Effect of breed and diet on the M. longissimus thoracis et lumborum transcriptome of steers divergent for residual feed intake.

Improving cattle feed efficiency through selection of residual feed intake (RFI) is a widely accepted approach to sustainable beef production. A greater understanding of the molecular control of RFI in various breeds offered contrasting diets is necessary for the accurate identification of feed efficient animals and will underpin accelerated genetic improvement of the trait. The aim of this study was to determine genes and biological processes contributing to RFI across varying breed type and dietary sources in skeletal muscle tissue. Residual feed intake was calculated in Charolais and Holstein-Friesian steers across multiple dietary phases (phase-1: high concentrate (growing-phase); phase-2: zero-grazed grass (growing-phase); phase-3: high concentrate (finishing-phase). Steers divergent for RFI within each breed and dietary phase were selected for muscle biopsy collection, and muscle samples subsequently subjected to RNAseq analysis. No gene was consistently differentially expressed across the breed and diet types examined. However, pathway analysis revealed commonality across breeds and diets for biological processes including fatty acid metabolism, immune function, energy production and muscle growth. Overall, the lack of commonality of individual genes towards variation in RFI both within the current study and compared to the published literature, suggests other genomic features warrant further evaluation in relation to RFI.

Metabolic influence of core ciliates within the rumen microbiome.

Protozoa comprise a major fraction of the microbial biomass in the rumen microbiome, of which the entodiniomorphs (order: Entodiniomorphida) and holotrichs (order: Vestibuliferida) are consistently observed to be dominant across a diverse genetic and geographical range of ruminant hosts. Despite the apparent core role that protozoal species exert, their major biological and metabolic contributions to rumen function remain largely undescribed in vivo. Here, we have leveraged (meta)genome-centric metaproteomes from rumen fluid samples originating from both cattle and goats fed diets with varying inclusion levels of lipids and starch, to detail the specific metabolic niches that protozoa occupy in the context of their microbial co-habitants. Initial proteome estimations via total protein counts and label-free quantification highlight that entodiniomorph species Entodinium and Epidinium as well as the holotrichs Dasytricha and Isotricha comprise an extensive fraction of the total rumen metaproteome. Proteomic detection of protozoal metabolism such as hydrogenases (Dasytricha, Isotricha, Epidinium, Enoploplastron), carbohydrate-active enzymes (Epidinium, Diplodinium, Enoploplastron, Polyplastron), microbial predation (Entodinium) and volatile fatty acid production (Entodinium and Epidinium) was observed at increased levels in high methane-emitting animals. Despite certain protozoal species having well-established reputations for digesting starch, they were unexpectedly less detectable in low methane emitting-animals fed high starch diets, which were instead dominated by propionate/succinate-producing bacterial populations suspected of being resistant to predation irrespective of host. Finally, we reaffirmed our abovementioned observations in geographically independent datasets, thus illuminating the substantial metabolic influence that under-explored eukaryotic populations have in the rumen, with greater implications for both digestion and methane metabolism.

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.

Uncertainties in direct N2O emissions from grazing ruminant excreta (EF3PRP) in national greenhouse gas inventories.

Excreta deposition onto pasture, range and paddocks (PRP) by grazing ruminant constitute a source of nitrous oxide (N2O), a potent greenhouse gas (GHG). These emissions must be reported in national GHG inventories, and their estimation is based on the application of an emission factor, EF3PRP (proportion of nitrogen (N) deposited to the soil through ruminant excreta, which is emitted as N2O). Depending on local data available, countries use various EF3PRPs and approaches to estimate N2O emissions from grazing ruminant excreta. Based on ten case study countries, this review aims to highlight the uncertainties around the methods used to account for these emissions in their national GHG inventories, and to discuss the efforts undertaken for considering factors of variation in the calculation of emissions. Without any local experimental data, 2006 the IPCC default (Tier 1) EF3PRPs are still widely applied although the default values were revised in 2019. Some countries have developed country-specific (Tier 2) EF3PRP based on local field studies. The accuracy of estimation can be improved through the disaggregation of EF3PRP or the application of models; two approaches including factors of variation. While a disaggregation of EF3PRP by excreta type is already well adopted, a disaggregation by other factors such as season of excreta deposition is more difficult to implement. Empirical models are a potential method of considering factors of variation in the establishment of EF3PRP. Disaggregation and modelling requires availability of sufficient experimental and activity data, hence why only few countries have currently adopted such approaches. Replication of field studies under various conditions, combined with meta-analysis of experimental data, can help in the exploration of influencing factors, as long as appropriate metadata is recorded. Overall, despite standard IPCC methodologies for calculating GHG emissions, large uncertainties and differences between individual countries' accounting remain to be addressed.

