Whole blood transcriptome analysis in dairy calves experimentally challenged with bovine herpesvirus 1 (BoHV-1) and comparison to a bovine respiratory syncytial virus (BRSV) challenge.

Bovine herpesvirus 1 (BoHV-1), is associated with several clinical syndromes in cattle, among which bovine respiratory disease (BRD) is of particular significance. Despite the importance of the disease, there is a lack of information on the molecular response to infection via experimental challenge with BoHV-1. The objective of this study was to investigate the whole-blood transcriptome of dairy calves experimentally challenged with BoHV-1. A secondary objective was to compare the gene expression results between two separate BRD pathogens using data from a similar challenge study with BRSV. Holstein-Friesian calves (mean age (SD) = 149.2 (23.8) days; mean weight (SD) = 174.6 (21.3) kg) were either administered BoHV-1 inoculate (1 × 107/mL × 8.5 mL) (n = 12) or were mock challenged with sterile phosphate buffered saline (n = 6). Clinical signs were recorded daily from day (d) -1 to d 6 (post-challenge), and whole blood was collected in Tempus RNA tubes on d six post-challenge for RNA-sequencing. There were 488 differentially expressed (DE) genes (p < 0.05, False Discovery rate (FDR) < 0.10, fold change ≥2) between the two treatments. Enriched KEGG pathways (p < 0.05, FDR <0.05); included Influenza A, Cytokine-cytokine receptor interaction and NOD-like receptor signalling. Significant gene ontology terms (p < 0.05, FDR <0.05) included defence response to virus and inflammatory response. Genes that are highly DE in key pathways are potential therapeutic targets for the treatment of BoHV-1 infection. A comparison to data from a similar study with BRSV identified both similarities and differences in the immune response to differing BRD pathogens.

Assessment of Rapid MinION Nanopore DNA Virus Meta-Genomics Using Calves Experimentally Infected with Bovine Herpes Virus-1.

Bovine respiratory disease (BRD), which is the leading cause of morbidity and mortality in cattle, is caused by numerous known and unknown viruses and is responsible for the widespread use of broad-spectrum antibiotics despite the use of polymicrobial BRD vaccines. Viral metagenomics sequencing on the portable, inexpensive Oxford Nanopore Technologies MinION sequencer and sequence analysis with its associated user-friendly point-and-click Epi2ME cloud-based pathogen identification software has the potential for point-of-care/same-day/sample-to-result metagenomic sequence diagnostics of known and unknown BRD pathogens to inform a rapid response and vaccine design. We assessed this potential using in vitro viral cell cultures and nasal swabs taken from calves that were experimentally challenged with a single known BRD-associated DNA virus, namely, bovine herpes virus 1. Extensive optimisation of the standard Oxford Nanopore library preparation protocols, particularly a reduction in the PCR bias of library amplification, was required before BoHV-1 could be identified as the main virus in the in vitro cell cultures and nasal swab samples within approximately 7 h from sample to result. In addition, we observed incorrect assignment of the bovine sequence to bacterial and viral taxa due to the presence of poor-quality bacterial and viral genome assemblies in the RefSeq database used by the EpiME Fastq WIMP pathogen identification software.

Elucidation of the Host Bronchial Lymph Node miRNA Transcriptome Response to Bovine Respiratory Syncytial Virus.

