Effect of carbohydrate type in silages and concentrates on feed intake, enteric methane and milk yield from dairy cows.

Dietary carbohydrate manipulation can be used to reduce enteric CH4 emission, but there is a lack of studies on the interaction of different types of carbohydrates that can affect feed intake and ruminal fermentation. Understanding this interaction is necessary to make the most out of CH4 mitigation feeding strategies using different dietary carbohydrates. The aim of this study was to test the effect on enteric CH4 emission, feed intake and milk production response when cows were fed either grass-clover (GCS) or corn silage (CS) as the sole forage source (55% of dry matter, DM), in combination with either barley (BAR) or dried beet pulp (DBP) as a concentrate (21.5% of DM). Twenty-four (half first and half second parity) cows were used in a crossover design with 2 periods of 21 d each, receiving 2 of 4 diets obtained from a 2 × 2 factorial arrangement of the experimental diet. Feed intake, CH4 emission metrics and milk production were recorded at the end of the experimental periods. The diets had NDF concentrations between 258 and 340 g/kg of DM, and starch concentrations between 340 and 7.45 g/kg of DM (CS-BAR and GCS-DBP, respectively). The effects of silage and concentrate on dry matter intake (DMI) were additive, with the highest feed intake in cows fed COR-BAR, followed by cows fed COR-DBP, GCS-BAR, and GCS-DBP (21.2, 19.9, 19.1, and 18.3 kg/d). Energy corrected milk (ECM) yield was not affected by silage source in first parity cows, but it was higher for cows fed CS than cows fed GCS in second parity. The effects of silage and concentrate on CH4 production (g/d), yield (g/kg of DMI) and intensity (g/kg of ECM) were not additive as cows fed GCS had similar responses regardless of the concentrate used, but cows fed CS had lower CH4 production, yield and intensity, when fed BAR instead of DBP. The lower CH4 production, yield and intensity in cows fed CS-BAR compared with other diets could be partially explained by the nonlinear relationship between ruminal VFA and carbohydrates (NDF and starch) concentration reported in literature, however, we observed a linear relationship between acetate:propionate ratio and CH4 yield, suggesting possible other effects. The effects of silage and concentrate on the ruminal VFA were additive in first parity cows, but not in second parity cows. The interaction between dietary CHO type and parity might indicate an effect of feed intake or the energy balance of the cow. Feeding cows silage and concentrate both rich in starch can result in the lowest enteric CH4 emission.

Rumen Lachnospiraceae isolate NK3A20 exhibits metabolic flexibility in response to substrate and coculture with a methanogen.

Hydrogen (H2) is the primary electron donor for methane formation in ruminants, but the H2-producing organisms involved are largely uncharacterized. This work integrated studies of microbial physiology and genomics to characterize rumen bacterial isolate NK3A20 of the family Lachnospiraceae. Isolate NK3A20 was the first recognized isolate of the NK3A20 group, which is among the ten most abundant bacterial genera in 16S rRNA gene surveys of rumen microbiota. NK3A20 produced acetate, butyrate, H2, and formate from glucose. The end product ratios varied when grown with different substrates and at different H2 partial pressures. NK3A20 produced butyrate as a major product using glucose or under high H2 partial pressures and switched to mainly acetate in the presence of galacturonic acid (an oxidized sugar) or in coculture with a methanogen. Growth with galacturonic acid was faster at elevated H2 concentrations, while elevated H2 slowed growth with glucose. Genome analyses revealed the presence of multiple hydrogenases including a membrane-bound Ech hydrogenase, an electron bifurcating butyryl-CoA dehydrogenase (Bcd-Etf), and an Rnf complex that may be involved in modulating the observed metabolic pathway changes, providing insight into H2 formation in the rumen. IMPORTANCE The genus-level NK3A20 group is one of the ten most abundant genera of rumen bacteria. Like most of the rumen bacteria that produce the hydrogen that is converted to methane in the rumen, it is understudied, without any previously characterized isolates. We investigated isolate NK3A20, a cultured member of this genus, and showed that it modulates hydrogen production in response to its growth substrates and the hydrogen concentration in its environment. Low-hydrogen concentrations stimulated hydrogen formation, while high concentrations inhibited its formation and shifted the fermentation to more reduced organic acid products. We found that growth on uronic acids, components of certain plant polymers, resulted in low hydrogen yields compared to glucose, which could aid in the selection of low-methane feeds. A better understanding of the major genera that produce hydrogen in the rumen is part of developing strategies to mitigate biogenic methane emitted by livestock agriculture.

Enteric methane emission of dairy cows supplemented with iodoform in a dose-response study.

