No abstract

The objective of this study was to investigate the effects of the concentrate proportion and Fusarium toxin-contaminated triticale (FCT) in the diet on nutrient degradation, microbial protein synthesis and structure of the microbial community, utilising a rumen simulation technique and single-strand conformation polymorphism (SSCP) profiles based on PCR-amplified small subunit ribosomal RNA genes. Four diets containing 60% or 30% concentrates on a dry matter basis with or without FCT were incubated. The fermentation of nutrients and microbial protein synthesis was measured. On the last day of incubation, microbial mass was obtained from the vessel liquid, DNA was extracted and PCR-primers targeting archaea, fibrobacter, clostridia, bifidobacteria, bacillii, fungi, and bacteria were applied to separately study the individual taxonomic groups with SSCP. The concentrate proportion affected the fermentation and the microbial community, but not the efficiency of microbial protein synthesis. Neither the fermentation of organic matter nor the synthesis and composition of microbial protein was affected by FCT. The fermentation of detergent fibre fractions was lower in diets containing FCT compared to diets with uncontaminated triticale. Except for the clostridia group, none of the microbial groups were affected by presence of FCT. In conclusion, our results give no indication that the supplementation of FCT up to a deoxynivalenol concentration in the diet of 5 mg per kg dry matter affects the fermentation of organic matter and microbial protein synthesis. These findings are independent of the concentrate level in the diets. A change in the microbial community composition of the genus Clostridia may be the reason for a reduction in the cellulolytic activity.

Recently, transgenic crops have been considered as possible donors of transgenes that could be taken up by micro-organisms under appropriate conditions. In an in vitro rumen simulation system, effects of ampicillin on microbial communities growing either on rumen contents with transgenic maize carrying a gene that confers resistance to ampicillin or its isogenic counterpart as substrates were examined continuously over 13 d. Rate of production of SCFA was measured to determine functional changes in the rumen model and single-strand conformational polymorphism was used to detect alterations in structure of the microbial community. Rumen contents treated with ampicillin displayed a marked decrease in the rate of production of SCFA and diversity of the microbial community was reduced severely. In the presence of transgenic maize, however, the patterns of change of rumen micro-organisms and their metabolic profiles were different from that of rumen fluid incorporating maize bred conventionally. Recovery of propionate production was observed both in the rumen fluid fed transgenic and conventional maize after a delay of several days but recovery occurred earlier in fermenters fed transgenic maize. Alterations in the microbial population structures resulting from the ampicillin challenge were not reversed during the experimental run although there was evidence of adaptation of the microbial communities over time in the presence of the antibiotic, showing that populations with different microbial structures could resume a pre-challenge metabolic profile following the introduction of ampicillin, irrespective of the source of the plant material in the growth medium.

The mycotoxin-producing fungus Fusarium culmorum causes major feed spoilages in agricultural livestock, but effects of F. culmorum-contaminated feed on the structural diversity of the rumen-inhabiting microbial community are not understood. Avoiding animal experiments, this study was conducted with the rumen simulating technique (Rusitec). Small subunit (SSU) rRNA gene copy numbers of bacteria and archaea, determined by quantitative polymerase chain reaction (PCR), indicated no differences between contaminated and non-contaminated digested feed, but fungal copy numbers, not attributable to F. culmorum itself, were elevated approximately fourfold in the contaminated feed, with 2.3 x 10(9) g(-1) dry weight. Single-strand conformation polymorphism profiles of PCR-amplified partial SSU rRNA genes revealed a single but clear difference between contaminated and non-contaminated feed in profiles encompassing the phylogenetic clusters of Fibrobacteriales and Clostridiales. Minor quantitative differences were also seen in profiles of archaea and fungi. Positive correlations were found between fungal rRNA gene copy numbers and the degradability of different nutrients, but there was no correlation with degradation rates of the major mycotoxin contaminant deoxynivalenol. Thus, the diversity responses of the microbial community to F. culmorum-contaminated feed were caused by a fungus-induced, altered feed quality rather than by direct mycotoxicity.

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.