• Electronic feeding behavioural data as indicators of health status in dairy calves

      Johnston, Dayle; Kenny, David A.; McGee, Mark; Waters, Sinead M.; Kelly, Alan K; Earley, Bernadette (Teagasc (Agriculture and Food Development Authority), Ireland, 2016-12-30)
      The objectives of this study were (i) to characterise clinical health in dairy calves on an Irish research farm during the artificial calf-rearing period and (ii) to determine whether calves’ pre-weaning intakes and feeding behaviour, recorded by electronic calf feeders, changes in response to incidents of bovine respiratory disease (BRD). Holstein-Friesian (H-F) and Jersey (J) calves were fed by automatic milk replacer (MR) and concentrate feeders. Feeding behaviour, including MR consumption, drinking speed, number of rewarded and unrewarded visits to the feeder as well as concentrate consumption, was recorded by the feeders. A modified version of the Wisconsin calf health scoring criteria chart was used to score calves’ clinical measurements and identify incidences of BRD. Thus, 40% of calves were found to have at least one incident of BRD. Feeding behaviour was altered during incidents of BRD. The number of unrewarded visits to the feeder was reduced, by approximately four visits, for calves with BRD during the 3 d prior to the identification of BRD (P < 0.05) and tended to be reduced during the 7 d following the identification of BRD (P = 0.05), compared with healthy calves. Additionally, calves with BRD had a tendency for reduced net energy intake (approximately 8%) during the 3 d prior to the identification of BRD, compared with healthy calves. Therefore, calf feeding behavioural data, recorded by electronic feeders during the pre-weaning period, can indicate cases of BRD.
    • Evaluating Established Methods for Rumen 16S rRNA Amplicon Sequencing With Mock Microbial Populations

      McGovern, Emily; Blackshields, Gordon; McCabe, Matthew; Waters, Sinead M.; FACCE-JPI grant; Walsh Fellowship award; 2014231 (Frontiers, 2018-06-25)
      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.
    • Evaluating Established Methods for Rumen 16S rRNA Amplicon Sequencing With Mock Microbial Populations

      McGovern, Emily; Waters, Sinead M.; Blackshields, Gordon; McCabe, Matthew; FACCE-JPI; Teagasc Walsh Fellowship Programme; 2014231 (Frontiers, 2018-06-25)
      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.
    • Examination of the bovine leukocyte environment using immunogenetic biomarkers to assess immunocompetence following exposure to weaning stress

      O'Loughlin, Aran; McGee, Mark; Waters, Sinead M.; Doyle, Sean; Earley, Bernadette; Teagasc Walsh Fellowship Programme (Biomed Central, 2011-08-11)
      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-1β, 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.
    • Examination of the molecular control of ruminal epithelial function in response to dietary restriction and subsequent compensatory growth in cattle

      O'Shea, Emma; Waters, Sinead M.; Keogh, Kate; Kelly, Alan K; Kenny, David A.; Science Foundation Ireland; 09/RFP/GEN2447 (2016-09-15)
      Background The objective of this study was to investigate the effect of dietary restriction and subsequent compensatory growth on the relative expression of genes involved in volatile fatty acid transport, metabolism and cell proliferation in ruminal epithelial tissue of beef cattle. Sixty Holstein Friesian bulls (mean liveweight 370 ± 35 kg; mean age 479 ± 15 d) were assigned to one of two groups: (i) restricted feed allowance (RES; n = 30) for 125 d (Period 1) followed by ad libitum access to feed for 55 d (Period 2) or (ii) ad libitum access to feed throughout (ADLIB; n = 30). Target growth rate for RES was 0.6 kg/d during Period 1. At the end of each dietary period, 15 animals from each treatment group were slaughtered and ruminal epithelial tissue and liquid digesta harvested from the ventral sac of the rumen. Real-time qPCR was used to quantify mRNA transcripts of 26 genes associated with ruminal epithelial function. Volatile fatty acid analysis of rumen fluid from individual animals was conducted using gas chromatography. Results Diet × period interactions were evident for genes involved in ketogenesis (BDH2, P = 0.017), pyruvate metabolism (LDHa, P = 0.048; PDHA1, P = 0.015) and cellular transport and structure (DSG1, P = 0.019; CACT, P = 0.027). Ruminal concentrations of propionic acid (P = 0.018) and n-valeric acid (P = 0.029) were lower in RES animals, compared with ADLIB, throughout the experiment. There was also a strong tendency (P = 0.064) toward a diet × period interaction for n-butyric with higher concentrations in RES animals, compared with ADLIB, during Period 1. Conclusions These data suggest that following nutrient restriction, the structural integrity of the rumen wall is compromised and there is upregulation of genes involved in the production of ketone bodies and breakdown of pyruvate for cellular energy. These results provide an insight into the potential molecular mechanisms regulating ruminal epithelial absorptive metabolism and growth following nutrient restriction and subsequent compensatory growth.
    • An examination of the molecular mechanisms controlling the tissue accumulation of conjugated linoleic acid (CLA) in cattle