Enteric methane (CH4) emission is one of the major greenhouse gasses originating from cattle. Iodoform has in studies been found to be a potent mitigator of rumen CH4 formation in vitro. This study aimed to quantify potential of iodoform as an anti-methanogenic feed additive for dairy cows and investigate effects on feed intake, milk production, feed digestibility, rumen microbiome, and animal health indicators. The experiment was conducted as a 4 × 4 Latin square design using four lactating rumen, duodenal, and ileal cannulated Danish Holstein dairy cows. The treatments consisted of four different doses of iodoform (1) 0 mg/day, (2) 320 mg/day, (3) 640 mg/day, and (4) 800 mg/day. Iodoform was supplemented intra-ruminally twice daily. Each period consisted of 7-days of adaptation, 3-days of digesta and blood sampling, and 4-days of gas exchange measurements using respiration chambers. Milk yield and dry matter intake (DMI) were recorded daily. Rumen samples were collected for microbial analyses and investigated for fermentation parameters. Blood was sampled and analyzed for metabolic and health status indicators. Dry matter intake and milk production decreased linearly by maximum of 48% and 33%, respectively, with increasing dose. Methane yield (g CH4/kg DMI) decreased by maximum of 66%, while up to 125-fold increases were observed in hydrogen yield (g H2/kg DMI) with increasing dose of iodoform. Total tract digestibility of DM, OM, CP, C, NDF, and starch were unaffected by treatments, but large shifts, except for NDF, were observed for ruminal to small intestinal digestion of the nutrients. Some indicators of disturbed rumen microbial activity and fermentation dynamics were observed with increasing dose, but total number of ruminal bacteria was unaffected by treatment. Serum and plasma biomarkers did not indicate negative effects of iodoform on cow health. In conclusion, iodoform was a potent mitigator of CH4 emission. However, DMI and milk production were negatively affected and associated with indications of depressed ruminal fermentation. Future studies might reveal if depression of milk yield and feed intake can be avoided if iodoform is continuously administered by mixing it into a total mixed ration.

Gastrointestinal Microbial Ecology of Weaned Piglets Fed Diets with Different Levels of Glyphosate.

Glyphosate possesses antimicrobial properties, and the present study investigated potential effects of feed glyphosate on piglet gastrointestinal microbial ecology. Weaned piglets were allocated to four diets (glyphosate contents [mg/kg feed]: 0 mg/kg control [CON; i.e., basal diet with no glyphosate added], 20 mg/kg as Glyphomax commercial herbicide [GM20], and 20 mg/kg [IPA20] and 200 mg/kg [IPA200] as glyphosate isopropylamine [IPA] salt). Piglets were sacrificed after 9 and 35 days of treatment, and stomach, small intestine, cecum, and colon digesta were analyzed for glyphosate, aminomethylphosphonic acid (AMPA), organic acids, pH, dry matter content, and microbiota composition. Digesta glyphosate contents reflected dietary levels (on day 35, 0.17, 16.2, 20.5, and 207.5 mg/kg colon digesta, respectively). Overall, we observed no significant glyphosate-associated effects on digesta pH, dry matter content, and-with few exceptions-organic acid levels. On day 9, only minor gut microbiota changes were observed. On day 35, we observed a significant glyphosate-associated decrease in species richness (CON, 462; IPA200, 417) and in the relative abundance of certain Bacteroidetes genera: CF231 (CON, 3.71%; IPA20, 2.33%; IPA200, 2.07%) and g_0.24 (CON, 3.69%; IPA20, 2.07%; IPA200, 1.75%) in cecum. No significant changes were observed at the phylum level. In the colon, we observed a significant glyphosate-associated increase in the relative abundance of Firmicutes (CON, 57.7%; IPA20, 69.4%; IPA200, 66.1%) and a decrease in Bacteroidetes (CON, 32.6%; IPA20, 23.5%). Significant changes were only observed for few genera, e.g., g_0.24 (CON, 7.12%; IPA20, 4.59%; IPA200, 4.00%). In conclusion, exposing weaned piglets to glyphosate-amended feed did not affect gastrointestinal microbial ecology to a degree that was considered actual dysbiosis, e.g., no potential pathogen bloom was observed. IMPORTANCE Glyphosate residues can be found in feed made from genetically modified glyphosate-resistant crops treated with glyphosate or from conventional crops, desiccated with glyphosate before harvest. If these residues affect the gut microbiota to an extent that is unfavorable to livestock health and productivity, the widespread use of glyphosate on feed crops may need to be reconsidered. Few in vivo studies have been conducted to investigate potential impact of glyphosate on the gut microbial ecology and derived health issues of animals, in particular livestock, when exposed to dietary glyphosate residues. The aim of the present study was therefore to investigate potential effects on the gastrointestinal microbial ecology of newly weaned piglets fed glyphosate-amended diets. Piglets did not develop actual gut dysbiosis when fed diets, containing a commercial herbicide formulation or a glyphosate salt at the maximum residue level, defined by the European Union for common feed crops, or at a 10-fold-higher level.

