incubation duration.