Characterization of the dynamics of initial bacterial colonization of nonconserved forage in the bovine rumen

Microbial colonization is central to ruminal degradation of dietary material yet little is known about the dynamics of this process. The aim of this study was to characterize the initial stages of bacterial colonization of forage, and to assess the impact that different postsample processing and analysis methods had on the results obtained. Bacterial 16S rRNA gene-based analysis of damaged, nonconserved perennial ryegrass, incubated in sacco in the bovine rumen, required the development and validation of new quantitative PCR and denaturing gradient gel electrophoresis (DGGE) primers. Analysis with previously available primer sets was compromised due to dominant amplification of forage-derived chloroplast 16S rRNA genes. DGGE analysis of incubated samples demonstrated that a diverse and consistent population of ruminal bacteria colonized rapidly. Postsampling methodologies did not affect overall population profiles whereas the washing method appeared to influence bacterial numbers. However, regardless of processing methodology, bacterial numbers increased rapidly within 5 min, stabilizing after 15 min of incubation. These findings reveal for the first time the dynamics of bacterial colonization of forage within the rumen.     

Dynamics of initial colonization of nonconserved perennial ryegrass by anaerobic fungi in the bovine rumen

Anaerobic fungi (Neocallimastigales) are active degraders of fibrous plant material in the rumen. However, only limited information is available relating to how quickly they colonize ingested feed particles. The aim of this study was to determine the dynamics of initial colonization of forage by anaerobic fungi in the rumen and the impact of different postsampling wash procedures used to remove loosely associated microorganisms. Neocallimastigales-specific molecular techniques were optimized to ensure maximal coverage before application to assess the population size (quantitative PCR) and composition (automated ribosomal intergenic spacer analysis) of the colonizing anaerobic fungi. Colonization of perennial ryegrass (PRG) was evident within 5 min, with no consistent effect of time or wash procedure on fungal population composition. Wash procedure had no effect on population size unlike time, which had a significant effect. Colonizing fungal population size continued to increase over the incubation period after an initial lag of c. 4 min. This dynamic differs from that reported previously for rumen bacteria, where substantial colonization of PRG occurred within 5 min. The observed delay in colonization of plant material by anaerobic fungi is suggested to be primarily mediated by the time taken for fungal zoospores to locate, attach and encyst on plant material.     

Ruminal Prevotella spp. May Play an Important Role in the Conversion of Plant Lignans into Human Health Beneficial Antioxidants

Secoisolariciresinol diglucoside (SDG), the most abundant lignan in flaxseed, is metabolized by the ruminal microbiota into enterolignans, which are strong antioxidants. Enterolactone (EL), the main mammalian enterolignan produced in the rumen, is transferred into physiological fluids, with potentially human health benefits with respect to menopausal symptoms, hormone-dependent cancers, cardiovascular diseases, osteoporosis and diabetes. However, no information exists to our knowledge on bacterial taxa that play a role in converting plant lignans into EL in ruminants. In order to investigate this, eight rumen cannulated cows were used in a double 4×4 Latin square design and fed with four treatments: control with no flax meal (FM), or 5%, 10% and 15% FM (on a dry matter basis). Concentration of EL in the rumen increased linearly with increasing FM inclusion. Total rumen bacterial 16S rRNA concentration obtained using Q-PCR did not differ among treatments. PCR-T-RFLP based dendrograms revealed no global clustering based on diet indicating between animal variation. PCR-DGGE showed a clustering by diet effect within four cows that had similar basal ruminal microbiota. DNA extracted from bands present following feeding 15% FM and absent with no FM supplementation were sequenced and it showed that many genera, in particular Prevotella spp., contributed to the metabolism of lignans. A subsequent in vitro study using selected pure cultures of ruminal bacteria incubated with SDG indicated that 11 ruminal bacteria were able to convert SDG into secoisolariciresinol (SECO), with Prevotella spp. being the main converters. These data suggest that Prevotella spp. is one genus playing an important role in the conversion of plant lignans to human health beneficial antioxidants in the rumen.     