Liquid fermented cereals with added Pediococcus acidilactici did not reduce post-weaning diarrhea in pigs - an Escherichia coli challenge study.

The effect of feeding fermented liquid feed (FLF) with added Pediococcus acidilactici to weaning piglets challenged with enterotoxigenic Escherichia coli (ETEC) F4 on aspects of diarrhea, performance, immune responses, and intestinal epithelial barrier function was investigated. A total of 46 weaners (weaning at 27-30 days of age) were assigned to four treatments: (1) Non-challenged and dry feed (Non-Dry); (2) Challenged and dry feed (Ch-Dry); (3) Non-challenged and FLF (Non-Ferm); (4) Challenged and FLF (Ch-Ferm). All groups received the same feed, either dry (Non-Dry and Ch-Dry), or in liquid form (Non-Ferm and Ch-Ferm) in which the cereals with added P. acidilactici (106 CFU/g cereals) had been fermented for 24 h at 30°C. On day 1 and 2 post weaning, Ch-Dry and Ch-Ferm were orally inoculated with 5 mL × 109 CFU ETEC F4/mL, whereas the Non-Dry and Non-Ferm received the same amount of saline. Fecal samples and blood samples were collected through the study period. The microbial composition, concentration of microbial metabolites and nutrient composition indicated that the quality of the FLF was high. In the first week, ADFI of both non-challenged groups was significantly higher (p < 0.05) than that of the Ch-Ferm group. The two challenged groups had higher fecal levels of FaeG gene (ETEC F4 fimbriae) from day 2 to 6 post weaning (p < 0.01), and higher risk of having ETEC F4 present in feces from day 3 to 5 post weaning (p < 0.05) compared to non-challenged groups, indicating the validity of the ETEC challenge model. Generally, ADG of the two groups fed FLF were numerically higher than those fed dry feed. Neither challenge nor FLF affected diarrhea. No significant differences were measured between Ch-Ferm and Ch-Dry regarding the level of plasma haptoglobin and C-reactive protein, hematological parameters or parameters related to epithelial barrier. The data indicated a low level of infection caused by the ETEC challenge, while recovery from weaning stress could be observed. The study showed that a strategy like this can be a way of providing a high level of probiotics to pigs by allowing their proliferation during fermentation.

Aristaeella hokkaidonensis gen. nov. sp. nov. and Aristaeella lactis sp. nov., two rumen bacterial species of a novel proposed family, Aristaeellaceae fam. nov.

Two strains of Gram-negative, anaerobic, rod-shaped bacteria, from an abundant but uncharacterized rumen bacterial group of the order 'Christensenellales', were phylogenetically and phenotypically characterized. These strains, designated R-7T and WTE2008T, shared 98.6-99.0 % sequence identity between their 16S rRNA gene sequences. R-7T and WTE2008T clustered together on a distinct branch from other Christensenellaceae strains and had <88.1 % sequence identity to the closest type-strain sequence from Luoshenia tenuis NSJ-44T. The genome sequences of R-7T and WTE2008T had 83.6 % average nucleotide identity to each other, and taxonomic assignment using the Genome Taxonomy Database indicates these are separate species within a novel family of the order 'Christensenellales'. Cells of R-7T and WTE2008T lacked any obvious appendages and their cell wall ultra-structures were characteristic of Gram-negative bacteria. The five most abundant cellular fatty acids of both strains were C16 : 0, C16 : 0 iso, C17 : 0 anteiso, C18 : 0 and C15 : 0 anteiso. The strains used a wide range of the 23 soluble carbon sources tested, and grew best on cellobiose, but not on sugar-alcohols. Xylan and pectin were fermented by both strains, but not cellulose. Acetate, hydrogen, ethanol and lactate were the major fermentation end products. R-7T produced considerably more hydrogen than WTE2008T, which produced more lactate. Based on these analyses, Aristaeellaceae fam. nov. and Aristaeella gen. nov., with type species Aristaeella hokkaidonensis sp. nov., are proposed. Strains R-7T (=DSM 112795T=JCM 34733T) and WTE2008T (=DSM 112788T=JCM 34734T) are the proposed type strains for Aristaeella hokkaidonensis sp. nov. and Aristaeella lactis sp. nov., respectively.

Prediction of protein and amino acid composition and digestibility in individual feedstuffs and mixed diets for pigs using near-infrared spectroscopy.

