Evaluating Established Methods for Rumen 16S rRNA Amplicon Sequencing With Mock Microbial Populations

peer-reviewedThe rumen microbiome scientific community has utilized amplicon sequencing as an aid in identifying potential community compositional trends that could be used as an estimation of various production and performance traits including methane emission, animal protein production efficiency, and ruminant health status. In order to translate rumen microbiome studies into executable application, there is a need for experimental and analytical concordance within the community. The objective of this study was to assess these factors in relation to selected currently established methods for 16S phylogenetic community analysis on a microbial community standard (MC) and a DNA standard (DS; ZymoBIOMICSTM). DNA was extracted from MC using the RBBC method commonly used for microbial DNA extraction from rumen digesta samples. 16S rRNA amplicon libraries were generated for the MC and DS using primers routinely used for rumen bacterial and archaeal community analysis. The primers targeted the V4 and V3–V4 region of the 16S rRNA gene and samples were subjected to both 20 and 28 polymerase chain reaction (PCR) cycles under identical cycle conditions. Sequencing was conducted using the Illumina MiSeq platform. As the bacteria contained in the microbial mock community were well-classified species, and for ease of explanation, we used the results of the Basic Local Alignment Search Tool classification to assess the DNA, PCR cycle number, and primer type. Sequence classification methodology was assessed independently. Spearman’s correlation analysis indicated that utilizing the repeated bead beating and column method for DNA extraction in combination with primers targeting the 16S rRNA gene using 20 first-round PCR cycles was sufficient for amplicon sequencing to generate a relatively accurate depiction of the bacterial communities present in rumen samples. These results also emphasize the requirement to develop and utilize positive mock community controls for all rumen microbiomic studies in order to discern errors which may arise at any step during a next-generation sequencing protocol

Effect of brown and green seaweeds on diet digestibility, ruminal fermentation patterns and enteric methane emissions using the rumen simulation technique

Inclusion of the red seaweed Asparagopsis taxiformis as a feed additive, has led to significant reductions in methane (CH4) production from ruminants. However, dietary supplementation with this seaweed is negatively associated with health and environmental concerns mainly due to its bromoform content, a compound with potential carcinogenic properties. Thus, there is renewed focus on ascertaining the anti-methanogenic potential of locally grown brown and green seaweeds, which typically do not contain bromoform. The objective of this study was to investigate the effects of selected brown and green seaweeds on diet digestibility, ruminal fermentation patterns, total gas (TGP) and CH4 production in vitro, using the rumen simulation technique system. In experiment 1, Pelvetia canaliculata (PEC) was examined. In experiment 2, Cystoseira tamariscifolia (CYT), Bifurcaria bifurcata (BIB), Fucus vesiculosus (FUV), Himanthalia elongata (HIM) and Ulva intestinalis (ULI) were analysed. Ascophyllum nodosum (ASC) was included in both experiments. A diet containing A. taxiformis (ASP1; ASP2) and an unsupplemented diet (CON) were included as positive and negative controls, respectively in both experiments. All seaweeds were included at a rate of 10 g/kg dry matter (DM) into a control diet of 50:50 (w:w) forage:concentrate. The seven brown and green seaweeds assessed failed to affect absolute CH4 emissions or alter fermentation patterns. In experiment 1, seaweed treatment had no effect on diet digestibility, CH4%, CH4 mmol/d or CH4 L/d (P>0.1), however ASP1 reduced CH4 mmol/g DOM by 49% (P<0.01) relative to the control. Both ASC and ASP1 tended to increase TGP (P<0.1) relative to the control. In addition to this, the inclusion of seaweed in experiment 1 reduced the production of NH3-N (P<.0001) compared to the control. In experiment 2, seaweed treatment had no effect on diet digestibility or TGP. Both ASP2 and FUV reduced CH4% (P<0.01) but only ASP2 significantly reduced CH4 mmol/d, CH4 L/d and CH4 mmol/g DOM (P<0.05). Daily mMol butyrate was reduced by ASP2 relative to the control and most other seaweeds (P<.0001). In both experiment 1 and 2, seaweed inclusion had no effect on daily total VFA, acetate or propionate production or the acetate:propionate ratio relative to the control. To conclude, including the bromoform-free brown and green seaweeds at 10g/kg DM has no negative effects on diet digestibility or fermentation patterns but also failed to reduce the production of enteric CH4in vitro