Bovine respiratory disease (BRD) causes substantial morbidity and mortality, affecting cattle of all ages. One of the main causes of BRD is an initial inflammatory response to bovine respiratory syncytial virus (BRSV). MicroRNAs are novel and emerging non-coding small RNAs that regulate many biological processes and are implicated in various inflammatory diseases. The objective of the present study was to elucidate the changes in the bovine bronchial lymph node miRNA transcriptome in response to BRSV following an experimental viral challenge. Holstein-Friesian calves were either administered a challenge dose of BRSV (103.5 TCID50/ml × 15 ml) (n = 12) or were mock inoculated with sterile phosphate buffered saline (n = 6). Daily scoring of clinical signs was performed and calves were euthanized at day 7 post-challenge. Bronchial lymph nodes were collected for subsequent RNA extraction and sequencing (75 bp). Read counts for known miRNAs were generated using the miRDeep2 package using the UMD3.1 reference genome and the bovine mature miRNA sequences from the miRBase database (release 22). EdgeR was used for differential expression analysis and Targetscan was used to identify target genes for the differentially expressed (DE) miRNAs. Target genes were examined for enriched pathways and gene ontologies using Ingenuity Pathway Analysis (Qiagen). Multi-dimensional scaling (MDS) based on miRNA gene expression changes, revealed a clearly defined separation between the BRSV challenged and control calves, although the clinical manifestation of disease was only mild. One hundred and nineteen DE miRNAs (P < 0.05, FDR < 0.1, fold change > 1.5) were detected between the BRSV challenged and control calves. The DE miRNAs were predicted to target 465 genes which were previously found to be DE in bronchial lymph node tissue, between these BRSV challenged and control calves. Of the DE predicted target genes, 455 had fold changes that were inverse to the corresponding DE miRNAs. There were eight enriched pathways among the DE predicted target genes with inverse fold changes to their corresponding DE miRNA including: granulocyte and agranulocyte adhesion and diapedesis, interferon signalling and role of pathogen recognition receptors in recognition of bacteria and viruses. Functions predicted to be increased included: T cell response, apoptosis of leukocytes, immune response of cells and stimulation of cells. Pathogen recognition and proliferation of cytotoxic T cells are vital for the recognition of the virus and its subsequent elimination.

Messenger RNA biomarkers of Bovine Respiratory Syncytial Virus infection in the whole blood of dairy calves.

Bovine Respiratory Syncytial Virus (BRSV) is a primary viral cause of Bovine Respiratory Disease (BRD) in young calves, which is responsible for substantial morbidity and mortality. Infection with BRSV induces global gene expression changes in respiratory tissues. If these changes are observed in tissues which are more accessible in live animals, such as whole blood, they may be used as biomarkers for diagnosis of the disease. Therefore, the objective of the current study was to elucidate the whole blood transcriptomic response of dairy calves to an experimental challenge with BRSV. Calves (Holstein-Friesian) were either administered BRSV inoculate (103.5 TCID50/ml × 15 ml) (n = 12) or sterile phosphate buffered saline (n = 6). Clinical signs were scored daily and whole blood was collected in Tempus RNA tubes immediately prior to euthanasia, at day 7 post-challenge. RNA was extracted from blood and sequenced (150 bp paired-end). The sequence reads were aligned to the bovine reference genome (UMD3.1) and EdgeR was subsequently employed for differential gene expression analysis. Multidimensional scaling showed that samples from BRSV challenged and control calves segregated based on whole blood gene expression changes, despite the BRSV challenged calves only displaying mild clinical symptoms of the disease. There were 281 differentially expressed (DE) genes (p < 0.05, FDR < 0.1, fold change > 2) between the BRSV challenged and control calves. The top enriched KEGG pathways and gene ontology terms were associated with viral infection and included "Influenza A", "defense response to virus", "regulation of viral life cycle" and "innate immune response". Highly DE genes involved in these pathways may be beneficial for the diagnosis of subclinical BRD from blood samples.

ATAC-Seq identifies regions of open chromatin in the bronchial lymph nodes of dairy calves experimentally challenged with bovine respiratory syncytial virus.