Knowledge of the amounts and digestibility of amino acids in pig feedstuffs is essential for calculating the appropriate inclusion level in a complete diet. Wet chemical analysis and in vivo digestibility trials are time-consuming and costly and cannot be used for routine assessment. Near-infrared spectroscopy (NIRS) offers a rapid, cost effective and environmentally friendly method for evaluating feedstuffs. Calibrations models were developed using NIRS to predict the content of crude protein and 18 amino acids from a wide range of feedstuffs used in pig production (n = 607). The samples ranged from single feed ingredients (containing amino acids from 0.3 to 129.8 g/kg of dry matter) to feed mixtures (containing amino acids from 1.2 to 53.2 g/kg of dry matter). The predictive ability of the calibrations was tested with an independent dataset (n = 150) and with cross-validation. Furthermore, we compare these calibrations with calibrations developed on more narrowly defined groups of samples and with predictions from regression analysis of crude protein. The models were able to predict the concentrations of crude protein and 18 amino acids with good levels of precision and high coefficients of determination for calibration (RSQ CAL) from 0.91 to 0.99 and validation (RSQVAL) from 0.87 to 0.97. Calibration models were able to predict all amino acids except tryptophan and valine with greater accuracy than those from protein regression. We also developed calibration models to predict the apparent ileal and total tract digestibility of protein and amino acids. With the exception of tryptophan, RSQ values (>0.7) and standard error of cross validation (SECV) values (<5%) were obtained for the digestibility of most of the amino acids. In conclusion, NIRS can be used to predict crude protein and amino acid concentrations from a wide range of single ingredients and feed mixtures used for pig diets without separate models for each feedstuff. The digestibility of protein and amino acids can be predicted with an acceptable accuracy to be useful in formulating pig diets.

Complete Genome Sequences of Three Clostridiales R-7 Group Strains Isolated from the Bovine Rumen in New Zealand.

Members of the Clostridiales R-7 group are abundant bacterial residents of the rumen microbiome; however, they are poorly characterized. We report the complete genome sequences of three members of the R-7 group, FE2010, FE2011, and XBB3002, isolated from the ruminal contents of pasture-grazed dairy cows in New Zealand.

Stability Assessment of the Rumen Bacterial and Archaeal Communities in Dairy Cows Within a Single Lactation and Its Association With Host Phenotype.

Better characterization of changes in the rumen microbiota in dairy cows over the lactation period is crucial for understanding how microbial factors may potentially be interacting with host phenotypes. In the present study, we characterized the rumen bacterial and archaeal community composition of 60 lactating Holstein dairy cows (33 multiparous and 27 primiparous), sampled twice within the same lactation with a 122 days interval. Firmicutes and Bacteroidetes dominated the rumen bacterial community and showed no difference in relative abundance between samplings. Two less abundant bacterial phyla (SR1 and Proteobacteria) and an archaeal order (Methanosarcinales), on the other hand, decreased significantly from the mid-lactation to the late-lactation period. Moreover, between-sampling stability assessment of individual operational taxonomic units (OTUs), evaluated by concordance correlation coefficient (C-value) analysis, revealed the majority of the bacterial OTUs (6,187 out of 6,363) and all the 79 archaeal OTUs to be unstable over the investigated lactation period. The remaining 176 stable bacterial OTUs were mainly assigned to Prevotella, unclassified Prevotellaceae, and unclassified Bacteroidales. Milk phenotype-based screening analysis detected 32 bacterial OTUs, mainly assigned to unclassified Bacteroidetes and Lachnospiraceae, associated with milk fat percentage, and 6 OTUs, assigned to Ruminococcus and unclassified Ruminococcaceae, associated with milk protein percentage. These OTUs were only observed in the multiparous cows. None of the archaeal OTUs was observed to be associated with the investigated phenotypic parameters, including methane production. Co-occurrence analysis of the rumen bacterial and archaeal communities revealed Fibrobacter to be positively correlated with the archaeal genus vadinCA11 (Pearson r = 0.76) and unclassified Methanomassiliicoccaceae (Pearson r = 0.64); vadinCA11, on the other hand, was negatively correlated with Methanobrevibacter (Pearson r = -0.56). In conclusion, the rumen bacterial and archaeal communities of dairy cows displayed distinct stability at different taxonomic levels. Moreover, specific members of the rumen bacterial community were observed to be associated with milk phenotype parameters, however, only in multiparous cows, indicating that dairy cow parity could be one of the driving factors for host-microbe interactions.

Predictive ability of host genetics and rumen microbiome for subclinical ketosis.