      Waters, Sinead M.; Hynes, A.C.; Killeen, Aideen P.; Moloney, Aidan P; Kenny, David A. (Teagasc, 01/12/2008)
      Long chain n-3 polyunsaturated fatty acids (n-3 PUFA) and conjugated linoleic acid (CLA) have demonstrable and potential human health benefits in terms of preventing cancer, diabetes, chronic inflammation, obesity and coronary heart disease. Supplementation of cattle diets with a blend of oils rich in n-3 PUFA and linoleic acid have a synergistic effect on the accumulation of ruminal and tissue concentrations of trans vaccenic acid (TVA), the main substrate for ?-9 desaturase which is responsible for de novo tissue synthesis of the cis 9, trans 11 isomer of CLA. This dietary strategy translates into increases in milk concentrations of CLA in dairy cows; however, concentrations in the muscle of beef animals have not always been increased. There is an apparent paradox in that n-3 PUFA supplementation enhances ruminal synthesis of trans-vaccenic acid (TVA), but then inhibits its conversion to CLA possibly through altering the activity of ?-9 desaturase. Recently, the promoter regions of the bovine ?- 9 desaturase gene has been isolated and analysed and has been shown to contain a conserved PUFA response region.
    • Experimental challenge with bovine respiratory syncytial virus in dairy calves: bronchial lymph node transcriptome response

      Johnston, Dayle; Earley, Bernadette; McCabe, Matthew S.; Lemon, Ken; Duffy, Catherine; McMenamy, Michael; Cosby, S. Louise; Kim, JaeWoo; Blackshields, Gordon; Taylor, Jeremy F.; et al. (Springer Science and Business Media LLC, 2019-10-14)
      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 (103.5TCID50/ml×15ml) (n=12) or mock challenged with phosphate bufered 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 (75bp paired-end) from bronchial lymph nodes. Sequence reads were aligned to the UMD3.1 bovine reference genome and diferential 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 benefcial for the diagnosis of subclinical BRD. There were 934 diferentially 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.
    • Gastrointestinal tract size, total-tract digestibility, and rumen microflora in different dairy cow genotypes

