Using 'Omic Approaches to Compare Temporal Bacterial Colonization of Lolium perenne, Lotus corniculatus, and Trifolium pratense in the Rumen.

Understanding rumen plant-microbe interactions is central for development of novel methodologies allowing improvements in ruminant nutrient use efficiency. This study investigated rumen bacterial colonization of fresh plant material and changes in plant chemistry over a period of 24 h period using three different fresh forages: Lolium perenne (perennial ryegrass; PRG), Lotus corniculatus (bird's foot trefoil; BFT) and Trifolium pratense (red clover; RC). We show using 16S rRNA gene ion torrent sequencing that plant epiphytic populations present pre-incubation (0 h) were substantially different to those attached post incubations in the presence of rumen fluid on all forages. Thereafter primary and secondary colonization events were evident as defined by changes in relative abundances of attached bacteria and changes in plant chemistry, as assessed using Fourier transform infrared (FTIR) spectroscopy. For PRG colonization, primary colonization occurred for up to 4 h and secondary colonization from 4 h onward. The changes from primary to secondary colonization occurred significantly later with BFT and RC, with primary colonization being up to 6 h and secondary colonization post 6 h of incubation. Across all 3 forages the main colonizing bacteria present at all time points post-incubation were Prevotella, Pseudobutyrivibrio, Ruminococcus, Olsenella, Butyrivibrio, and Anaeroplasma (14.2, 5.4, 1.9, 2.7, 1.8, and 2.0% on average respectively), with Pseudobutyrivibrio and Anaeroplasma having a higher relative abundance during secondary colonization. Using CowPI, we predict differences between bacterial metabolic function during primary and secondary colonization. Specifically, our results infer an increase in carbohydrate metabolism in the bacteria attached during secondary colonization, irrespective of forage type. The CowPI data coupled with the FTIR plant chemistry data suggest that attached bacterial function is similar irrespective of forage type, with the main changes occurring between primary and secondary colonization. These data suggest that the sward composition of pasture may have major implications for the temporal availability of nutrients for animal.

The Effects of Irrigants on the Survival of Human Stem Cells of the Apical Papilla, Including Endocyn.

INTRODUCTION: Endocyn, a pH-neutral solution of hypochlorous acid and hypochlorite has been developed for use as an endodontic irrigant. The purpose of this study was to evaluate the effect of Endocyn on human periodontal ligament (PDL) fibroblasts, rat osteosarcoma cells (UMR-106), and stem cells of the apical papilla (SCAP) compared with other commonly used endodontic irrigants. METHODS: To determine cytotoxicity, cells were exposed to various concentrations of Endocyn, 6% sodium hypochlorite (NaOCl), 17% EDTA, and 2% chlorhexidine for 10 minutes, 1 hour, or 24 hours. Cell survival was measured fluorescently using calcein AM. Endocyn also was tested for its ability to inhibit SCAP proliferation and alkaline phosphatase activity. Finally, SCAP transcript expression was examined via reverse-transcriptase polymerase chain reaction. RESULTS: Endocyn was no more toxic to PDL and UMR cells than water for up to 24 hours. Endocyn concentrations of 50% were toxic to SCAP after 1 hour of exposure. Endocyn concentrations of >20% inhibited SCAP proliferation, whereas concentrations of ≥10% inhibited alkaline phosphatase activity. Exposure of SCAP to 10% Endocyn for 3 days did not alter most transcript expression, but did significantly reduce the expression of alkaline phosphatase, fibromodulin, and osteomodulin. CONCLUSION: Endocyn was significantly less cytotoxic to PDL, UMR-106, and SCAP cells compared with other commonly used endodontic irrigants. High concentrations of Endocyn did inhibit some transcript expression and alkaline phosphatase activity, indicating a potential reduction in the osteogenic potential of stems cells exposed to Endocyn.

Potential of chlorophyll-rich feed ingredients to improve detection of fecal contamination in the abattoir.