BACKGROUND: Bovine Respiratory Syncytial Virus (BRSV) is a cause of Bovine Respiratory Disease (BRD). DNA-based biomarkers contributing to BRD resistance are potentially present in non-protein-coding regulatory regions of the genome, which can be determined using ATAC-Seq. The objectives of this study were to: (i) identify regions of open chromatin in DNA extracted from bronchial lymph nodes (BLN) of healthy dairy calves experimentally challenged with BRSV and compare them with those from non-challenged healthy control calves, (ii) elucidate the chromatin regions that were differentially or uniquely open in the BRSV challenged relative to control calves, and (iii) compare the genes found in regions proximal to the differentially open regions to the genes previously found to be differentially expressed in the BLN in response to BRSV and to previously identified BRD susceptibility loci. This was achieved by challenging clinically healthy Holstein-Friesian calves (mean age 143 ± 14 days) with either BRSV inoculum (n = 12) or with sterile phosphate buffered saline (PBS) (n = 6) and preparing and sequencing ATAC-Seq libraries from fresh BLN tissues. RESULTS: Using Diffbind, 9,144 and 5,096 differentially accessible regions (P < 0.05, FDR < 0.05) were identified between BRSV challenged and control calves employing DeSeq2 and EdgeR, respectively. Additionally, 8,791 chromatin regions were found to be uniquely open in BRSV challenged calves. Seventy-six and 150 of the genes that were previously found to be differentially expressed using RNA-Seq, were located within 2 kb downstream of the differentially accessible regions, and of the regions uniquely open in BRSV challenged calves, respectively. Pathway analyses within ClusterProfiler indicated that these genes were involved in immune responses to infection and participated in the Th1 and Th2 pathways, pathogen recognition and the anti-viral response. There were 237 differentially accessible regions positioned within 40 previously identified BRD susceptibility loci. CONCLUSIONS: The identified open chromatin regions are likely to be involved in the regulatory response of gene transcription induced by infection with BRSV. Consequently, they may contain variants which impact resistance to BRD that could be used in breeding programmes to select healthier, more robust cattle.

Synthetic Sequencing Standards: A Guide to Database Choice for Rumen Microbiota Amplicon Sequencing Analysis.

Our understanding of complex microbial communities, such as those residing in the rumen, has drastically advanced through the use of high throughput sequencing (HTS) technologies. Indeed, with the use of barcoded amplicon sequencing, it is now cost effective and computationally feasible to identify individual rumen microbial genera associated with ruminant livestock nutrition, genetics, performance and greenhouse gas production. However, across all disciplines of microbial ecology, there is currently little reporting of the use of internal controls for validating HTS results. Furthermore, there is little consensus of the most appropriate reference database for analyzing rumen microbiota amplicon sequencing data. Therefore, in this study, a synthetic rumen-specific sequencing standard was used to assess the effects of database choice on results obtained from rumen microbial amplicon sequencing. Four DADA2 reference training sets (RDP, SILVA, GTDB, and RefSeq + RDP) were compared to assess their ability to correctly classify sequences included in the rumen-specific sequencing standard. In addition, two thresholds of phylogenetic bootstrapping, 50 and 80, were applied to investigate the effect of increasing stringency. Sequence classification differences were apparent amongst the databases. For example the classification of Clostridium differed between all databases, thus highlighting the need for a consistent approach to nomenclature amongst different reference databases. It is hoped the effect of database on taxonomic classification observed in this study, will encourage research groups across various microbial disciplines to develop and routinely use their own microbiome-specific reference standard to validate analysis pipelines and database choice.

Sward type alters the relative abundance of members of the rumen microbial ecosystem in dairy cows.

The performance of ruminant livestock has been shown to benefit from the enhanced nutritive value and herbage yield associated with clover incorporation in the grazing sward. However, little research to date has been conducted investigating the effects of mixed swards containing white clover on the composition of the rumen microbiome. In this study, the rumen microbial composition of late lactation dairy cows grazing perennial ryegrass only (PRG; n = 20) or perennial ryegrass and white clover (WCPRG; n = 19) swards, was characterised using 16S rRNA amplicon sequencing. PERMANOVA analysis indicated diet significantly altered the composition of the rumen microbiome (P = 0.024). Subtle shifts in the relative abundance of 14 bacterial genera were apparent between diets, including an increased relative abundance of Lachnospira (0.04 vs. 0.23%) and Pseudobutyrivibrio (1.38 vs. 0.81%) in the WCPRG and PRG groups, respectively. The composition of the archaeal community was altered between dietary groups, with a minor increase in the relative abundance of Methanosphaera in the WCPRG observed. Results from this study highlight the potential for sward type to influence the composition of the rumen microbial community.

