2.6#x2010;Diaminopimelic acid (DAPA), ribonucleic acid (RNA),15N, D#x2010;alanine (D#x2010;ALA) and the amino acid profiles (AAP) were compared as microbial markers for determination of the microbial protein synthesis in the rumen. Three dairy cows (Schwarzbuntes Milchrind, LW 602 kg), each fitted with a rumen cannula and a re#x2010;entrant cannula in the proximal duodenum, were offered four isoenergetic and isonitrogenous diets (mean daily intake 15.0 #xB1; 0.45 kg DM; forage: concentrate = 50:50) in a periodic experiment. The diets contained soyabean extracted meal, meat and bone meal, pea meal and dried clover as major sources of protein. On the 4th day after administration of 9 g15N#x2010;labelled urea (95 atom#x2010;15N#x2010;excess) per day, samples of rumen fluid and duodenal digesta were obtained 3 h after feeding. The bacteria were isolated by differential centrifugation. Bacteria harvested from the rumen had significantly higher15N enrichment and D#x2010;ALA: N ratio than #x2018;duodenal#x2019; bacteria. However, DAPA: N ratio was higher in #x2018;duodenal#x2019; bacteria compared to rumen bacteria. There were no differences in RNA: N ratio between rumen and #x2018;duodenal#x2019; bacteria. The source of the bacteria in the digestive tract has an influence on the ratio of microbial N: total N, especially when15N, AAP, DAPA and D#x2010;ALA but not RNA were used as markers. The most reproducible method was D#x2010;ALA (C.V. 4.7 for rumen and 6.8 for #x2018;duodenal#x2019; bacteria) followed by15N (10.8 resp. 4.8) and RNA (9.7 resp. 8.2). The results obtained with15N and D#x2010;ALA agreed closely at the same source of bacteria. The RNA method reached the level of these markers (15N, D#x2010;ALA) when the bacteria were isolated from the duodenum. It is concluded that D#x2010;ALA (bacteria isolated from rumen and duodenum) and also15N (bacteria isolated from duodenum) were the best markers for estimation of the microbial protein synthesis.
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