Evolution of the rumen fluid enzymatic activity during in vitro incubation

The aim of this study was to evaluate the evolution of the rumen fluid enzymatic activity (EA) during in vitro incubation. Rumen fluids (RFs) were collected from dairy cows in 2 physiological stages fed 4 diets (DT): dry cows administered 100% hay (TH) or 80:20 forage:concentrate ratio (F:C) diets; and lactating cows fed hay ad libitum and concentrate separately at about 60:40 F:C or total mixed rations (TMR) with a 60:40 F:C. Three farms per each DT were involved and rumen collection was performed on 3 donor cows/farm. RFs were pooled by farm, divided in 2 flasks, inoculated at a ratio of 1:4 with medium and 5 g of a common substrate –a TMR diet- in an in vitro batch fermentation system. Flasks were maintained at 39 °C under anaerobic conditions for 48 hours. During the incubation process, diluted RFs were sampled in duplicate at 0, 1, 2, 4, 8, 24 and 48 h of incubation. Samples were centrifuged and filtered through 0.45 µm porosity filters for the EA determination. EA was evaluated through the radial enzyme diffusion method. Petri dishes with the specific substrate were inoculated with RFs, incubated for 16 h. Halos dimension was measured and expressed as area of the halos surface. Statistical analysis was performed through the repeated measures procedure of the general linear model using DT as a fixed factor, farm as random effect and intervals as repeated measures. With the exception of Xyl, starting from different EA, after an initial irregular peak of activity, a gradual reduction of EA was observed over time. RFs derived from TH showed an opposite trend for both cellulose and xylanase. RFs showed similar A activity at 4 h. Overall, DT showed significant effect for C and A (P ≤0.001) while only a trend was observed for Xyl (P=0.065). At 48 h considering A and at 24h considering C and Xyl, EA were similar between DT and only TH rumen fluid was different compared to the other RFs. In general it appears that the incubation of RFs of different origin with a common substrate tend to homogenise their EA