Links between the rumen microbiota, methane emissions and feed efficiency of finishing steers offered dietary lipid and nitrate supplementation.

Ruminant methane production is a significant energy loss to the animal and major contributor to global greenhouse gas emissions. However, it also seems necessary for effective rumen function, so studies of anti-methanogenic treatments must also consider implications for feed efficiency. Between-animal variation in feed efficiency represents an alternative approach to reducing overall methane emissions intensity. Here we assess the effects of dietary additives designed to reduce methane emissions on the rumen microbiota, and explore relationships with feed efficiency within dietary treatment groups. Seventy-nine finishing steers were offered one of four diets (a forage/concentrate mixture supplemented with nitrate (NIT), lipid (MDDG) or a combination (COMB) compared to the control (CTL)). Rumen fluid samples were collected at the end of a 56 d feed efficiency measurement period. DNA was extracted, multiplexed 16s rRNA libraries sequenced (Illumina MiSeq) and taxonomic profiles were generated. The effect of dietary treatments and feed efficiency (within treatment groups) was conducted both overall (using non-metric multidimensional scaling (NMDS) and diversity indexes) and for individual taxa. Diet affected overall microbial populations but no overall difference in beta-diversity was observed. The relative abundance of Methanobacteriales (Methanobrevibacter and Methanosphaera) increased in MDDG relative to CTL, whilst VadinCA11 (Methanomassiliicoccales) was decreased. Trimethylamine precursors from rapeseed meal (only present in CTL) probably explain the differences in relative abundance of Methanomassiliicoccales. There were no differences in Shannon indexes between nominal low or high feed efficiency groups (expressed as feed conversion ratio or residual feed intake) within treatment groups. Relationships between the relative abundance of individual taxa and feed efficiency measures were observed, but were not consistent across dietary treatments.

Investigating temporal microbial dynamics in the rumen of beef calves raised on two farms during early life.

Manipulation of the rumen microorganisms during early life has emerged as a promising strategy for persistent improvement of nutrient utilisation and lowering of enteric methanogenesis. However, limited understanding of the dynamics of rumen microbial colonisation has prevented the identification of the optimum timeframe for such interventions. The present study used DNA amplicon sequencing of the 16S rRNA gene to assess bacterial and archaeal dynamics in the rumen digesta of beef calves raised on two farms from birth through to post-weaning. The colonisation patterns of both communities were influenced by age (P < 0.05) and farm of origin (P < 0.05). The bacterial community exhibited an age-wise progression during the first month of life which appeared to be partly related to diet, and settled by day 21, indicating that this may mark the boundary of a timeframe for intervention. The archaeal community appeared less sensitive to age/diet than bacteria in the first month of life but was more sensitive to farm environment. These data show that ruminal microbial composition during early life is driven by calf age, diet and local environment, and provide important fundamental information concerning the ontogeny of the rumen microbiota from birth.

Experimental challenge with bovine respiratory syncytial virus in dairy calves: bronchial lymph node transcriptome response.