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.

Effects of dietary n-3-PUFA supplementation, post-insemination plane of nutrition and pregnancy status on the endometrial transcriptome of beef heifers.

Supplementation of cattle diets with n-3-polyunsaturated fatty acids (PUFA) can improve reproductive efficiency. Conversely, short-term fluctuations in feed supply can impact pregnancy establishment. The objectives of this study were to examine the effects of (1) dietary supplementation with n-3-PUFA and (2) post-insemination plane of nutrition on the endometrial transcriptome. Beef crossbred heifers were offered concentrate based diets fortified with n-3-PUFA (PUFA; n = 32) or not (CONT; n = 28) for 30 days prior to breeding at a synchronised oestrous. Following artificial insemination, heifers were allocated within treatment to either a high or low plane of nutrition. Heifers were maintained on these diets for 16 days following which endometrial tissue was harvested at slaughter for subsequent RNAseq analysis. The influence of pregnancy status on the endomentrial transcriptome, within each dietary treatment group, was also examined. Post-insemination diet affected (P < 0.05) the endometrial transcriptome. Specifically, within n-3-PUFA-supplemented heifers, genes involved in embryonic development and mTOR signalling pathways, important in pregnancy establishment, were identified as differentially expressed. Results indicate that dietary supplementation of cattle diets with n-3-PUFA may have a positive effect on the expression of key fertility-related genes and pathways, during the critical window of maternal recognition of pregnancy, particularly where animals are underfed.

The Structural and Functional Capacity of Ruminal and Cecal Microbiota in Growing Cattle Was Unaffected by Dietary Supplementation of Linseed Oil and Nitrate.

Microorganisms in the digestive tract of ruminants differ in their functionality and ability to use feed constituents. While cecal microbiota play an important role in post-rumen fermentation of residual substrates undigested in the rumen, limited knowledge exists regarding its structure and function. In this trial we investigated the effect of dietary supplementation with linseed oil and nitrate on methane emissions and on the structure of ruminal and cecal microbiota of growing bulls. Animals were allocated to either a CTL (control) or LINNIT (CTL supplemented with 1.9% linseed and 1.0% nitrates) diet. Methane emissions were measured using the GreenFeed system. Microbial diversity was assessed using amplicon sequencing of microbial genomic DNA. Additionally, total RNA was extracted from ruminal contents and functional mcrA and mtt genes were targeted in amplicon sequencing approach to explore the diversity of functional gene expression in methanogens. LINNIT had no effect on methane yield (g/kg DMI) even though it decreased methane production by 9% (g/day; P < 0.05). Methanobrevibacter- and Methanomassiliicoccaceae-related OTUs were more abundant in cecum (72 and 24%) compared to rumen (60 and 11%) irrespective of the diet (P < 0.05). Feeding LINNIT reduced the relative abundance of Methanomassiliicoccaceae mcrA cDNA reads in the rumen. Principal component analysis revealed significant differences in taxonomic composition and abundance of bacterial communities between rumen and cecum. Treatment decreased the relative abundance of a few Ruminococcaceae genera, without affecting global bacterial community structure. Our research confirms a high level of heterogeneity in species composition of microbial consortia in the main gastrointestinal compartments where feed is fermented in ruminants. There was a parallel between the lack of effect of LINNIT on ruminal and cecal microbial community structure and functions on one side and methane emission changes on the other. These results suggest that the sequencing strategy used here to study microbial diversity and function accurately reflected the absence of effect on methane phenotypes in bulls treated with linseed plus nitrate.

Endometrial gene expression in high- and low-fertility heifers in the late luteal phase of the estrous cycle and a comparison with midluteal gene expression.