Subclinical metabolic disorders such as ketosis cause substantial economic losses for dairy farmers in addition to the serious welfare issues they pose for dairy cows. Major hurdles in genetic improvement against metabolic disorders such as ketosis include difficulties in large-scale phenotype recording and low heritability of traits. Milk concentrations of ketone bodies, such as acetone and ß-hydroxybutyric acid (BHB), might be useful indicators to select cows for low susceptibility to ketosis. However, heritability estimates reported for milk BHB and acetone in several dairy cattle breeds were low. The rumen microbial community has been reported to play a significant role in host energy homeostasis and metabolic and physiologic adaptations. The current study aims at investigating the effects of cows' genome and rumen microbial composition on concentrations of acetone and BHB in milk, and identifying specific rumen microbial taxa associated with variation in milk acetone and BHB concentrations. We determined the concentrations of acetone and BHB in milk using nuclear magnetic resonance spectroscopy on morning milk samples collected from 277 Danish Holstein cows. Imputed high-density genotype data were available for these cows. Using genomic and microbial prediction models with a 10-fold resampling strategy, we found that rumen microbial composition explains a larger proportion of the variation in milk concentrations of acetone and BHB than do host genetics. Moreover, we identified associations between milk acetone and BHB with some specific bacterial and archaeal operational taxonomic units previously reported to have low to moderate heritability, presenting an opportunity for genetic improvement. However, higher covariation between specific microbial taxa and milk acetone and BHB concentrations might not necessarily indicate a causal relationship; therefore further validation is needed before considering implementation in selection programs.

Rumen and Fecal Microbial Community Structure of Holstein and Jersey Dairy Cows as Affected by Breed, Diet, and Residual Feed Intake.

Identifying factors that influence the composition of the microbial population in the digestive system of dairy cattle will be key in regulating these populations to reduce greenhouse gas emissions. In this study, we analyzed rumen and fecal samples from five high residual feed intake (RFI) Holstein cows, five low RFI Holstein cows, five high RFI Jersey cows and five low RFI Jersey cows, fed either a high-concentrate diet (expected to reduce methane emission) or a high-forage diet. Bacterial communities from both the rumen and feces were profiled using Illumina sequencing on the 16S rRNA gene. Rumen archaeal communities were profiled using Terminal-Restriction Fragment Length Polymorphism (T-RFLP) targeting the mcrA gene. The rumen methanogen community was influenced by breed but not by diet or RFI. The rumen bacterial community was influenced by breed and diet but not by RFI. The fecal bacterial community was influenced by individual animal variation and, to a lesser extent, by breed and diet but not by RFI. Only the bacterial community correlated with methane production. Community differences seen in the rumen were reduced or absent in feces, except in the case of animal-to-animal variation, where differences were more pronounced. The two cattle breeds had different levels of response to the dietary intervention; therefore, it may be appropriate to individually tailor methane reduction strategies to each cattle breed.

Impact of the rumen microbiome on milk fatty acid composition of Holstein cattle.

BACKGROUND: Fatty acids (FA) in bovine milk derive through body mobilization, de novo synthesis or from the feed via the blood stream. To be able to digest feedstuff, the cow depends on its rumen microbiome. The relative abundance of the microbes has been shown to differ between cows. To date, there is little information on the impact of the microbiome on the formation of specific milk FA. Therefore, in this study, our aim was to investigate the impact of the rumen bacterial microbiome on milk FA composition. Furthermore, we evaluated the predictive value of the rumen microbiome and the host genetics on the composition of individual FA in milk. RESULTS: Our results show that the proportion of variance explained by the rumen bacteria composition (termed microbiability or [Formula: see text]) was generally smaller than that of the genetic component (heritability), and that rumen bacteria influenced most C15:0, C17:0, C18:2 n-6, C18:3 n-3 and CLA cis-9, trans-11 with estimated [Formula: see text] ranging from 0.26 to 0.42. For C6:0, C8:0, C10:0, C12:0, C16:0, C16:1 cis-9 and C18:1 cis-9, the variance explained by the rumen bacteria component was close to 0. In general, both the rumen microbiome and the host genetics had little value for predicting FA phenotype. Compared to genetic information only, adding rumen bacteria information resulted in a significant improvement of the predictive value for C15:0 from 0.22 to 0.38 (P = 9.50e-07) and C18:3 n-3 from 0 to 0.29 (P = 8.81e-18). CONCLUSIONS: The rumen microbiome has a pronounced influence on the content of odd chain FA and polyunsaturated C18 FA, and to a lesser extent, on the content of the short- and medium-chain FA in the milk of Holstein cattle. The accuracy of prediction of FA phenotypes in milk based on information from either the animal's genotypes or rumen bacteria composition was very low.

Host genetics and the rumen microbiome jointly associate with methane emissions in dairy cows.