Bovine Respiratory Disease (BRD) is the leading cause of mortality in calves. The objective of this study was to examine the response of the host's bronchial lymph node transcriptome to Bovine Respiratory Syncytial Virus (BRSV) in a controlled viral challenge. Holstein-Friesian calves were either inoculated with virus (103.5 TCID50/ml × 15 ml) (n = 12) or mock challenged with phosphate buffered saline (n = 6). Clinical signs were scored daily and blood was collected for haematology counts, until euthanasia at day 7 post-challenge. RNA was extracted and sequenced (75 bp paired-end) from bronchial lymph nodes. Sequence reads were aligned to the UMD3.1 bovine reference genome and differential gene expression analysis was performed using EdgeR. There was a clear separation between BRSV challenged and control calves based on gene expression changes, despite an observed mild clinical manifestation of the disease. Therefore, measuring host gene expression levels may be beneficial for the diagnosis of subclinical BRD. There were 934 differentially expressed genes (DEG) (p < 0.05, FDR <0.1, fold change >2) between the BRSV challenged and control calves. Over-represented gene ontology terms, pathways and molecular functions, among the DEG, were associated with immune responses. The top enriched pathways included interferon signaling, granzyme B signaling and pathogen pattern recognition receptors, which are responsible for the cytotoxic responses necessary to eliminate the virus.

Long-term effects of prior diets, dietary transition and pregnancy on adipose gene expression in dairy heifers.

Adipose tissue is highly involved in whole-body metabolism and is the main site for lipid synthesis, storage and mobilization in ruminants. Therefore, knowledge about adipose tissue responses to different diets is important, especially in growing heifers as the feeding regimes of replacement heifers affect their future success as dairy cows. However, at gene expression level such knowledge is limited. As part of a larger feed trial, adipose tissue biopsies from 24 Norwegian Red heifers were collected at 12 months of age (12MO) and at month seven of gestation (PREG) and analyzed by next-generation mRNA sequencing. Between these two sampling points, all heifers had gone through a successful conception and a feed change from four dietary treatments of high or low energy (HE/LE) and protein (HP/LP) content (treatments LPHE, HPHE, LPLE and HPLE) to a low-energy, low-protein pregnancy feed given to all animals. Gene expression differences between different feed treatments at 12MO are described in an earlier publication from our group. The main objectives of this study were to investigate the long-term effects of diets differing in protein and energy density level on gene expression in adipose tissue of growing replacement dairy heifers. To achieve this, we examined the post-treatment effects between the treatment groups at month seven of gestation; 6 months after the termination of experimental feeding, and the long-term gene expression changes occurring in the adipose tissue between 12MO and PREG. Post-treatment group comparisons showed evidence of long-term effects of dietary treatment on adipose gene expression. Differences between protein treatments were smaller than between energy treatments. Adipose gene expression changes from 12MO to PREG were much larger for the HE than the LE treatments and seemed to mostly be explained by the characteristics of the diet change. 97 genes displayed a unidirectional expression change for all groups from 12MO to PREG, and are considered to be treatment-independent, possibly caused by pregnancy or increased age. This study provides candidate genes and key regulators for further studies on pregnancy preservation (TGFB1, CFD) and metabolic regulation and efficiency (PI3K, RICTOR, MAP4K4,) in dairy cattle.

Concurrent and long-term associations between the endometrial microbiota and endometrial transcriptome in postpartum dairy cows.

BACKGROUND: Fertility in dairy cows depends on ovarian cyclicity and on uterine involution. Ovarian cyclicity and uterine involution are delayed when there is uterine dysbiosis (overgrowth of pathogenic bacteria). Fertility in dairy cows may involve a mechanism through which the uterine microbiota affects ovarian cyclicity as well as the transcriptome of the endometrium within the involuting uterus. The hypothesis was that the transcriptome of the endometrium in postpartum cows would be associated with the cyclicity status of the cow as well as the microbiota during uterine involution. The endometrium of first lactation dairy cows was sampled at 1, 5, and 9 weeks postpartum. All cows were allowed to return to cyclicity without intervention until week 5 and treated with an ovulation synchronization protocol so that sampling at week 9 was on day 13 of the estrous cycle. The endometrial microbiota was measured by 16S rRNA gene sequencing and principal component analysis. The endometrial transcriptome was measured by mRNA sequencing, differential gene expression analysis, and Ingenuity Pathway Analysis. RESULTS: The endometrial microbiota changed from week 1 to week 5 but the week 5 and week 9 microbiota were similar. The endometrial transcriptome differed for cows that were either cycling or not cycling at week 5 and cyclicity status depended in part on the endometrial microbiota. Compared with cows cycling at week 5, there were large changes in the transcriptome of cows that progressed from non-cycling at week 5 to cycling at week 9. There was evidence for concurrent and longer-term associations between the endometrial microbiota and transcriptome. The week 1 endometrial microbiota had the greatest effect on the subsequent endometrial transcriptome and this effect was greatest at week 5 and diminished by week 9. CONCLUSIONS: The cumulative response of the endometrial transcriptome to the microbiota represented the combination of past microbial exposure and current microbial exposure. The endometrial transcriptome in postpartum cows, therefore, depended on the immediate and longer-term effects of the uterine microbiota that acted directly on the uterus. There may also be an indirect mechanism through which the microbiome affects the transcriptome through the restoration of ovarian cyclicity postpartum.

