A new in vitro system to study the effect of liquid phase turnover and pH on microbial fermentation of concentrate diets for ruminants

A simple and inexpensive in vitro feed evaluation system which estimates microbial fermentation by measuring gas production and allows for adjustment of liquid dilution rate has been design. Working conditions, in terms of substrate and inoculum proportions (experiment 1, EXP 1) and the liquid replacement schedule (experiment 2, EXP 2) were initially set. In EXP 1, three substrate concentrations (10, 20 and 30 mg/ml: S1, S2 and S3) and three inoculum proportions (0.10, 0.20 and 0.30; I10, I20 and I30) were assayed in a 3 × 3 factorial design, in three incubations of 24 h. Gas production (ml/g organic matter) with S1 was the highest, avoiding to be in excess for microbial colonisation and activity and inoculum I20 limited inoculum contribution by self-fermentation; they were hence considered for further trials. In EXP 2, two liquid replacement schedules (every hour from 0 to 12 h and every 3 h from 12 to 24 h, HF, and every 2 h from 0 to 12 h and every 4 h from 12 to 24 h, LF) were compared with a non-replacement (NR) treatment, using three bottles per treatment. No differences among treatments were detected, and thus the LF schedule was chosen because it was easier to carry out. Once these working conditions were adjusted in EXP 1 and 2, the effect of fluid turnover and incubation pH on the extent of microbial fermentation in a mixed diet (experiment 3, EXP 3) was studied in two incubations. Two liquid turnover rates (0.06 and 0.10 h−1) were compared with a non-replacement treatment at two different pH (6.8 and 6.1) in a 3 × 2 factorial design. Liquid outflow increased gas production, dry matter disappearance (DMd) and total volatile fatty acid (VFA) concentration at both pH (P>0.01), but protozoal numbers were unaffected. Liquid replacement at pH 6.8 did not affect the VFA profile, but the acetate proportion diminished (P=0.04) and the propionate increased (P<0.001), with replacement at pH 6.1. Increasing the fluid turnover rate from 0.06 to 0.10 h−1 reduced gas production from 8 to 20 h of incubation (P<0.05) but it did not alter the VFA profile. The volume of gas produced decreased (P<0.05) with incubation pH, as did DMd (P=0.04) and protozoal (P=0.03) and total VFA (P=0.002) concentration. The acetate proportion was lower (P<0.001) and that of propionate and butyrate was higher (P<0.001) at pH 6.1 than at pH 6.8. Both liquid turnover and pH must be taken into consideration for in vitro evaluation of mixed and concentrate diets for ruminants and this system provides a simple and inexpensive method for doing this.