Cattle and other ruminants produce large quantities of methane (~110 million metric tonnes per annum), which is a potent greenhouse gas affecting global climate change. Methane (CH4) is a natural by-product of gastro-enteric microbial fermentation of feedstuffs in the rumen and contributes to 6% of total CH4 emissions from anthropogenic-related sources. The extent to which the host genome and rumen microbiome influence CH4 emission is not yet well known. This study confirms individual variation in CH4 production was influenced by individual host (cow) genotype, as well as the host's rumen microbiome composition. Abundance of a small proportion of bacteria and archaea taxa were influenced to a limited extent by the host's genotype and certain taxa were associated with CH4 emissions. However, the cumulative effect of all bacteria and archaea on CH4 production was 13%, the host genetics (heritability) was 21% and the two are largely independent. This study demonstrates variation in CH4 emission is likely not modulated through cow genetic effects on the rumen microbiome. Therefore, the rumen microbiome and cow genome could be targeted independently, by breeding low methane-emitting cows and in parallel, by investigating possible strategies that target changes in the rumen microbiome to reduce CH4 emissions in the cattle industry.

Changes in rumen bacterial and archaeal communities over the transition period in primiparous Holstein dairy cows.

In the present study, we hypothesized that the rumen bacterial and archaeal communities would change significantly over the transition period of dairy cows, mainly as an adaptation to the classical use of low-grain prepartum and high-grain postpartum diets. Bacterial 16S rRNA gene amplicon sequencing of rumen samples from 10 primiparous Holstein dairy cows revealed no changes over the transition period in relative abundance of genera such as Ruminococcus, Butyrivibrio, Clostridium, Coprococcus, and Pseudobutyrivibrio. However, other dominant genus-level taxa, such as Prevotella, unclassified Ruminococcaceae, and unclassified Succinivibrionaceae, showed distinct changes in relative abundance from the prepartum to the postpartum period. Overall, we observed individual fluctuation patterns over the transition period for a range of bacterial taxa that, in some cases, were correlated with observed changes in the rumen short-chain fatty acids profile. Combined results from clone library and terminal-restriction fragment length polymorphism (T-RFLP) analyses, targeting the methyl-coenzyme M reductase α-subunit (mcrA) gene, revealed a methanogenic archaeal community dominated by the Methanobacteriales and Methanomassiliicoccales orders, particularly the genera Methanobrevibacter, Methanosphaera, and Methanomassiliicoccus. As observed for the bacterial community, the T-RFLP patterns showed significant shifts in methanogenic community composition over the transition period. Together, the composition of the rumen bacterial and archaeal communities exhibited changes in response to particularly the dietary changes of dairy cows over the transition period.

Holistic Assessment of Rumen Microbiome Dynamics through Quantitative Metatranscriptomics Reveals Multifunctional Redundancy during Key Steps of Anaerobic Feed Degradation.

Ruminant livestock is a major source of the potent greenhouse gas methane. The complex rumen microbiome, consisting of bacteria, archaea, and microbial eukaryotes, facilitates anaerobic plant biomass degradation in the cow rumen, leading to methane emissions. Using an integrated approach combining multidomain quantitative metatranscriptomics with gas and volatile fatty acid (VFA) profiling, we aimed at obtaining the most comprehensive picture of the active rumen microbiome during feed degradation to date. Bacterial, archaeal, and eukaryotic biomass, but also methane emissions and VFA concentrations, increased drastically within an hour after feed intake. mRNA profiling revealed a dynamic response of carbohydrate-active enzyme transcripts, transcripts involved in VFA production and methanogenesis. While the relative abundances of functional transcripts did not mirror observed processes, such as methane emissions, transformation to mRNA abundance per gram of rumen fluid echoed ruminant processes. The microbiome composition was highly individual, with, e.g., ciliate, Neocallimastigaceae, Prevotellaceae, Succinivibrionaceae, and Fibrobacteraceae abundances differing between cows. Microbiome individuality was accompanied by inter- and intradomain multifunctional redundancy among microbiome members during feed degradation. This likely enabled the robust performance of the anaerobic degradation process in each rumen. Neocallimastigaceae and ciliates contributed an unexpectedly large share of transcripts for cellulose- and hemicellulose-degrading enzymes, respectively. Methyl-reducing but not CO2-reducing methanogens were positively correlated with methane emissions. While Methanomassiliicoccales switched from methanol to methylamines as electron acceptors, Methanosphaera became the dominating methanol-reducing methanogen. This study for the first time linked rumen meta-omics with processes and enabled holistic insights into the contribution of all microbiome members to feed degradation. IMPORTANCE Ruminant animals, such as cows, live in a tight symbiotic association with microorganisms, allowing them to feed on otherwise indigestible plant biomass as food sources. Methane is produced as an end product of the anaerobic feed degradation in ruminants and is emitted to the atmosphere, making ruminant animals among the major anthropogenic sources of the potent greenhouse gas methane. Using newly developed quantitative metatranscriptomics for holistic microbiome analysis, we here identified bacterial, archaeal, and eukaryotic key players and the short-term dynamics of the rumen microbiome during anaerobic plant biomass degradation and subsequent methane emissions. These novel insights might pave the way for novel ecologically and economically sustainable methane mitigation strategies, much needed in times of global climate change.