      Beecher, Marion; Buckley, Frank; Waters, Sinead M.; Boland, T. M.; Enriquez-Hidalgo, D.; Deighton, M. H.; O'Donovan, Michael; Lewis, Eva (Elsevier Inc and American Dairy Science Association, 2014-04-03)
      The superior milk production efficiency of Jersey (JE) and Jersey × Holstein-Friesian (JE × HF) cows compared with Holstein-Friesian (HF) has been widely published. The biological differences among dairy cow genotypes, which could contribute to the milk production efficiency differences, have not been as widely studied however. A series of component studies were conducted using cows sourced from a longer-term genotype comparison study (JE, JE × HF, and HF). The objectives were to (1) determine if differences exist among genotypes regarding gastrointestinal tract (GIT) weight, (2) assess and quantify whether the genotypes tested differ in their ability to digest perennial ryegrass, and (3) examine the relative abundance of specific rumen microbial populations potentially relating to feed digestibility. Over 3 yr, the GIT weight was obtained from 33 HF, 35 JE, and 27 JE × HF nonlactating cows postslaughter. During the dry period the cows were offered a perennial ryegrass silage diet at maintenance level. The unadjusted GIT weight was heavier for the HF than for JE and JE × HF. When expressed as a proportion of body weight (BW), JE and JE × HF had a heavier GIT weight than HF. In vivo digestibility was evaluated on 16 each of JE, JE × HF, and HF lactating dairy cows. Cows were individually stalled, allowing for the total collection of feces and were offered freshly cut grass twice daily. During this time, daily milk yield, BW, and dry matter intake (DMI) were greater for HF and JE × HF than for JE; milk fat and protein concentration ranked oppositely. Daily milk solids yield did not differ among the 3 genotypes. Intake capacity, expressed as DMI per BW, tended to be different among treatments, with JE having the greatest DMI per BW, HF the lowest, and JE × HF being intermediate. Production efficiency, expressed as milk solids per DMI, was higher for JE than HF and JE × HF. Digestive efficiency, expressed as digestibility of dry matter, organic matter, N, neutral detergent fiber, and acid detergent fiber, was higher for JE than HF. In grazing cows (n = 15 per genotype) samples of rumen fluid, collected using a transesophageal sampling device, were analyzed to determine the relative abundance of rumen microbial populations of cellulolytic bacteria, protozoa, and fungi. These are critically important for fermentation of feed into short-chain fatty acids. A decrease was observed in the relative abundance of Ruminococcus flavefaciens in the JE rumen compared with HF and JE × HF. We can deduce from this study that the JE genotype has greater digestibility and a different rumen microbial population than HF. Jersey and JE × HF cows had a proportionally greater GIT weight than HF. These differences are likely to contribute to the production efficiency differences among genotypes previously reported.
    • Genetic merit for fertility traits in Holstein cows: III. Hepatic expression of somatotropic axis genes during pregnancy and lactation

      Cummins, Sean B; Waters, Sinead M.; Evans, A.C.O.; Lonergan, P.; Butler, Stephen T.; National Development Plan Ireland; Dairy Levy Research Trust Ireland (American Dairy Science Association and Elsevier Inc., 2012-07)
      The objective of this study was to characterize the circulating concentrations of insulin-like growth factor-I (IGF-I) and the hepatic expression of key genes regulating the somatotropic axis in cows divergent in genetic merit for fertility traits but with similar genetic merit for milk production traits. A total of 11 cows with good genetic merit for fertility (Fert+) and 12 cows with poor genetic merit for fertility (Fert−) underwent liver biopsy by percutaneous punch technique on d 20 (± 6.7 d) prepartum and on d 2 (± 1.5 d), d 58 (± 3.7 d), d 145 (± 13 d), and d 245 (± 17.1 d) postpartum. Total RNA was isolated and the mRNA expression of growth hormone receptor (GHR 1A and GHRtot), IGF-I, janus tyrosine kinase 2 (JAK2), signal transducer and activator of transcription 5B (STAT5B), suppressor of cytokine signaling 3 (SOCS-3), acid-labile subunit (ALS), and IGF-binding proteins (IGFBP1 to IGFBP6) were measured by real-time quantitative PCR. During lactation, the circulating concentrations of IGF-I were 34% greater in Fert+ cows. The Fert+ cows had increased mean expression of IGF-I mRNA during the study; however, the difference in IGF-I mRNA abundance between Fert+ and Fert− cows was most pronounced at d 145 and 245. The expression of IGFBP3 and ALS transcript was similar in Fert+ and Fert− cows for the duration of the study. The Fert− cows, however, had greater expression of IGFBP2, IGFBP4, IGFBP5, and IGFBP6. Genotype had no effect on mRNA abundance of GHR 1A, STAT5B, JAK2, or SOCS-3. Genetic merit for fertility traits affects hepatic expression of key genes of the somatotropic axis regulating the synthesis, bioavailability, and stability of circulating IGF-I.
    • Genome-Wide microRNA Binding Site Variation between Extinct Wild Aurochs and Modern Cattle Identifies Candidate microRNA-Regulated Domestication Genes

