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Query: UMLS:C0040822 (tremor)
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Three detachment procedures (DP) were evaluated for their ability to remove particle-associated microbes from digesta in Rusitec fermenters fed a 30:70 alfalfa hay:concentrate diet. Forage and concentrate were incubated in separate nylon bags, and incubation residues were treated independently. Microbial biomass was labeled with (15)NH(4)Cl. Treatments were 1) MET: residues were incubated at 38 degrees C for 15 min with saline solution (0.9% NaCl) containing 0.1% methylcellulose with continuous shaking; 2) STO: residues were mixed with cold saline solution and homogenized with a stomacher for 5 min at 230 revolutions per min; and 3) FRE: residues were immediately frozen at -20 degrees C for 72 h, thawed at 4 degrees C, mixed with saline solution, and subjected to STO procedure. Common to all treatments was storing at 4 degrees C for 24 h after the treatment, homogenization, filtration, and resuspension of residues 2 times in the treatment solutions. Microbial pellets were obtained by centrifugation, and microbial removal was estimated indirectly by measuring removal of (15)N. The PCR-single-stranded conformation polymorphism analysis of the 16S ribosomal DNA was used to analyze the similarity between microbial communities attached to the substrate and those in the pellet obtained after each DP. There were no feed x DP interactions (P = 0.16 to 0.96) for any variable, except for N content in microbial pellets (P = 0.02). Detaching efficiency (P = 0.004) and total recovery (P = 0.01) were affected by DP, with STO showing the greatest values (mean values across substrates of 64.1% for detaching efficiency and 58.3% for total recovery) and MET the least values (57.0 and 51.8%). Similarity index between the microbes attached to substrates and those in the pellets were affected (P = 0.02) by DP, with MET showing greater (P < 0.02) values (84.0 and 86.4% for forage and concentrate, respectively) than FRE (72.5 and 67.8%) and STO having intermediate values (77.1 and 82.4%). There were no differences (P = 0.70) among particle-associated microbe pellets in their N content, but MET pellets had greater (P < 0.05) (15)N enrichments than those obtained by STO and FRE. Although STO was the most effective method to detach ruminal microbes from concentrate and forage, MET produced pellets with the greatest similarity to the microbial communities attached to the substrates and therefore could be considered the most appropriate DP method for treating digesta from Rusitec fermenters.
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PMID:Evaluation of procedures for detaching particle-associated microbes from forage and concentrate incubated in Rusitec fermenters: efficiency of recovery and representativeness of microbial isolates. 1925 33

Six ruminally and duodenally cannulated sheep were used in a partially replicated 4 x 4 Latin square experiment designed to evaluate the efficiency of 3 detachment procedures (DP) to recover solid-associated bacteria (SAB) from ruminal digesta. The 4 experimental diets contained forage to concentrate (F:C) ratios of 70:30 or 30:70 with either alfalfa hay or grass hay as the forage. Bacterial biomass was labeled with 15NH4Cl. The DP were 1) MET: digesta was incubated at 38 degrees C for 15 min with saline solution (0.9% NaCl) containing 0.1% methylcellulose under continuous shaking; 2) STO: digesta was mixed with cold saline solution and homogenized with a stomacher for 5 min at 230 rpm; 3) FRE: digesta was immediately frozen at -20 degrees C for 72 h, thawed at 4 degrees C, mixed with saline solution and subjected to STO procedure. Common to all treatments was storing at 4 degrees C for 24 h after the treatment, homogenization, filtration, and resuspension of digesta 2 times in the treatment solutions. The automated ribosomal intergenic spacer analysis of the 16S ribosomal DNA was used to analyze the similarity between bacterial communities attached to the digesta and those in the pellet obtained after each DP. There were no significant F:C x DP or forage x DP interactions for any variable. On average, STO treatment detached 65.8% of SAB from ruminal digesta, about 1.2 and 1.5 times more than FRE and MET treatments, respectively. Total recovery of SAB in STO pellets (48.9%) was greater compared with FRE (31.7%) and MET (33.1%), values being greater for high-forage compared with high-concentrate diets. Similarity index between the bacteria attached to digesta and those in the pellets were lower for FRE (48.2%) compared with MET (54.1%) and STO (54.1%), which suggests that FRE could have destroyed cell integrity of some bacterial species, thus reducing the bacterial diversity present in the pellets. The STO method was the most effective removing SAB from digesta, but only a moderate similarity between the bacterial communities attached to digesta and those recovered in the bacterial pellets was obtained. Values of duodenal microbial flow estimated using SAB as reference bacteria were greater with FRE compared with STO and MET, but all DP detected similar differences between diets, and therefore did not influence the interpretation of results.
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PMID:Influence of detachment procedure and diet on recovery of solid-associated bacteria from sheep ruminal digesta and representativeness of bacterial isolates as assessed by automated ribosomal intergenic spacer analysis-polymerase chain reaction. 1984 Dec 25