Embryonic mortality is a major constraint to improving reproductive efficiency and profitability in livestock enterprises. We previously reported differential expression of genes with identified roles in cellular growth and proliferation, lipid metabolism, endometrial remodeling, inflammation, angiogenesis, and metabolic exchange in endometrial tissue on day 7 of the estrous cycle (D7), between heifers ranked as either high (HF) or low (LF) for fertility. The aim of the current study was to further elucidate the underlying molecular mechanisms contributing to early embryo loss by examining differential endometrial gene expression in HF or LF heifers at a later stage of the estrous cycle;day 14(D14). A second objective was to compare these expression profiles with those from midluteal HF and LF endometrium. Using the same animal model as employed in the previous study, we slaughtered HF and LF animals on D14, harvested endometrial tissue, and carried out global gene expression analysis using the Affymetrix Bovine GeneChip. Microarray analysis detected 430 differentially expressed genes (DEG) between HF and LF animals. Ingenuity Pathway Analysis revealed enrichment for a host of biological pathways including lipid metabolism, molecular transport, immune response, cell morphology and development, and cell growth and proliferation. Important DEG includedALB, BMPR2, CCL28, COL4A3/4, FADS1, ITGA6, LDLR, PLCB3, PPARG, PTGS2, and SLC27A4 Furthermore, DEG expressed on both D7 and D14 included:PCCB,SLC25A24,DAP, and COL4A4 This study highlights some of the pathways and mechanisms underpinning late luteal bovine endometrial physiology and endometrial-related conception rate variance.

Global gene expression in endometrium of high and low fertility heifers during the mid-luteal phase of the estrous cycle.

BACKGROUND: In both beef and dairy cattle, the majority of early embryo loss occurs within the first 14 days following insemination. During this time-period, embryos are completely dependent on their maternal uterine environment for development, growth and ultimately survival, therefore an optimum uterine environment is critical to their survival. The objective of this study was to investigate whether differences in endometrial gene expression during the mid-luteal phase of the estrous cycle exist between crossbred beef heifers ranked as either high (HF) or low fertility (LF) (following four rounds of artificial insemination (AI)) using the Affymetrix® 23 K Bovine Gene Chip. RESULTS: Conception rates for each of the four rounds of AI were within a normal range: 70-73.3%. Microarray analysis of endometrial tissue collected on day 7 of the estrous cycle detected 419 differentially expressed genes (DEG) between HF (n = 6) and LF (n = 6) animals. The main gene pathways affected were, cellular growth and proliferation, angiogenesis, lipid metabolism, cellular and tissue morphology and development, inflammation and metabolic exchange. DEG included, FST, SLC45A2, MMP19, FADS1 and GALNT6. CONCLUSIONS: This study highlights, some of the molecular mechanisms potentially controlling uterine endometrial function during the mid-luteal phase of the estrous cycle, which may contribute to uterine endometrial mediated impaired fertility in cattle. Differentially expressed genes are potential candidate genes for the identification of genetic variation influencing cow fertility, which may be incorporated into future breeding programmes.

Effect of dietary n-3 polyunsaturated fatty acids on transcription factor regulation in the bovine endometrium.

Dietary n-3 polyunsaturated fatty acid (n-3 PUFA) supplementation is postulated to have positive effects on fertility. The impact of dietary n-3 PUFA supplementation on physiological and biochemical processes involved in reproduction is likely to be associated with significant alterations in gene expression in key reproductive tissues which is in turn regulated by transcription factors. Beef heifers were supplemented with a rumen protected source of either a saturated fatty acid or high n-3 PUFA diet per animal per day for 45 days and uterine endometrial tissue was harvested post slaughter. A microarray analysis was conducted and bioinformatic tools were employed to evaluate the effect of n-3 PUFA supplementation on gene expression in the bovine endometrium. Clustering of microarray gene expression data was performed to identify co-expressed genes. Functional annotation of each cluster of genes was carried out using Ingenuity Pathway Analysis. Furthermore, oPOSSUM was employed to identify transcription factors involved in gene expression changes due to supplementary PUFA. Gene functions which showed a significant response to n-3 PUFA supplementation included tissue development, immune function and reproductive function. Numerous transcription factors such as FOXD1, FOXD3, NFKB1, ESR1, PGR, FOXA2, NKX3-1 and PPARα were identified as potential regulators of gene expression in the endometrium of cattle supplemented with n-3 PUFA. This study demonstrates the complex nature of the alterations in the transcriptional regulation process in the uterine endometrium of cattle following dietary supplementation which may positively influence the uterine environment.