Development of a species-specific TaqMan-MGB real-time PCR assay to quantify Olsenella scatoligenes in pigs offered a chicory root-based diet.

Olsenella scatoligenes is the only skatole-producing bacterium isolated from the pig gut. Skatole, produced from microbial degradation of l-tryptophan, is the main contributor to boar taint, an off-odor and off-flavor taint, released upon heating meat from some entire male pigs. An appropriate method for quantifying O. scatoligenes would help investigating the relationship between O. scatoligenes abundance and skatole concentration in the pig gut. Thus, the present study aimed at developing a TaqMan-MGB probe-based, species-specific qPCR assay for rapid quantification of O. scatoligenes. The use of a MGB probe allowed discriminating O. scatoligenes from other closely related species. Moreover, the assay allowed quantifying down to three target gene copies per PCR reaction using genomic DNA-constructed standards, or 1.5 × 103 cells/g digesta, using O. scatoligenes-spiked digesta samples as reference standards. The developed assay was applied to assess the impact of dietary chicory roots on O. scatoligenes in the hindgut of pigs. Olsenella scatoligenes made up < 0.01% of the microbial population in the pig hindgut. Interestingly, the highest number of O. scatoligenes was found in young entire male pigs fed high levels of chicory roots. This indicates that the known effect of chicory roots for reducing skatole production is not by inhibiting the growth of this skatole-producing bacterium in the pig hindgut. Accordingly, the abundance of O. scatoligenes in the hindgut does not seem to be an appropriate indicator of boar taint. The present study is the first to describe a TaqMan-MGB probe qPCR assay for detection and quantification of O. scatoligenes in pigs.

Community structure of the metabolically active rumen bacterial and archaeal communities of dairy cows over the transition period.

Dairy cows experience dramatic changes in host physiology from gestation to lactation period and dietary switch from high-forage prepartum diet to high-concentrate postpartum diet over the transition period (parturition +/- three weeks). Understanding the community structure and activity of the rumen microbiota and its associative patterns over the transition period may provide insight for e.g. improving animal health and production. In the present study, rumen samples from ten primiparous Holstein dairy cows were collected over seven weeks spanning the transition period. Total RNA was extracted from the rumen samples and cDNA thereof was subsequently used for characterizing the metabolically active bacterial (16S rRNA transcript amplicon sequencing) and archaeal (qPCR, T-RFLP and mcrA and 16S rRNA transcript amplicon sequencing) communities. The metabolically active bacterial community was dominated by three phyla, showing significant changes in relative abundance range over the transition period: Firmicutes (from prepartum 57% to postpartum 35%), Bacteroidetes (from prepartum 22% to postpartum 18%) and Proteobacteria (from prepartum 7% to postpartum 32%). For the archaea, qPCR analysis of 16S rRNA transcript number, revealed a significant prepartum to postpartum increase in Methanobacteriales, in accordance with an observed increase (from prepartum 80% to postpartum 89%) in relative abundance of 16S rRNA transcript amplicons allocated to this order. On the other hand, a significant prepartum to postpartum decrease (from 15% to 2%) was observed in relative abundance of Methanomassiliicoccales 16S rRNA transcripts. In contrast to qPCR analysis of the 16S rRNA transcripts, quantification of mcrA transcripts revealed no change in total abundance of metabolically active methanogens over the transition period. According to T-RFLP analysis of the mcrA transcripts, two Methanobacteriales genera, Methanobrevibacter and Methanosphaera (represented by the T-RFs 39 and 267 bp), represented more than 70% of the metabolically active methanogens, showing no significant changes over the transition period; minor T-RFs, likely to represent members of the order Methanomassiliicoccales and with a relative abundance below 5% in total, decreased significantly over the transition period. In accordance with the T-RFLP analysis, the mcrA transcript amplicon sequencing revealed Methanobacteriales to cover 99% of the total reads, dominated by the genera Methanobrevibacter (75%) and Methanosphaera (24%), whereas the Methanomassiliicoccales order covered only 0.2% of the total reads. In conclusion, the present study showed that the structure of the metabolically active bacterial and archaeal rumen communities changed over the transition period, likely in response to the dramatic changes in physiology and nutritional factors like dry matter intake and feed composition. It should be noted however that for the methanogens, the observed community changes were influenced by the analyzed gene (mcrA or 16S rRNA).