Diversity and Community Composition of Methanogenic Archaea in the Rumen of Scottish Upland Sheep Assessed by Different Methods

Acknowledgments We thank Bob Mayes and Dave Hamilton of the James Hutton Institute for their permission and help in sampling the sheep digesta. Gillian Campbell and Pauline Young provided an excellent DNA sequencing service. We also thank Dr Matthew McCabe for preparing V6–V8 amplicon libraries. Author Contributions Conceived and designed the experiments: RJW. Performed the experiments: RJW BG NM SMW CJC. Analyzed the data: TJS MW SMW CJC RJW. Contributed reagents/materials/analysis tools: NM RJW MW SMW CJC. Contributed to the writing of the manuscript: TJS MW SMW CJC RJW.Peer reviewedPublisher PD

Enteric methane research and mitigation strategies for pastoral-based beef cattle production systems

peer-reviewedRuminant livestock play a key role in global society through the conversion of lignocellulolytic plant matter into high-quality sources of protein for human consumption. However, as a consequence of the digestive physiology of ruminant species, methane (CH4), which originates as a byproduct of enteric fermentation, is accountable for 40% of global agriculture's carbon footprint and ~6% of global greenhouse gas (GHG) emissions. Therefore, meeting the increasing demand for animal protein associated with a growing global population while reducing the GHG intensity of ruminant production will be a challenge for both the livestock industry and the research community. In recent decades, numerous strategies have been identified as having the potential to reduce the methanogenic output of livestock. Dietary supplementation with antimethanogenic compounds, targeting members of the rumen methanogen community and/or suppressing the availability of methanogenesis substrates (mainly H2 and CO2), may have the potential to reduce the methanogenic output of housed livestock. However, reducing the environmental impact of pasture-based beef cattle may be a challenge, but it can be achieved by enhancing the nutritional quality of grazed forage in an effort to improve animal growth rates and ultimately reduce lifetime emissions. In addition, the genetic selection of low-CH4-emitting and/or faster-growing animals will likely benefit all beef cattle production systems by reducing the methanogenic potential of future generations of livestock. Similarly, the development of other mitigation technologies requiring minimal intervention and labor for their application, such as anti-methanogen vaccines, would likely appeal to livestock producers, with high uptake among farmers if proven effective. Therefore, the objective of this review is to give a detailed overview of the CH4 mitigation solutions, both currently available and under development, for temperate pasture-based beef cattle production systems. A description of ruminal methanogenesis and the technologies used to estimate enteric emissions at pastures are also presented.Funding and support from the FACCE ERA-GAS RumenPredict grant (16/RD/ERAGAS/1RUMENPREDICTROI 2017) and Horizon 2020 MASTER grant (818368) is acknowledged. PS was funded by a Teagasc Walsh Scholarship (RMIS 0364)

Genome wide association study of passive immunity and disease traits in beef-suckler and dairy calves on Irish farms

peer reviewedCalves with lower concentrations of immunoglobulin G (IgG) in their blood, have a greater risk of developing diseases. There is a lack of knowledge on genetic markers known to be associated with immunological variability or disease resistance. Therefore, the objective of this study was to identify SNP markers associated with passive immunity measures (serum IgG, serum protein, albumin, globulin and total protein concentrations, total solids Brix percentage, zinc sulphate turbidity units) and disease (pneumonia, diarrhoea, crude illness) traits in Irish commercial beef-suckler and dairy calves through genome wide association studies (GWAS). Genotyping was performed on DNA samples from beef-suckler (n = 698) and dairy (n = 1178) calves, using the IDBv3 chip. Heritability of passive immunity associated traits (range 0.02–0.22) and the disease traits (range 0.03–0.20) were low-to-moderate. Twenty-five and fifteen SNPs approached genome wide significance (P < 5 × 10−5) for the passive immunity and the disease traits, respectively. One SNP “ARS-BFGL-BAC-27914” reached Bonferroni genome wide significance (P < 1.15 × 10−6) for an association with serum IgG concentration in beef calves. Further work will evaluate these SNPs in larger cattle populations and assess their contribution to genomic selection breeding strategies, aimed towards producing more disease resistant livestock.Department of Agriculture, Food and the Marine, Irelan