Dietary n-3 polyunsaturated fatty acid supplementation alters the expression of genes involved in the control of fertility in the bovine uterine endometrium.

The potential for dietary supplementation with n-3 polyunsaturated fatty acids (n-3 PUFA) to improve reproductive efficiency in cattle has received much interest. The mechanisms by which n-3 PUFA may affect physiological and biochemical processes in key reproductive tissues are likely to be mediated by significant alterations in gene expression. The objective of this study was to examine the effects of dietary n-3 PUFA supplementation on global uterine endometrial gene expression in cattle. Beef heifers were supplemented with a rumen protected source of either a saturated fatty acid (CON; palmitic acid) or high n-3 PUFA (n-3 PUFA; 275 g) diet per animal per day for 45 days and global gene expression was determined in uterine endometrial tissue using an Affymetrix oligonucleotide bovine array. A total of 1,807 (946 up- and 861 downregulated) genes were differentially expressed following n-3 PUFA supplementation. Dietary n-3 PUFA altered numerous cellular processes potentially important in the control of reproduction in cattle. These included prostaglandin biosynthesis, steroidogenesis and transcriptional regulation, while effects on genes involved in maternal immune response and tissue remodeling were also observed. This study provides new insights into the effects of n-3 PUFA supplementation on the regulation of gene expression in the bovine uterus.

Analysis of multiple polymorphisms in the bovine neuropeptide Y5 receptor gene and structural modelling of the encoded protein.

The neuropeptide Y 5 receptor (NPY5R) plays an important role in the regulation of appetite and feeding behaviour in mammals by modulating the effect of the neurotransmitter neuropeptide Y. As single nucleotide polymorphism (SNP) variation in the bovine NPY5R gene is likely to influence the expression and/or function of this gene, the objectives of this study were to identify SNPs in the bovine NPY5R gene and to predict their functional role in the expression and physico-chemical characteristics of the protein product. Nineteen novel SNPs were identified in a 2.1 kb genomic region of the NPY5R gene in a total of 419 beef cattle from 13 Bos taurus breeds and eight Bos indicus animals. Four of these SNPs were non-synonymous (Met â†’ Ile, Leu â†’ Phe, Pro â†’ Leu, Arg â†’ Stop codon), while 10 were synonymous. Of particular interest was one non-synonymous SNP (c.1090C>T) that introduced a stop codon in the third intracellular loop of the NPY5R molecule. This stop codon is predicted to create a truncated NPY5R molecule with different physico-chemical properties compared to the native NPY5R protein. A further four SNPs were located in the 5' untranslated region (UTR) and one in the 3'UTR. Two of the 5'UTR SNPs affected putative transcription factor binding sites (GATA binding factor and snRNA-activating protein complex). In conclusion, regulatory and functional SNPs were identified in the bovine NPY5R gene. These include SNPs which potentially modify transcription factor binding sites as well as SNPs that cause amino acid changes and premature termination of the NPY5R protein. Such polymorphisms are likely to play vital physiological roles in the neuropeptide Y mediated appetite, feed intake and energy homeostasis in cattle.

Examination of the bovine leukocyte environment using immunogenetic biomarkers to assess immunocompetence following exposure to weaning stress.