The use of fecal fluorescence to improve detection of contamination of carcasses in the abattoir was previously reported. However, incidents of false negatives can result when animals are offered diets that contain little chlorophyll (e.g., concentrate). Here, we investigated the potential of incorporating a high-chlorophyll-containing feed ingredient (concentrated alfalfa extract; CAE) into the diets of sheep and cattle to improve fecal fluorescence intensity. The sheep experiment evaluated the fecal fluorescence of animals from pasture, when fed a concentrate-barley straw diet and when the concentrate diet incorporated CAE (100 g of dry matter a day). Fecal chlorophyll and metabolite content was highest on the pasture-fed animals and increased significantly over the concentrate diet when CAE was included. Subsequently fluorescent intensity was increased from 15,000 to 36,000 arbitrary units for concentrate and CAE-concentrate diets, respectively, compared with 59,000 for the pasture-fed animals. The cattle experiment investigated the potential of CAE to improve fluorescence of feces from a concentrate diet as well as a silage diet at two levels of incorporation (75 and 150 g CAE/kg of dry matter intake). This study also determined the fluorescence of digesta and carcass contamination in the abattoir on a subset of carcasses. In agreement with the sheep study, CAE significantly improved fluorescence of feces and digesta when added to a concentrate diet, but had little effect on improving fecal fluorescence from the silage-fed animals. This was thought to be related to greater chlorophyll degradation in the rumen or/and the dark nature of the silage feces acting as a quencher of emitted fluoresced light. Incorporating high-chlorophyll-containing plant ingredients into ruminant concentrate diets will improve detection of fecal contamination by reducing false-negative readings. However, they will have little effect on false-positive readings due to the range of wavelengths emitted by natural chlorophyll and its metabolites. Implications and potential solutions for this are discussed.

As yet uncultured bacteria phylogenetically classified as Prevotella, Lachnospiraceae incertae sedis and unclassified Bacteroidales, Clostridiales and Ruminococcaceae may play a predominant role in ruminal biohydrogenation.

Microbial biohydrogenation of dietary poly-unsaturated fatty acids (PUFA) to saturated fatty acids (SFA) in the rumen results in the high ratio of SFA/PUFA in ruminant products, such as meat and milk. In vitro, Butyrivibrio proteoclasticus-related bacteria extensively biohydrogenate PUFA to SFA, yet their contribution in the rumen has not been confirmed. The aim of this study was to evaluate the role of Butyrivibrio proteoclasticus group bacteria in ruminal biohydrogenation and to assess the possible role of other bacteria. Fish oil at 0%, 1.5% and 3% dry matter intake was fed to eight Holstein × Friesian steers, in order to elicit changes in the extent of PUFA biohydrogenation. Fatty acid and B. proteoclasticus group 16S rRNA concentrations in rumen digesta were determined. Correlation between digesta 18:0 concentration and B. proteoclasticus group 16S rRNA concentration was low. Terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis (DGGE) coupled with multivariate statistics revealed that many terminal restriction fragments (T-RFs) and DGGE bands were linked to cis-9, trans-11 conjugated linoleic acid (CLA), 18:1 trans-11 and 18:0 ruminal concentrations. MiCA T-RF predictive identification software showed that these linked T-RFs were likely to originate from as yet uncultured bacteria classified as Prevotella, Lachnospiraceae incertae sedis, and unclassified Bacteroidales, Clostridiales and Ruminococcaceae. Sequencing of linked DGGE bands also revealed that as yet uncultured bacteria classified as Prevotella, Anaerovoax (member of the Lachnospiraceae incertae sedis family), and unclassified Clostridiales and Ruminococcaceae may play a role in biohydrogenation.

Forage type and fish oil cause shifts in rumen bacterial diversity.

Despite evidence supporting improved incorporation of beneficial polyunsaturated fatty acids (PUFA) into ruminant products, such as meat and milk, following red clover and fish oil (FO) inclusion in the ruminant diet, little is known regarding the concomitant bacterial diversity. We evaluated the effects of feeding grass vs. red clover silage with incremental FO inclusion on known lipolytic, biohydrogenating, cellulolytic and proteolytic rumen bacterial communities of steers. Following 14 days of dietary adaptation, liquid-associated (LAB) and solid-associated (SAB) bacterial communities were harvested, DNA extracted and bacterial denaturing gradient gel electrophoresis (DGGE) and specific-bacterial quantitative PCR (QPCR) were undertaken. DGGE-derived dendrograms showed that diet caused the greatest change in LAB and SAB bacterial diversity, with FO inclusion at the 2% and 3% dry matter intake also causing some changes. QPCR revealed that diet resulted in changes in the DNA concentration of Anaerovibrio lipolytica, the Butyrivibrio proteoclasticus group, Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. FO inclusion caused changes in A. lipolytica, F. succinogenes and R. flavefaciens DNA concentration only. In the B. proteoclasticus group, which are the only known bacteria with the capacity to biohydrogenate PUFA to 18:0, DNA concentration did not correlate to 18:0 flow to the duodenum, however, suggesting that other bacteria may play a role in biohydrogenation. A greater understanding of microbial changes that accompany beneficial dietary changes will lead to novel strategies to improve ruminant product quality.