Rumen fluid handling affects measurements of its enzymatic activity and in vitro digestibility

•Blending released more amylase, lichenase,endoglucanase and xylanase but not protease enzymes.•Blending is simpler and an adequate alternative to quantify amylase, lichenase,endoglucanase and xylanase activity, when compared to sonication.•Blending cannot be replaced by sonication for the extraction of cell-associated protease enzymes.•Blending rumen fluid before an in vitro trial does not necessarily result in higher digestibility values and reduced lag. Traditionally, rumen fluid without the particulate matter is used in in vitro studies, however, a higher concentration of rumen microbes is associated with particulate fraction than the liquid fraction of the rumen content. The objectives of this study were to determine if blending or sonication could increase the enzymatic activity of rumen fluid and to determine the effect of blending rumen fluid on in vitro digestibility of starch, neutral detergent fiber and protein when compared to filtration (control). Rumen fluid collected from two cannulated dairy cows was filtrated, blended or sonicated prior to enzymatic assay. In vitro digestibility of starch (7 h, corn), NDF (6, 12 and 24 h, oat hay) and nitrogen (16 h, soya oilcake) was determined. Both blending and sonication had the ability to consistently release more amylase, lichenase, endoglucanase and xylanase enzymes. Blending was not able to release more protease enzymes (P = 0.1515) and sonication cannot be replaced by blending for the extraction of cell-associated protease enzymes (P = 0.0322). For rumen in vitro digestibility, blending was not successful in liberating amylolytic microorganisms or enzymes associated with the particulate fraction and might have negatively affected the microbial population responsible for fiber and protein digestion. Therefore, as opposed to what is sometimes suggested, blending rumen fluid before an in vitro trial does not necessarily result in higher digestibility values and reduced lag (i.e. higher microbial activity) and could result in opposite results.