      Braud, Martin; Magee, David A.; Park, Stephen D. E.; Sonstegard, Tad S.; Waters, Sinead M.; MacHugh, David E.; Spillane, Charles; Teagasc Walsh Fellowship Programme; Science Foundation Ireland; Department of Agriculture, Food and the Marine; et al. (Frontiers, 2017-01-31)
      The domestication of cattle from the now-extinct wild aurochs (Bos primigenius) involved selection for physiological and behavioral traits, with underlying genetic factors that remain largely unknown. Non-coding microRNAs have emerged as key regulators of the spatio-temporal expression of target genes controlling mammalian growth and development, including in livestock species. During the domestication process, selection of mutational changes in miRNAs and/or miRNA binding sites could have provided a mechanism to generate some of the traits that differentiate domesticated cattle from wild aurochs. To investigate this, we analyzed the open reading frame DNA sequence of 19,994 orthologous protein-coding gene pairs from extant Bos taurus genomes and a single extinct B. primigenius genome. We identified miRNA binding site polymorphisms in the 3′ UTRs of 1,620 of these orthologous genes. These 1,620 genes with altered miRNA binding sites between the B. taurus and B. primigenius lineages represent candidate domestication genes. Using a novel Score Site ratio metric we have ranked these miRNA-regulated genes according to the extent of divergence between miRNA binding site presence, frequency and copy number between the orthologous genes from B. taurus and B. primigenius. This provides an unbiased approach to identify cattle genes that have undergone the most changes in miRNA binding (i.e., regulation) between the wild aurochs and modern-day cattle breeds. In addition, we demonstrate that these 1,620 candidate domestication genes are enriched for roles in pigmentation, fertility, neurobiology, metabolism, immunity and production traits (including milk quality and feed efficiency). Our findings suggest that directional selection of miRNA regulatory variants was important in the domestication and subsequent artificial selection that gave rise to modern taurine cattle.
    • Global gene expression in endometrium of high and low fertility heifers during the mid-luteal phase of the estrous cycle

      Killeen, Aideen P.; Morris, Dermot G.; Kenny, David A.; Mullen, Michael P.; Diskin, Michael G.; Waters, Sinead M. (Biomed Central, 2014-03-26)
      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.
    • GWAS and eQTL analysis identifies a SNP associated with both residual feed intake and GFRA2 expression in beef cattle

      Higgins, Marc G.; Fitzsimons, Clare; McClure, Matthew C.; McKenna, Clare; Conroy, S.B.; Kenny, David A.; McGee, Mark; Waters, Sinead M.; Morris, Derek W.; Department of Agriculture, Food and the Marine; et al. (Nature Publishing Group, 2018-09-24)
      Residual feed intake (RFI), a measure of feed efficiency, is an important economic and environmental trait in beef production. Selection of low RFI (feed efficient) cattle could maintain levels of production, while decreasing feed costs and methane emissions. However, RFI is a difficult and expensive trait to measure. Identification of single nucleotide polymorphisms (SNPs) associated with RFI may enable rapid, cost effective genomic selection of feed efficient cattle. Genome-wide association studies (GWAS) were conducted in multiple breeds followed by meta-analysis to identify genetic variants associated with RFI and component traits (average daily gain (ADG) and feed intake (FI)) in Irish beef cattle (n = 1492). Expression quantitative trait loci (eQTL) analysis was conducted to identify functional effects of GWAS-identified variants. Twenty-four SNPs were associated (P < 5 × 10−5) with RFI, ADG or FI. The variant rs43555985 exhibited strongest association for RFI (P = 8.28E-06). An eQTL was identified between this variant and GFRA2 (P = 0.0038) where the allele negatively correlated with RFI was associated with increased GFRA2 expression in liver. GFRA2 influences basal metabolic rates, suggesting a mechanism by which genetic variation may contribute to RFI. This study identified SNPs that may be useful both for genomic selection of RFI and for understanding the biology of feed efficiency.
    • Illumina MiSeq 16S amplicon sequence analysis of bovine respiratory disease associated bacteria in lung and mediastinal lymph node tissue

