Study of the in vivo and in vitro variations of the enzymatic activity of bovine rumen fluid inocula

Aim of the present work was to quantify the enzymatic activities (EAs) of rumen fluids (RFs) collected as inoculum in “in vitro” digestibility tests, in the attempt to assess the possible variations of RFs degradative capacity. A further objective was to evaluate the effects of the incubation of RFs on a standard substrate on enzyme expression by rumen microbes. In a first trial the activities of cellulase (C), amilase (A) and xylanase (X) were tested on 7 rumen fluid pools collected from two cows fed a hay-forage based diet in the interval June-September 2015. The EAs were tested using the radial enzyme diffusion method (RED) using substrate concentrations of 0.5%, 0.5% and 0.1% for C, A and X respectively. Wide variations of rumen fluid EA were observed with values (area of the halo surface; mm2 ) ranging from 295.75 to 621.70 for C, from 310.26 to 495.40 for A and from 95.00 to 723.17 for X. The differences observed between the rumen fluids examined were significant for all the enzymes (p<.001) The EAs decreased in August, probably in relationship to heat/dietary stress. In a second trial, 3 different pools of (RFs), collected from cows fed no (NC), medium (MC) or high concentrate (HC) diets, were incubated in triplicate “in vitro” on a standard substrate (lactating cows total mixed ration). The relative EA were tested at different intervals (h: 1, 2, 4, 8, 24 and 48) to evaluate the dynamic of C, A and X enzymes expression pattern by ruminal microbiota, and to individuate the interval at which the different inoculums showed similar and/or maximum EAs. Initial EA were NC ¼ 226.12; MC ¼ 198.49 and HC ¼ 282.93 mm2 for A activity; NC ¼ 250.32, MC ¼ 365.67 and HC ¼ 578.79 mm2 for C; NC ¼ 227.83; MC ¼ 95.00; HC ¼ 124.10 mm2 for X. An increase of EA was observed for A from 4 to 8 hours, with maximum values of 321.71 for NC, 270.99 for MC and 304.27 mm2 for HC. EA peaks were observed for C at 2 and 8 h. X activity showed a maximum value at 8 h for HC (208.67 mm2 ) and after 48 h for NC (343.5 mm2 ) and MC (288.07 mm2 ). Similar values of EA in the 3 pools (NC, MC and HC) were observed at 24 h for C and at 4 h for X. Concerning A, two (MC and HC) out of 3 pools showed similar EA values at 24 h. The RFs EA can vary widely across inoculums from the same animals. Incubations affects EA, generally inducing an increase in RFs coming from NC diet, and a decrease in RFs derived from HC diet, with similar values at variable intervals depending on the enzyme