The effects of exogenous proteolytic enzyme (EPE) on intake, digestibility, ruminal fermentation, and lactational performance were determined using 8 lactating Holstein cows in a double 4 ×4 Latin square experiment with a 2 ×2 factorial arrangement of treatments. Diets based on barley silage and alfalfa hay as the forage sources were formulated to maintain different forage to concentrate ratios [60:40 vs. 34:66, dry matter (DM) basis]. Four dietary treatments were tested: high forage (HF) without EPE (HF−EPE), HF with EPE (HF+EPE), low forage (LF) without EPE (LF–EPE), and LF with EPE (LF+EPE). The EPE, which contained proteolytic activity but negligible fibrolytic activity, was added to the concentrate portion of the diets after pelleting at a rate of 1.25 mL/kg of DM. Adding EPE to the diet increased total tract digestibilities of DM, organic matter, N, acid detergent fiber, and neutral detergent fiber, with larger increases in digestibility observed for cows fed LF+EPE. Effects of added EPE on in vivo digestibility were consistent with improvements in gas production and degradability of the individual components of the TMR observed in vitro. Ruminal enzymic activities of xylanase and endoglucanase increased with addition of EPE to the diet, which may have accounted for improvements in fiber digestion. However, feeding EPE unexpectedly decreased feed intake of cows, which offset the benefits of improved feed digestibility. Consequently, milk yield of cows fed high or low forage diets decreased with adding EPE. Nevertheless, dairy efficiency, expressed as milk/DM intake, was highest for the LF+EPE diet. Addition of EPE to the diet increased milk fat and milk lactose percentages, but decreased milk protein percentage of cows fed a low forage diet. For cows fed high forage diets, EPE only increased milk lactose percentage. Efficiency of N use for milk production was decreased for both the high and low forage diets when EPE was added to the diet. Mean ruminal pH was lowered when EPE was added a low forage diet, likely due to the increased degradation of forage and concentrate, but there was no effect of EPE on rumen pH when cows were fed high forage diets. Profiles of VFA and microbial yield were not affected by adding EPE to the diets. Adding EPE to a total mixed ration containing alfalfa hay, barley silage, and concentrate improved nutrient digestibility in the total tract, and the response was maximized with a high concentrate diet. However, improvements in digestibility were offset by decreased feed intake, likely due to increased ruminal acidosis.
