Determination of the optimal priming interval of rumen fluids used as inocula for the in vitro digestibility trials through radial enzyme diffusion method

Context: Determination of the neutral detergent fibre digestibility is one of the important parameters to consider when formulating diets. However, the in vitro determination shows low repeatability because of the source of rumen-fluid inoculum. Priming of the rumen fluid inocula, obtained through an oesophageal probe, has been proposed to overcome this issue. Aim: The objective of the study was to investigate the evolution of the microbial enzymatic activities of different rumen fluids during a priming procedure, to establish the fermentation interval that minimises the differences among rumen-fluid degradative potentials. Methods: Three farms for each type of diet were involved in the study. Rumen fluids were obtained from dry and lactating cows fed the following four diet types: 100% hay or a diet with 80: 20 forage: Concentrate ratio (F: C) as dry-cow diets, and ad libitum hay and concentrate, or a total mixed ration (both at 60: 40 F: C) as lactating-cow diets. On each farm, rumen fluid was collected from three Holstein cows by using an oesophageal probe, and mixed. Two aliquots of each rumen fluid mix were added to the medium containing the same priming substrate in an in vitro batch-fermentation system. During the incubation, the fermentation fluids were sampled in duplicate at 0-, 1-, 2-, 4-, 8-, 24- A nd 48-h intervals. Enzymatic activities of amylase, cellulase and xylanase were determined by radial enzyme diffusion method. Key results: Initial enzymatic activities were quite variable and increased with an increasing incubation time. By 24 h, amylase showed similar values among high-concentrate diet fermentation fluids, and a lower data dispersion in comparison to the other intervals cellulase was characterised by similar values in all the fermentation fluids derived from diets including concentrates, and xylanase showed similar activity in the fermentation fluids derived from high-concentrate diets. Development of the enzymatic activity of the fermentation fluids derived from the 100% hay diet differed from the others. Conclusions: A 24-h priming procedure was needed to stabilise and equalise the enzymatic activity of the rumen fluid from cows fed high-concentrate diets. This was not observed in rumen fluid from cows fed hay-based diets. Implications: The 24-h-primed rumen fluid can be used to increase the repeatability of neutral detergent fibre digestibility determination.