      Johnston, Dayle; Earley, Bernadette; Cormican, Paul; Murray, Gerard; Kenny, David A.; Waters, Sinead M.; McGee, Mark; Kelly, Alan K; McCabe, Matthew; Department of Agriculture, Food and the Marine; et al. (Biomed Central, 2017-05-02)
      Background Bovine respiratory disease (BRD) is caused by growth of single or multiple species of pathogenic bacteria in lung tissue following stress and/or viral infection. Next generation sequencing of 16S ribosomal RNA gene PCR amplicons (NGS 16S amplicon analysis) is a powerful culture-independent open reference method that has recently been used to increase understanding of BRD-associated bacteria in the upper respiratory tract of BRD cattle. However, it has not yet been used to examine the microbiome of the bovine lower respiratory tract. The objective of this study was to use NGS 16S amplicon analysis to identify bacteria in post-mortem lung and lymph node tissue samples harvested from fatal BRD cases and clinically healthy animals. Cranial lobe and corresponding mediastinal lymph node post-mortem tissue samples were collected from calves diagnosed as BRD cases by veterinary laboratory pathologists and from clinically healthy calves. NGS 16S amplicon libraries, targeting the V3-V4 region of the bacterial 16S rRNA gene were prepared and sequenced on an Illumina MiSeq. Quantitative insights into microbial ecology (QIIME) was used to determine operational taxonomic units (OTUs) which corresponded to the 16S rRNA gene sequences. Results Leptotrichiaceae, Mycoplasma, Pasteurellaceae, and Fusobacterium were the most abundant OTUs identified in the lungs and lymph nodes of the calves which died from BRD. Leptotrichiaceae, Fusobacterium, Mycoplasma, Trueperella and Bacteroides had greater relative abundances in post-mortem lung samples collected from fatal cases of BRD in dairy calves, compared with clinically healthy calves without lung lesions. Leptotrichiaceae, Mycoplasma and Pasteurellaceae showed higher relative abundances in post-mortem lymph node samples collected from fatal cases of BRD in dairy calves, compared with clinically healthy calves without lung lesions. Two Leptotrichiaceae sequence contigs were subsequently assembled from bacterial DNA-enriched shotgun sequences. Conclusions The microbiomes of the cranial lung lobe and mediastinal lymph node from calves which died from BRD and from clinically healthy H-F calves have been characterised. Contigs corresponding to the abundant Leptotrichiaceae OTU were sequenced and found not to be identical to any known bacterial genus. This suggests that we have identified a novel bacterial species associated with BRD.
    • Illumina MiSeq Phylogenetic Amplicon Sequencing Shows a Large Reduction of an Uncharacterised Succinivibrionaceae and an Increase of the Methanobrevibacter gottschalkii Clade in Feed Restricted Cattle

      McCabe, Matthew; Cormican, Paul; Keogh, Kate; O'Connor, Aaron; O'Hara, Eoin; Palladino, Rafael Alejandro; Kenny, David A.; Waters, Sinead M.; Science Foundation Ireland; Teagasc Walsh Fellowship Programme; et al. (PLOS, 2015-07-30)
      Periodic feed restriction is used in cattle production to reduce feed costs. When normal feed levels are resumed, cattle catch up to a normal weight by an acceleration of normal growth rate, known as compensatory growth, which is not yet fully understood. Illumina Miseq Phylogenetic marker amplicon sequencing of DNA extracted from rumen contents of 55 bulls showed that restriction of feed (70% concentrate, 30% grass silage) for 125 days, to levels that caused a 60% reduction of growth rate, resulted in a large increase of relative abundance of Methanobrevibacter gottschalkii clade (designated as OTU-M7), and a large reduction of an uncharacterised Succinivibrionaceae species (designated as OTU-S3004). There was a strong negative Spearman correlation (ρ = -0.72, P = <1x10-20) between relative abundances of OTU-3004 and OTU-M7 in the liquid rumen fraction. There was also a significant increase in acetate:propionate ratio (A:P) in feed restricted animals that showed a negative Spearman correlation (ρ = -0.69, P = <1x10-20) with the relative abundance of OTU-S3004 in the rumen liquid fraction but not the solid fraction, and a strong positive Spearman correlation with OTU-M7 in the rumen liquid (ρ = 0.74, P = <1x10-20) and solid (ρ = 0.69, P = <1x10-20) fractions. Reduced A:P ratios in the rumen are associated with increased feed efficiency and reduced production of methane which has a global warming potential (GWP 100 years) of 28. Succinivibrionaceae growth in the rumen was previously suggested to reduce methane emissions as some members of this family utilise hydrogen, which is also utilised by methanogens for methanogenesis, to generate succinate which is converted to propionate. Relative abundance of OTU-S3004 showed a positive Spearman correlation with propionate (ρ = 0.41, P = <0.01) but not acetate in the liquid rumen fraction.
    • Long-term effects of prior diets, dietary transition and pregnancy on adipose gene expression in dairy heifers

