Description of the structural diversity of rumen microbial communitiesin vitrousing single-strand conformation polymorphism profiles

Abstract

Changes of the rumen microbial community structure, as it can be established with a rumen simulation technique (RUSITEC) were studied using PCR and single-strand conformation polymorphism (SSCP) of small subunit rDNA genes (SSU rDNA). Four total mixed rations were incubated and two ammonia levels in the artificial saliva were applied. Three replicated vessels were used for each treatment. Mixed microbial fractions were isolated by stepwise centrifugation from the liquid fraction (reference microbes, RM) and from the solids of the feed residues (solid-associated microbes, SAM). PCR-primers targeting archaea, fibrobacter, clostridia, and bacteria, respectively, were applied to represent the individual taxonomic groups by SSCP profiles. These SSCP profiles were converted into a binary matrix and distances among treatments were visualised by non-metric multidimensional scaling. Between replicates belonging to one treatment only small differences were found, indicating a high reproducibility of the RUSITEC and the chosen SSCP method. The ammonia concentration seems to be affecting the SSCP profiles. Great differences occurred between RM and SAM, especially for profiles targeting bacteria and clostridia. Differences in the profiles of RM were also found between mixed rations that contained the same feedstuffs in different ratios and between rations with similar nutrient content but based on different feedstuffs. In conclusion, the PCR-SSCP-based technique in conjunction with non-metric multidimensional scaling was sufficiently sensitive to detect and compare changes in composition of rumen microbial community structure in vitro as affected by diet and other environmental factors.

Influence of transgenic Bt176 and non-transgenic corn silage on the structure of rumen bacterial communities

“…Variation in bacterial species composition between individuals fed with different diets has been demonstrated for various ruminant species (Ferme et al 2004;Lanyasunya et al 2007;Boguhn et al 2008;Goel et al 2008), and even small changes in chemical composition of forages were strong enough to alter the bacterial population structure in the rumen, both in artificial systems and in vivo (Ferme et al 2004;Grabber et al 2009). Several authors have demonstrated that transgenic Bt-corn plants have an unexpectedly different chemical composition with respect to the parental non-genetically modified cultivars, having a higher lignin content (Masoero et al 1999;Saxena and Stotzky 2001;Poerschmann et al 2005), higher starch content, and lower soluble nitrogen and protein contents (Masoero et al 1999).…”

Evaluation of the effects of tropical tanniferous plants on rumen microbiota using qRT PCR and DGGE analysis

“…Although the influence of the host cannot be reproduced in the in vitro systems, Boguhn et al (2008) demonstrated that fluctuations in the microbial community structure due to changes in the composition of the feed can be detected in vitro. In this study, DGGE analysis showed differences regarding treatments, time, and repetition (Figures 2A and 2B).…”

“…Christophersen et al (2004) applying DGGE reported that methanogenic diversity was influenced by diet, passage rate, and the interaction to protozoa species. Boguhn et al (2008) testing four diets using PCR-SSCP and comparing reference microbes isolated from liquid and solid-associate microbes, found strong differences with primers targeting bacteria, fewer differences for Archaea and clostridia, and no clearly distinguishable differences with primers for Fibrobacter sp.…”

Effects of concentrate proportion in the diet with or withoutFusariumtoxin-contaminated triticale on ruminal fermentation and the structural diversity of rumen microbial communitiesin vitro

“…Each PCR was performed with a TGradient Thermocycler (Biometra Biomedizinische Analytik GmbH, Go¨ttingen, Germany) in a total volume of 25 ml, as described in detail by Boguhn et al (2008). The cycling conditions were as follows: initial denaturation at 958C for 15 min; 25-30 cycles of denaturation at 948C for 45 s (for archaea 30 s and bacillii 60 s); annealing for 45 s (for archaea 30 s and bacillii 60 s); elongation at 728C for 60 s (for bacillii 70 s); and a final elongation at 728C for 5 min.…”