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

Abstract

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.

Comparative Metabolite Fingerprinting of the Rumen System during Colonisation of Three Forage Grass (Lolium perenne L.) Varieties

“…In the work by Allwood et al [23], bacteria were effectively removed from the cell culture samples by sequential washing in 0.2 % NaCl. Although there would be merit in separation of plant and attached bacteria in future studies, separation of colonising rumen bacteria from the forage substrate is difficult, usually requiring some form of chemical and physical treatment to enable detachment of a representative bacterial pool [41–43]. In the current study it was considered that such processing could substantially alter the plant metabolome and so provide an erroneous impression of the interactome under investigation.…”

Influence of rumen contents’ processing method on microbial populations in the fluid and subsequent in vitro fermentation of substrates of variable composition

“…A great proportion of the ruminal microorganisms can be attached to feed particles, especially in animals fed high-forage diets (Cheng et al, 1995;Yáñez-Ruiz et al, 2016). Previous studies (Martínez et al, 2009;Ramos et al, 2009a) showed that treating the ruminal contents with a Stomacher ® detached a greater percentage of solid-associated bacteria compared with other treatments, and produced ruminal fluid with the greatest similarity index between the bacterial communities in the rumen contents and in the resulting fluid. Our hypothesis was that including solid-associated bacteria in the inoculum (STO treatment) would result in improved fermentation, especially for high-fiber substrates.…”

“…To test this hypothesis we used substrates of variable composition, and determined the main fermentation parameters at two incubation times (8 and 24 h), as differences between substrates may decrease or even disappear with increasing incubation time (Kamel et al, 2008;Mateos et al, 2014). Previous studies have investigated the effects of the processing method of rumen contents on gas production profiles (Cone et al, 1996;Lee et al, 2004;Pell and Schofield, 1993) and fermentation parameters (Lee et al, 2004;Rymer et al, 1999;Senshu et al, 1980), but few have looked into the effects on rumen microbial composition (Mackie et al, 1983;Martínez et al, 2009;Ramos et al, 2009a), and to our knowledge none has addressed both of them.…”

“…Previous studies have shown that stomaching the ruminal digesta has an impact on both the amount and profile of bacteria recovered in the fluid. Ramos et al (2009a) and Martínez et al (2009) compared different methods of detaching solid-associated bacteria from ruminal digesta from sheep and Rusitec fermenters, respectively. In both studies the treatment with Stomacher ® for five min at 230 rpm detached a greater percentage of solid-associated bacteria (68.7%, averaged value) compared with either incubating the digesta with a solution of methylcellulose (54.4%) or freezing before treating with Stomacher ® (62.6%).…”

Improved Method for Isolation of Microbial RNA from Biofuel Feedstock for Metatranscriptomics

“…The control method (MC) used to evaluate the efficiency of our new method was based on the conventional TRIzol-based RNA isolation technique that has been successfully used to extract RNA from various biological samples [22,23]. As undigested feed particle have been shown to prevent the TRIzol reagent from rapid sample penetration and to introduce a bias in the RNA recovered from microbial populations, samples are manually disrupted in liquid nitrogen using a mortar and pestle prior to exposing the samples to the TRIzol reagent, which enhances unbiased RNA recovery [21,24]. Using this control method we consistently obtained RNA yields >300 µg (352 µg and 538 µg total RNA from 1 gram rumen incubated corn stover and switchgrass, respectively), which is approximately four times more RNA than the 85 μg RNA from 1 gram of solid rumen content that was reported in a recent study in which the authors utilized a slightly altered version of this standard procedure [21].…”