      Waerp, Hilde K.; Waters, Sinead M.; McCabe, Matthew S.; Cormican, Paul; Salte, Ragnar; The Research Council of Norway; 199448 (Public Library of Science (PLoS), 2019-07-03)
      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.
    • Long-term effects of prior diets, dietary transition and pregnancy on adipose gene expression in dairy heifers

      Wærp, Hilde K. L.; Waters, Sinead M.; McCabe, Matthew S.; Cormican, Paul; Salte, Ragnar; Research Council of Norway; TINE SA Norwegian dairies; Felleskjøpet agricultural cooperative; Animalia AS; 199448 (Public Library of Science (PLoS), 2019-07-03)
      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.
    • Plane of nutrition affects the phylogenetic diversity and relative abundance of transcriptionally active methanogens in the bovine rumen

      McGovern, Emily; McCabe, Matthew; Cormican, Paul; Popova, Milka; Keogh, Kate; Kelly, Alan K; Kenny, David A.; Waters, Sinead M. (Springer Nature, 2017-10-12)
      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.
    • Polymorphism discovery and allele frequency estimation using high-throughput DNA sequencing of target-enriched pooled DNA samples.

      Mullen, Michael P.; Creevey, Christopher J.; Berry, Donagh; McCabe, Matthew; Magee, David A; Howard, Dawn J.; Killeen, Aideen P.; Park, Stephen D. E.; McGettigan, Paul; Lucy, Matt C.; et al. (Biomed Central, 2012-01-11)
      Background: The central role of the somatotrophic axis in animal post-natal growth, development and fertility is well established. Therefore, the identification of genetic variants affecting quantitative traits within this axis is an attractive goal. However, large sample numbers are a pre-requisite for the identification of genetic variants underlying complex traits and although technologies are improving rapidly, high-throughput sequencing of large numbers of complete individual genomes remains prohibitively expensive. Therefore using a pooled DNA approach coupled with target enrichment and high-throughput sequencing, the aim of this study was to identify polymorphisms and estimate allele frequency differences across 83 candidate genes of the somatotrophic axis, in 150 Holstein-Friesian dairy bulls divided into two groups divergent for genetic merit for fertility. Results: In total, 4,135 SNPs and 893 indels were identified during the resequencing of the 83 candidate genes. Nineteen percent (n = 952) of variants were located within 5' and 3' UTRs. Seventy-two percent (n = 3,612) were intronic and 9% (n = 464) were exonic, including 65 indels and 236 SNPs resulting in non-synonymous substitutions (NSS). Significant (P < 0.01) mean allele frequency differentials between the low and high fertility groups were observed for 720 SNPs (58 NSS). Allele frequencies for 43 of the SNPs were also determined by genotyping the 150 individual animals (Sequenom® MassARRAY). No significant differences (P > 0.1) were observed between the two methods for any of the 43 SNPs across both pools (i.e., 86 tests in total). Conclusions: The results of the current study support previous findings of the use of DNA sample pooling and high-throughput sequencing as a viable strategy for polymorphism discovery and allele frequency estimation. Using this approach we have characterised the genetic variation within genes of the somatotrophic axis and related pathways, central to mammalian post-natal growth and development and subsequent lactogenesis and fertility. We have identified a large number of variants segregating at significantly different frequencies between cattle groups divergent for calving interval plausibly harbouring causative variants contributing to heritable variation. To our knowledge, this is the first report describing sequencing of targeted genomic regions in any livestock species using groups with divergent phenotypes for an economically important trait.
    • Quantitative analysis of ruminal methanogenic microbial populations in beef cattle divergent in phenotypic residual feed intake (RFI) offered contrasting diets

