Enzymatic extracts were prepared from rumen fluid (RF) either directly after collection or after incubation in enriched media and their proteolytic activities were measured and compared The objective of RF in vitro cultivation was to augment enzymatic activity by using diverse incubation media containing different feed substrates to selectively induce microorganisms with essentially unrestricted sources of energy and N The effects on protein degradation in bovine serum albumin (BSA) and soybean meal rapeseed meal dehydrated alfalfa meal and perennial ryegrass were determined Enzymatic extracts were characterized by means of protease zymograms Two adult Holstein Friesian rumen fistulated cows were used as RF donors Standard anaerobic incubation techniques in buffer-mineral media were used and protein degradation was measured as residual true protein after enzymatic incubations Results reveal an effect of RF pre-incubation on protein degradation (P<0 0001) measured in BSA and in feed substrates and also on the hydrolysis rate (P<0 001) measured with native substrates only The enzymatic extracts showed a high potential for proteolytic degradation reaching 785 mg/g crude protein (CP) degraded in BSA after 12h of incubation and 830 mg/g CP degraded in soybean meal with 48h of incubation compared to only 654 mg/g and 414 mg/g in controls The zymograms of these extracts revealed six zones of proteolytic activity with the highest concentration of peptidases in the zone of high molecular weight (117 and 130 kDa) In vitro pre-incubation of rumen fluid in culture media enriched with energy and N sources allowed collection of enzymatic extracts with higher proteolytic activity than extracts obtained directly from fresh rumen fluid (C) 2010 Elsevier B V All rights reserved