Complete Genome Sequence of Mannheimia varigena Isolated from Bovine Milk.

Mannheimia varigena is a pathogen of cattle that has been isolated from diseased lung and udder. There are currently complete genome sequences for 4 M. varigena isolates, all from lungs of cattle in the United States. We report a complete genome sequence of M. varigena isolated from bovine milk in Ireland.

Effect of a butyrate-fortified milk replacer on gastrointestinal microbiota and products of fermentation in artificially reared dairy calves at weaning.

Enrichment of calf diets with exogenous butyrate has shown promise as a promotor of calf growth and intestinal development. However, the impact of dietary derived butyrate on the gut microbiota and their potential role, in turn, as mediators of its effect on calf growth and development is not known. Here, the effects of butyrate supplementation on rumen and hindgut microbiota and fermentation profiles were assessed in 16 Holstein-Friesian bull calves randomly assigned to one of two groups: Control (CON) fed conventional milk replacer or Sodium-Butyrate (SB - added to milk replacer) from days 7 to 56 of life. In the colon, total short chain fatty acid (SCFA), propionate and acetate concentrations were increased by SB (P < 0.05). 16S rRNA gene amplicon sequencing showed cecal abundance of butyrate producers Butyrivibrio and Shuttleworthia were decreased by SB (P < 0.05), while that of the propionate producer Phascolarctobacterium was higher (P < 0.05). Mogibacterium is associated with impaired gut health and was reduced in the cecum of SB calves (P < 0.05). These data show that the beneficial effects of SB on growth and performance occur in tandem with changes in the abundance of important SCFA producing and health-associated bacteria in the hindgut in milk-fed calves.

Evaluation of Microbial Communities Associated With the Liquid and Solid Phases of the Rumen of Cattle Offered a Diet of Perennial Ryegrass or White Clover.

Rumen microbiota plays an important role in animal productivity, methane production and health. Several different locations have been used to obtain rumen samples (i.e., liquid-phase samples, solid-phase samples, buccal swabs) in previous studies. Here we assess differences in the rumen microbiota between solid- and liquid-phases of the rumen under differing dietary conditions (white clover vs. perennial ryegrass); there were 4 sample types: liquid-associated/grass (LG), solid-associated/grass (SG), liquid-associated/clover (LC), and solid-associated/clover (SC). Four Holstein-Friesian cows were strip grazed on pure stands of perennial ryegrass or white clover in a change-over design experiment with 3 periods (each lasting for 3 weeks). Solid- and liquid- phase microbes were obtained following total rumen evacuation on the penultimate day of each period. DNA was extracted and multiplexed libraries sequenced using 16S next generation sequencing (Illumina MiSeq). Demultiplexed sequences underwent quality control and taxonomic profiles were generated for each sample. Statistical analysis for the effects of diet and phase was conducted both overall [using non-metric multidimensional scaling (NMDS) and diversity indices] and for individual taxa. Separation of both diet and phase was observed NMDS, with significant effects of diet (P < 0.001) and phase (P < 0.001) being observed. Regardless of diet, Prevotella was most abundant in the liquid samples. When assessing differences between phases, the majority of statistically significant taxa (predominantly from Archaea and the order Clostridiales) were found at higher relative abundances in solid-phase samples. Diversity (Shannon Index) was lower in the liquid-phase samples, possibly because of the higher relative abundance of Prevotella. A presence vs. absence approach, followed by Chi-squared testing, was adopted. Differences between phases (LG vs. LC, LC vs. LG, SG vs. SC, and SC vs. SG) and differences between phases for the clover diet (LC vs. SC and SC vs. LC) were significant (P < 0.001); differences between phases for the grass diet were non-significant. Sampling technique has a profound impact on reported microbial communities, which must be taken into consideration, particularly as archaea may be underestimated in the liquid-phase.