      Carberry, Ciara A; Kenny, David A.; Kelly, Alan K; Waters, Sinead M.; Department of Agriculture, Food and the Marine; RSF 05 224 (Biomed Central, 2014-08-22)
      Background Methane (CH4) emissions in cattle are an undesirable end product of rumen methanogenic fermentative activity as they are associated not only with negative environmental impacts but also with reduced host feed efficiency. The aim of this study was to quantify total and specific rumen microbial methanogenic populations in beef cattle divergently selected for residual feed intake (RFI) while offered (i) a low energy high forage (HF) diet followed by (ii) a high energy low forage (LF) diet. Ruminal fluid was collected from 14 high (H) and 14 low (L) RFI animals across both dietary periods. Quantitative real time PCR (qRT-PCR) analysis was conducted to quantify the abundance of total and specific rumen methanogenic microbes. Spearman correlation analysis was used to investigate the association between the relative abundance of methanogens and animal performance, rumen fermentation variables and diet digestibility. Results Abundance of methanogens, did not differ between RFI phenotypes. However, relative abundance of total and specific methanogen species was affected (P < 0.05) by diet type, with greater abundance observed while animals were offered the LF compared to the HF diet. Conclusions These findings suggest that differences in abundance of specific rumen methanogen species may not contribute to variation in CH4 emissions between efficient and inefficient animals, however dietary manipulation can influence the abundance of total and specific methanogen species.
    • Residual feed intake phenotype and gender affect the expression of key genes of the lipogenesis pathway in subcutaneous adipose tissue of beef cattle

      McKenna, Clare; Porter, Richard K.; Keogh, Kate; Waters, Sinead M.; McGee, Mark; Kenny, David A.; Teagasc Walsh Fellowship Programme; RMIS: 6092 (Springer Science and Business Media LLC, 2018-09-20)
      Background Feed accounts for up to 75% of costs in beef production systems, thus any improvement in feed efficiency (FE) will benefit the profitability of this enterprise. Residual feed intake (RFI) is a measure of FE that is independent of level of production. Adipose tissue (AT) is a major endocrine organ and the primary metabolic energy reservoir. It modulates a variety of processes related to FE such as lipid metabolism and glucose homeostasis and thus measures of inter-animal variation in adiposity are frequently included in the calculation of the RFI index. The aim of this study was to determine the effect of phenotypic RFI status and gender on the expression of key candidate genes related to processes involved in energy metabolism within AT. Dry matter intake (DMI) and average daily gain (ADG) were measured over a period of 70 d for 52 purebred Simmental heifers (n = 24) and bulls (n = 28) with an initial BW±SD of 372±39.6 kg and 387±50.6 kg, respectively. Residual feed intake was calculated and animals were ranked within gender by RFI into high (inefficient; n = 9 heifers and n = 8 bulls) and low (efficient; n = 9 heifers and n = 8 bulls) groups. Results Average daily gain ±SD and daily DMI ±SD for heifers and bulls were 1.2±0.4 kg and 9.1±0.5 kg, and 1.8±0.3 kg and 9.5±1 kg respectively. High RFI heifers and bulls consumed 10% and 15% more (P < 0.05) than their low RFI counterparts, respectively. Heifers had a higher expression of all genes measured than bulls (P < 0.05). A gender × RFI interaction was detected for HMGCS2(P < 0.05) in which high RFI bulls tended to have lower expression of HMGCS2 than low RFI bulls (P < 0.1), whereas high RFI heifers had higher expression than low RFI heifers (P < 0.05) and high RFI bulls (P < 0.05). SLC2A4 expression was consistently higher in subcutaneous AT of low RFI animals across gender. Conclusion The findings of this study indicate that low RFI cattle exhibit upregulation of the molecular mechanisms governing glucose metabolism in adipose tissue, in particular, glucose clearance. The decreased expression of SLC2A4 in the inefficient cattle may result in less efficient glucose metabolism in these animals. We conclude that SLC2A4 may be a potential biomarker for RFI in cattle.