Seasonal changes in the digesta-adherent rumen bacterial communities of dairy cattle grazing pasture.

The complex microbiota that resides within the rumen is responsible for the break-down of plant fibre. The bacteria that attach to ingested plant matter within the rumen are thought to be responsible for initial fibre degradation. Most studies examining the ecology of this important microbiome only offer a 'snapshot' in time. We monitored the diversity of rumen bacteria in four New Zealand dairy cows, grazing a rye-grass and clover pasture over five consecutive seasons, using high throughput pyrosequencing of bacterial 16S rRNA genes. We chose to focus on the digesta-adherent bacterial community to learn more about the stability of this community over time. 16S rRNA gene sequencing showed a high level of bacterial diversity, totalling 1539 operational taxonomic units (OTUs, grouped at 96% sequence similarity) across all samples, and ranging from 653 to 926 OTUs per individual sample. The nutritive composition of the pasture changed with the seasons as did the production phase of the animals. Sequence analysis showed that, overall, the bacterial communities were broadly similar between the individual animals. The adherent bacterial community was strongly dominated by members of Firmicutes (82.1%), followed by Bacteroidetes (11.8%). This community differed between the seasons, returning to close to that observed in the same season one year later. These seasonal differences were only small, but were statistically significant (p < 0.001), and were probably due to the seasonal differences in the diet. These results demonstrate a general invariability of the ruminal bacterial community structure in these grazing dairy cattle.

Fabrication and surface modification of poly lactic acid (PLA) scaffolds with epidermal growth factor for neural tissue engineering.

In an effort to design biomaterials that may promote repair of the central nervous system, 3-dimensional scaffolds made of electrospun poly lactic acid nanofibers with interconnected pores were fabricated. These scaffolds were functionalized with polyallylamine to introduce amine groups by wet chemistry. Experimental conditions of the amination protocol were thoroughly studied and selected to introduce a high amount of amine group while preserving the mechanical and structural properties of the scaffold. Subsequent covalent grafting of epidermal growth factor was then performed to further tailor these aminated structures. The scaffolds were then tested for their ability to support Neural Stem-Like Cells (NSLCs) culture. Of interest, NSLCs were able to proliferate on these EGF-grafted substrates and remained viable up to 14 d even in the absence of soluble growth factors in the medium.

Co-immobilization of adhesive peptides and VEGF within a dextran-based coating for vascular applications.

UNLABELLED: Multifunctional constructs providing a proper environment for adhesion and growth of selected cell types are needed for most tissue engineering and regenerative medicine applications. In this context, vinylsulfone (VS)-modified dextran was proposed as a matrix featuring low-fouling properties as well as multiple versatile moieties. The displayed VS groups could indeed react with thiol, amine or hydroxyl groups, be it for surface grafting, crosslinking or subsequent tethering of biomolecules. In the present study, a library of dextran-VS was produced, grafted to aminated substrates and characterized in terms of degree of VS modification (%VS), cell-repelling properties and potential for the oriented grafting of cysteine-tagged peptides. As a bioactive coating of vascular implants, ECM peptides (e.g. RGD) as well as vascular endothelial growth factor (VEGF) were co-immobilized on one of the most suitable dextran-VS coating (%VS=ca. 50% of saccharides units). Both RGD and VEGF were efficiently tethered at high densities (ca. 1nmol/cm(2) and 50fmol/cm(2), respectively), and were able to promote endothelial cell adhesion as well as proliferation. The latter was enhanced to the same extent as with soluble VEGF and proved selective to endothelial cells over smooth muscle cells. Altogether, multiple biomolecules could be efficiently incorporated into a dextran-VS construct, while maintaining their respective biological activity. STATEMENT OF SIGNIFICANCE: This work addresses the need for multifunctional coatings and selective cell response inherent to many tissue engineering and regenerative medicine applications, for instance, vascular graft. More specifically, a library of dextrans was first generated through vinylsulfone (VS) modification. Thoroughly selected dextran-VS provided an ideal platform for unbiased study of cell response to covalently grafted biomolecules. Considering that processes such as healing and angiogenesis require multiple factors acting synergistically, vascular endothelial growth factor (VEGF) was then co-immobilized with the cell adhesive RGD peptide within our dextran coating through a relevant strategy featuring orientation and specificity. Altogether, both adhesive and proliferative cues could be incorporated into our construct with additive, if not synergetic, effects.