RNA-seq analysis of bovine adipose tissue in heifers fed diets differing in energy and protein content.

Adipose tissue is no longer considered a mere energy reserve, but a metabolically and hormonally active organ strongly associated with the regulation of whole-body metabolism. Knowledge of adipose metabolic regulatory function is of great importance in cattle management, as it affects the efficiency and manner with which an animal converts feedstuff to milk, meat and fat. However, the molecular mechanisms regulating metabolism in bovine adipose tissue are still not fully elucidated. The emergence of next-generation sequencing technologies has facilitated the analysis of metabolic function and regulation at the global gene expression level. The aim of this study was to investigate the effect of diets differing in protein and energy density level on gene expression in adipose tissue of growing replacement dairy heifers using next-generation RNA sequencing (RNAseq). Norwegian Red heifers were fed either a high- or low-protein concentrate (HP/LP) and a high- or low-energy roughage (HE/LE) diet from 3 months of age until confirmed pregnancy to give four treatments (viz, HPHE, HPLE, LPHE, LPLE) with different growth profiles. Subcutaneous adipose tissue sampled at 12 months of age was analyzed for gene expression differences using RNAseq. The largest difference in gene expression was found between LPHE and LPLE heifers, for which 1092 genes were significantly differentially expressed, representing an up-regulation of mitochondrial function, lipid, carbohydrate and amino acid metabolism as well as changes in the antioxidant system in adipose tissue of LPHE heifers. Differences between HPHE and HPLE heifers were much smaller, and dominated by genes representing NAD biosynthesis, as was the significantly differentially expressed genes (DEG) common to both HE-LE contrasts. Differences between HP and LP groups within each energy treatment were minimal. This study emphasizes the importance of transcriptional regulation of adipose tissue energy metabolism, and identifies candidate genes for further studies on early-stage obesity and glucose load in dairy cattle.

16S rRNA Sequencing Reveals Relationship Between Potent Cellulolytic Genera and Feed Efficiency in the Rumen of Bulls.

The rumen microbial population dictates the host's feed degradation capacity and subsequent nutrient supply. The rising global human population and intensifying demand for animal protein is creating environmental challenges. As a consequence, there is an increasing requirement for livestock with enhanced nutrient utilization capacity in order to more efficiently convert plant material to high quality edible muscle. In the current study, residual feed intake (RFI), a widely used and a highly accepted measure of feed efficiency in cattle, was calculated for a combination of three cohorts of Simmental bulls. All animals were managed similarly from birth and offered concentrate ad libitum in addition to 3 kg of grass silage daily during the finishing period. Solid and liquid rumen digesta samples collected at slaughter and were analyzed using amplicon sequencing targeting the 16S rRNA gene utilizing the Illumina MiSeq platform. Volatile fatty acid analysis was also conducted on the liquid digesta samples. Spearman's correlation coefficient was utilized to determine the association between RFI and bacterial and archaeal taxa and inter-taxonomic relationships. The data indicate a tendency toward an increase in butyrate (P = 0.06), which corresponds with an increase in plasma ß-hydroxybutyrate concentration in low RFI (LRFI) bulls in comparison to their high RFI (HRFI) contemporaries (P < 0.05). A decrease in propionate (P < 0.05) was also recorded in the rumen of LRFI in comparison to HRFI bulls. These results indicate alternate fermentation patterns in the rumen of LRFI bulls. The data also identified that OTUs within the phyla Tenericutes, Fibrobacteres, and Cyanobacteria may potentially influence RFI phenotype. In particular, a negative association between F. succinogenes and RFI was evident. The unique cellulolytic metabolism of F. succinogenes suggests it could contribute to host efficiency by providing substrate to the host ruminant and other microbial populations (e.g., Selenomonas ruminantium, Methanobrevibacter, and Methanomassiliicoccaceae) in the rumen. This study provides evidence that bacterial OTUs within common phyla could influence ruminant feed efficiency phenotype through their role in ruminal degradation of complex plant polysaccharides or increased capability to harvest nutrients from ingested feed.