Liver transcriptome profiling of beef steers with divergent growth rate, feed intake, or metabolic body weight phenotypes

peer-reviewedAverage daily gain (ADG) and daily dry matter intake (DMI) are key determinants of beef industry profitability. These traits together with metabolic body weight (MWT) are combined as component traits to calculate residual feed intake (RFI), a common measure of feed efficiency in beef cattle. Recently, there have been significant efforts towards molecular genetic characterization of RFI through transcriptomic studies in different breeds and tissues. However, molecular mechanisms of RFI component traits still remain predominately unexplored. Therefore, in the current study, we investigated the hepatic transcriptomic profiles and their associations with ADG, DMI, and MWT in Angus, Charolais, and Kinsella Composite (KC) populations through global RNAseq analyses. In each population and for each trait, 12 steers with extreme phenotypes (n = 6 low and n = 6 high) were analyzed for differential gene expression. These animals were from 20 beef steers of each Angus, Charolais, and KC breed population that were initially selected for a transcriptome study of RFI. At a false discovery rate 1.5, we identified 123, 102, and 78 differentially expressed (DE) genes between high- and lowADG animals of Angus, Charolais, and KC populations, respectively. For DMI, 108, 180, and 156 DE genes were identified between high- and low-DMI from Angus, Charolais, and KC populations, respectively, while for MWT, 80, 82, and 84 genes were differentially expressed between high- and low-MWT animals in Angus, Charolais, and KC populations, respectively. The identified DE genes were largely breed specific (81.7% for ADG, 82.7% for DMI, and 83% for MWT), but were largely involved in the same biological functions across the breeds. Among the most enriched biological functions included metabolism of major nutrients (lipids, carbohydrates, amino acids, vitamins, and minerals), small molecule biochemistry, cellular movement, cell morphology, and cell-to-cell signaling and interaction. Notably, we identified multiple DE genes that are involved in cholesterol biosynthesis, and immune response pathways for the 3 studied traits. Thus, our findings present potential molecular genetic mechanisms and candidate genes that influence feed intake, growth, and MWT of beef cattle

Effect of early calf-hood nutrition on the transcriptomic profile of subcutaneous adipose tissue in Holstein-Friesian bulls

peer-reviewedBackground: Adipose tissue is a major endocrine organ and is thought to play a central role in the metabolic control of reproductive function in cattle. Plane of nutrition during early life has been shown to influence the timing of puberty in both male and female cattle, though the exact biological mechanisms involved are currently unknown. The aim of this study was to investigate the effect of early calf-hood nutrition on the transcriptomic profile of subcutaneous adipose tissue in Holstein-Friesian bulls to identify possible downstream effects on reproductive physiology. Results: Holstein-Friesian bull calves with a mean (±S.D.) age and bodyweight of 19 (±8.2) days and 47.5 (±5.3) kg, respectively, were assigned to either a high (n = 10) or low (n = 10) plane of nutrition. Calves were fed in order to achieve an overall growth rate of 1.08 and 0.57 kg/day for the high and low plane of nutrition treatments, respectively. At 126 days of age, the bulls were euthanized, subcutaneous adipose tissue samples were harvested and RNAseq analysis was performed. There were 674 genes differentially expressed in adipose tissue of calves on the low compared with the high plane of nutrition (P 2.0). High plane of nutrition positively altered the expression of genes across an array of putative biological processes but the most dominant cellular processes affected were cellular energy production and branched chain amino acid degradation. A high plane of nutrition caused upregulation of genes such as leptin (LEP) and adiponectin (ADIPOQ), which are known to directly affect reproductive function. Conclusions: These results provide an insight into the effect of augmenting the plane of nutrition of Holstein-Friesian bull calves in the prepubertal period on the transcriptome of adipose tissue