Ruminal, post-ruminal and total tract crude protein disappearance of alfalfa hay and barley grain were measured using in vivo, in situ mobile bag and three-step in situ/in vitro enzyme procedures (3-step). For in vivo, four Baluchi lambs (49.4 ± 3.5 kg, body weight) were used in a 2 × 2 Latin square design within 2 periods (24 days of each period). Experimental diets were made of two alfalfa hay: barley grain ratios (DM basis) as 1.0:0.0 and 0.5:0.5. Diets were fed to animals for 24 days, with 7 days of feces collection. In situ mobile bag technique was followed by 12 h rumen incubation and then intestinal movement of the bags in rumen and post-rumen cannulated Holstein steers. The three-step procedure was followed by rumen incubation of samples for 12 h (using polyester bags) and enzymatic incubation of ruminal undegradable samples. In vivo total tract crude protein disappearance of alfalfa hay and barley grain (0.74 and 0.69, respectively) was significantly (P<0.01) lower than in situ mobile nylon bag (0.89 and 0.96, respectively) and the 3-step procedure (0.81 and 0.89, respectively). Total tract crude protein disappearance from in situ mobile bag was significantly (P<0.01) higher than the 3-step technique. Post-ruminal disappearance of ruminal undegradable crude protein from alfalfa hay and barley grain using in situ mobile bag method (0.69 and 0.86, respectively) was significantly (P<0.01) higher than the 3-step enzymatic method (0.49 and 0.56, respectively). Results of the present study showed that there was a significant difference between in vivo, in situ mobile bag and 3-step methods when total tract crude protein disappearance of barley grain and alfalfa hay was evaluated.

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