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

The 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.

Plane of nutrition affects the phylogenetic diversity and relative abundance of transcriptionally active methanogens in the bovine rumen.

Methane generated during enteric fermentation in ruminant livestock species is a major contributor to global anthropogenic greenhouse gas emissions. A period of moderate feed restriction followed by ad libitum access to feed is widely applied in cattle management to exploit the animal's compensatory growth potential and reduce feed costs. In the present study, we utilised microbial RNA from rumen digesta samples to assess the phylogenetic diversity of transcriptionally active methanogens from feed-restricted and non-restricted animals. To determine the contribution of different rumen methanogens to methanogenesis during dietary restriction of cattle, we conducted high-throughput mcrA cDNA amplicon sequencing on an Illumina MiSeq and analysed both the abundance and phylogenetic origin of different mcrA cDNA sequences. When compared to their unrestricted contemporaries, in feed-restricted animals, the methanogenic activity, based on mcrA transcript abundance, of Methanobrevibacter gottschalkii clade increased while the methanogenic activity of the Methanobrevibacter ruminantium clade and members of the Methanomassiliicoccaceae family decreased. This study shows that the quantity of feed consumed can evoke large effects on the composition of methanogenically active species in the rumen of cattle. These data potentially have major implications for targeted CH4 mitigation approaches such as anti-methanogen vaccines and/or tailored dietary management.

The transcriptome of the endometrium and placenta is associated with pregnancy development but not lactation status in dairy cows.

Infertility in lactating dairy cows is explained partially by the metabolic state associated with high milk production. The hypothesis was that lactating and nonlactating cows would differ in endometrial and placental transcriptomes during early pregnancy (day 28 to 42) and this difference would explain the predisposition for lactating cows to have embryonic loss at that time. Cows were either milked or not milked after calving. Reproductive [endometrium (caruncular and intercaruncular) and placenta] and liver tissues were collected on day 28, 35, and 42 of pregnancy. The hypothesis was rejected because no effect of lactation on mRNA abundance within reproductive tissues was found. Large differences within liver demonstrated the utility of the model to test an effect of lactation on tissue gene expression. Major changes in gene expression in reproductive tissues across time were found. Greater activation of the transcriptome for the recruitment and activation of macrophages was found in the endometrium and placenta. Changes in glucose metabolism between day 28 and 42 included greater mRNA abundance of rate-limiting genes for gluconeogenesis in intercaruncular endometrium and evidence for the establishment of aerobic glycolysis (Warburg effect) in the placenta. Temporal changes were predicted to be controlled by CSF1, PDGFB, TGFB1, and JUN. Production of nitric oxide and reactive oxygen species by macrophages was identified as a mechanism to promote angiogenesis in the endometrium. Reported differences in pregnancy development for lactating vs. nonlactating cows could be explained by systemic glucose availability to the conceptus and appeared to be independent of the endometrial and placental transcriptomes.