BACKGROUND: The molecular mechanisms by which stress induces the development of pathologies remains unclear, although it is recognised that one of the major factors affecting health as a consequence of stress is the involvement of the neuroendocrine system. In cattle, a number of necessary husbandry practices have been shown to activate the stress response, yet very little is known about the impact these have at the molecular level. The objectives of the study were to characterise, in male and female beef calves, the immune response to weaning stress in bovine leukocytes at the physiological and molecular levels and to assess the difference between calves weaned in the presence of the dam and those weaned and penned away from the dam. RESULTS: Following exposure to weaning stress, total neutrophil number and neutrophil:lymphocyte (N:L) ratio increased (P < 0.01) in calves. Additionally, expression of pro-inflammatory cytokine genes, including IL-1ß, IL-8, IFN-γ and TNFα, were up-regulated (P < 0.01). Furthermore, there was increased (P < 0.001) expression of the glucocorticoid receptor, GRα, the pro-apoptotic gene, Fas and the Gram-negative pattern recognition receptor, TLR4. Calves penned away from the dam post-weaning had increased (P < 0.01) neutrophil number and N:L ratio compared with calves penned next to the dam, and female calves had higher (P < 0.05) expression levels of IL-2, IL-8, IFN-γ and TNFα than male calves. CONCLUSIONS: Weaning elicits an immediate and somewhat short-lived acute stress response in the calf. The effects serve to enhance, rather than suppress, the immune response by means of a heightened inflammatory response and cellular mobilization. The earlier and more profound increase in neutrophil number and N:L ratio together with reduced lymphocyte number in calves penned away compared with calves penned near their dams post-weaning suggests that the former may be more sensitive to weaning stress. The data also show a clear effect of gender in differential gene expression in response to stress with IFN-γ having increased expression in female calves compared with male calves over the course of the study. Additionally, this study has helped to characterise the inflammatory response to stress in calves and identify a number of novel candidate biomarkers suitable for investigation in future studies of stress.

Effect of dietary n-3 polyunsaturated fatty acid supplementation on bovine uterine endometrial and hepatic gene expression of the insulin-like growth factor system.

Supplementation of cattle diets with n-3 polyunsaturated fatty acids (n-3 PUFA) has been suggested to have positive effects on fertility. In addition, the actions of the insulin-like growth factor (IGF) system both systemically and locally have been shown to influence reproductive processes. The objective of this study was to evaluate the effect of dietary n-3 PUFA supplementation on hepatic and endometrial expression of IGF signalling genes in cattle. Beef heifers were supplemented with a rumen protected source of either a saturated fatty acid (palmitic acid; CON) or high n-3 PUFA diet (n-3 PUFA) for 45 days before slaughter and tissue recovery. Transcription level of candidate IGF signalling genes was measured by reverse transcription quantitative real-time PCR (RT-qPCR) in total RNA isolated from uterine endometrial and liver tissue from seven CON and seven n-3 PUFA supplemented animals. Compared to controls, mRNA abundance in n-3 PUFA liver tissues was higher for IGF-2R, IGFBP-1 and IGFBP-5 (P < 0.05); lower for GHR-1A (P < 0.05); and unchanged for IGF-1, IGF-2, IGF-1R, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, ALS and GHR(total) (P > 0.05). Compared to controls, mRNA abundance in n-3 PUFA endometrial tissues was higher for IGF-2, IGF-1R, IGF-2R and IGFBP-2 (P < 0.05); lower for IGF-1, IGFBP-3 and IGFBP-6 (P < 0.05); and unchanged for IGFBP-1, IGFBP-4, IGFBP-5 and GHR(total) (P > 0.05). Thus, dietary supplementation of cattle with n-3 PUFA affects transcription of genes involved in IGF signalling, in a tissue dependent fashion.

Development of a molecular methodology to quantify Staphylococcus epidermidis in surgical wash-out samples from prosthetic joint replacement surgery.

Prosthetic joint infection is a serious complication of total joint arthroplasty that causes great morbidity in affected individuals. The most common cause of prosthesis associated infections are members of Staphylococcus spp., including Staphylococcus epidermidis. Culture has served as the gold standard for diagnosis, despite obvious shortcomings in terms of sensitivity and time. Bacterial genomic DNA extraction methodologies were evaluated for optimal recovery of genomic DNA from sterilised wash-out samples, spiked with S. epidermidis. Real time Polymerase chain reaction (PCR) assays targeting the S. epidermidis specific gseA gene were designed to reliably detect and quantify S. epidermidis. Sixty post-operative wash-out samples from primary hip and knee arthroplasties were taken aseptically. All were shown to be culture negative using the culture-dependent approach. These were samples were subjected to S. epidermidis-specific real time PCR. Standard curve showed good linearity. Sensitivity limit of the assay was <10 CFU S. epidermidis per sample. Reproducibility of the assay was confirmed. S. epidermidis was not identified in any of these samples using the novel species specific SYBR Green real time PCR technique. Results indicated that wash-out samples were true negatives and did not harbour S. epidermidis. To support this, patients displayed no symptoms of infection. To illustrate the full effectiveness of the novel real time PCR assay, a larger number of samples need to be tested (>1,000 patients).