Characterization of the rumen lipidome and microbiome of steers fed a diet supplemented with flax and echium oil

Developing novel strategies for improving the fatty acid composition of ruminant products relies upon increasing our understanding of rumen bacterial lipid metabolism. This study investigated whether flax or echium oil supplementation of steer diets could alter the rumen fatty acids and change the microbiome. Six Hereford × Friesian steers were offered grass silage/sugar beet pulp only (GS), or GS supplemented either with flax oil (GSF) or echium oil (GSE) at 3% kg⁻¹ silage dry matter in a 3 × 3 replicated Latin square design with 21-day periods with rumen samples taken on day 21 for the analyses of the fatty acids and microbiome. Flax oil supplementation of steer diets increased the intake of polyunsaturated fatty acids, but a substantial degree of rumen biohydrogenation was seen. Likewise, echium oil supplementation of steer diets resulted in increased intake of 18:4n-3, but this was substantially biohydrogenated within the rumen. Microbiome pyrosequences showed that 50% of the bacterial genera were core to all diets (found at least once under each dietary intervention), with 19.10%, 5.460% and 12.02% being unique to the rumen microbiota of steers fed GS, GSF and GSE respectively. Higher 16S rDNA sequence abundance of the genera Butyrivibrio, Howardella, Oribacterium, Pseudobutyrivibrio and Roseburia was seen post flax feeding. Higher 16S rDNA abundance of the genus Succinovibrio and Roseburia was seen post echium feeding. The role of these bacteria in biohydrogenation now requires further study.     

Rumen protozoa are rich in polyunsaturated fatty acids due to the ingestion of chloroplasts

Within this study, we investigated whether the polyunsaturated fatty acids (PUFA)-rich nature of rumen protozoa is a consequence of ingestion of PUFA-rich chloroplasts. Four Hereford × Friesian steers were offered hay [low 18:3 (n-3) and low chlorophyll concentration] followed by freshly cut perennial ryegrass [high 18:3 (n-3) and high chlorophyll concentration] for 16 days. On the 14th and 16th days, rumen protozoa as well as attached and planktonic bacteria were fractionated 1 h before (−1 h), 2 and 6 h postfeeding, and their fatty acid concentrations determined. Protozoa fractionated from fresh grass-fed steers were richer ( P <0.05) in PUFA, except conjugated linoleic acid, for all time points compared with those from hay-fed steers. Protozoal density was higher ( P <0.05) for grass compared with hay. Entodinomorphid abundance was 3.4 times higher on fresh grass ( P <0.01) compared with hay. Confocal microscopy and transmission electron microscopy confirmed that Epidinium spp. were commonly saturated with intracellular cytoplasmic chloroplasts. These data suggest that engulfment of chloroplasts is a major contributor to the high 18:3 (n-3) concentration of protozoa.     

Effects of feeding Mediterranean buffalo sorghum silage versus maize silage on the rumen microbiota and milk fatty acid content

Sorghum presents a sustainable feedstock for Mediterranean buffaloes due to its reduced water and nitrogen requirements compared with maize, which is currently fed primarily. We investigated the effects of feeding sorghum as opposed to maize on Mediterranean buffalo rumen microbial diversity and milk fatty acid content. Four cannulated lactating Mediterranean buffalo cows were fed a basal diet for one month before switching either to maize or sorghum-silage based diets for a 3-month period. Buffaloes were then changed over to the contrasting diet for a further one month. Rumen and milk samples were collected at the end of each month. DGGE- and T-RFLP-based dendrograms generated from rumen samples did not show an effect of diet on rumen bacterial diversity. Milk samples also did not differ in terms of their fatty acid content post sorghum feeding as compared with maize feeding. Thus, sorghum provides an environmentally beneficial alternative to maize for feeding Mediterranean buffalo with little effect on rumen microbial diversity or milk fatty acid composition compared with maize feeding.