• Birth delivery method affects expression of immune genes in lung and jejunum tissue of neonatal beef calves

      Surlis, Carla; McNamara, Keelan; O’Hara, Eoin; Waters, Sinead; Beltman, Marijke; Cassidy, Joseph; Kenny, David (Biomed Central, 2017-12-14)
      Background Caesarean section is a routine veterinary obstetrical procedure employed to alleviate dystocia in cattle. However, CS, particularly before the onset of labour, is known to negatively affect neonatal respiration and metabolic adaptation in humans, though there is little published information for cattle. The aim of this study was to investigate the effect of elective caesarean section (ECS) or normal trans-vaginal (TV) delivery, on lung and jejunal gene expression profiles of neonatal calves. Results Paternal half-sib Angus calves (gestation length 278 + 1.8 d) were delivered either transvaginally (TV; n = 8) or by elective caesarean section (ECS; n = 9) and immediately euthanized. Lung and jejunum epithelial tissue was isolated and snap frozen. Total RNA was extracted using Trizol reagent and reverse transcribed to generate cDNA. For lung tissue, primers were designed to target genes involved in immunity, surfactant production, cellular detoxification, membrane transport and mucin production. Primers for jejunum tissue were chosen to target mucin production, immunoglobulin uptake, cortisol reaction and membrane trafficking. Quantitative real-time PCR reactions were performed and data were statistically analysed using mixed models ANOVA. In lung tissue the expression of five genes were affected (p < 0.05) by delivery method. Four of these genes were present at lower (LAP, CYP1A1, SCN11α and SCN11β) and one (MUC5AC) at higher abundance in ECS compared with TV calves. In jejunal tissue, expression of TNFα, Il-1β and 1 l-6 was higher in ECS compared with TV calves. Conclusions This novel study shows that ECS delivery affects the expression of key genes involved in the efficiency of the pulmonary liquid to air transition at birth, and may lead to an increased inflammatory response in jejunal tissue, which could compromise colostral immunoglobulin absorption. These findings are important to our understanding of the viability and management of neonatal calves born through ECS.
    • Detection and characterisation of Complement protein activity in bovine milk by bactericidal sequestration assay

      Maye, Susan; Stanton, Catherine; Fitzgerald, Gerald F.; Kelly, Philip M. (Cambridge University Press for the Institute of Food Research and the Hannah Research Institute, 2015-06-29)
      While the Complement protein system in human milk is well characterised, there is little information on its presence and activity in bovine milk. Complement forms part of the innate immune system, hence the importance of its contribution during milk ingestion to the overall defences of the neonate. A bactericidal sequestration assay, featuring a Complement sensitive strain, Escherichia coli 0111, originally used to characterise Complement activity in human milk was successfully applied to freshly drawn bovine milk samples, thus, providing an opportunity to compare Complement activities in both human and bovine milks. Although not identical in response, the levels of Complement activity in bovine milk were found to be closely comparable with that of human milk. Differential counts of Esch. coli 0111 after 2 h incubation were 6·20 and 6·06 log CFU/ml, for raw bovine and human milks, respectively – the lower value representing a stronger Complement response. Exposing bovine milk to a range of thermal treatments e.g. 42, 45, 65, 72, 85 or 95 °C for 10 min, progressively inhibited Complement activity by increasing temperature, thus confirming the heat labile nature of this immune protein system. Low level Complement activity was found, however, in 65 and 72 °C heat treated samples and in retailed pasteurised milk which highlights the outer limit to which high temperature, short time (HTST) industrial thermal processes should be applied if retention of activity is a priority. Concentration of Complement in the fat phase was evident following cream separation, and this was also reflected in the further loss of activity recorded in low fat variants of retailed pasteurised milk. Laboratory-based churning of the cream during simulated buttermaking generated an aqueous (buttermilk) phase with higher levels of Complement activity than the fat phase, thus pointing to a likely association with the milk fat globule membrane (MFGM) layer.
    • Effect of genotype on duodenal expression of nutrient transporter genes in dairy cows

      Waters, Sinead M.; Keogh, Kate; Buckley, Frank; Kenny, David A (Biomed Central, 2013-12-09)
      Background Studies have shown clear differences between dairy breeds in their feed intake and production efficiencies. The duodenum is critical in the coordination of digestion and absorption of nutrients. This study examined gene transcript abundance of important classes of nutrient transporters in the duodenum of non lactating dairy cows of different feed efficiency potential, namely Holstein-Friesian (HF), Jersey (JE) and their F1 hybrid. Duodenal epithelial tissue was collected at slaughter and stored at -80°C. Total RNA was extracted from tissue and reverse transcribed to generate cDNA. Gene expression of the following transporters, namely nucleoside; amino acid; sugar; mineral; and lipid transporters was measured using quantitative real-time RT-PCR. Data were statistically analysed using mixed models ANOVA in SAS. Orthogonal contrasts were used to test for potential heterotic effects and spearman correlation coefficients calculated to determine potential associations amongst gene expression values and production efficiency variables. Results While there were no direct effects of genotype on expression values for any of the genes examined, there was evidence for a heterotic effect (P < 0.05) on ABCG8, in the form of increased expression in the F1 genotype compared to either of the two parent breeds. Additionally, a tendency for increased expression of the amino acid transporters, SLC3A1 (P = 0.072), SLC3A2 (P = 0.081) and SLC6A14 (P = 0.072) was also evident in the F1 genotype. A negative (P < 0.05) association was identified between the expression of the glucose transporter gene SLC5A1 and total lactational milk solids yield, corrected for body weight. Positive correlations (P < 0.05) were also observed between the expression values of genes involved in common transporter roles. Conclusion This study suggests that differences in the expression of sterol and amino acid transporters in the duodenum could contribute towards the documented differences in feed efficiency between HF, JE and their F1 hybrid. Furthermore, positive associations between the expression of genes involved in common transporter roles suggest that these may be co-regulated. The study identifies potential candidates for investigation of genetic variants regulating nutrient transport and absorption in the duodenum in dairy cows, which may be incorporated into future breeding programmes.
    • Effect of short term diet restriction on gene expression in the bovine hypothalamus using next generation RNA sequencing technology

      Matthews, Daragh; Diskin, Michael G; Kenny, David A; Creevey, Christopher J; Keogh, Kate; Waters, Sinead M (Biomed Central, 2017-11-09)
      Background Negative energy balance (NEB) is an imbalance between energy intake and energy requirements for lactation and body maintenance affecting high-yielding dairy cows and is of considerable economic importance due to its negative impact on fertility and health in dairy herds. It is anticipated that the cow hypothalamus experiences extensive biochemical changes during the early post partum period in an effort to re-establish metabolic homeostasis. However, there is variation in the tolerance to NEB between individual cows. In order to understand the genomic regulation of ovulation in hypothalamic tissue during NEB, mRNA transcriptional patterns between tolerant and sensitive animals were examined. A short term dietary restriction heifer model was developed which induced abrupt onset of anoestrus in some animals (Restricted Anovulatory; RA) while others maintained oestrous cyclicity (Restricted Ovulatory; RO). A third control group (C) received a higher level of normal feeding. Results A total of 15,295 genes were expressed in hypothalamic tissue. Between RA and C groups 137 genes were differentially expressed, whereas between RO and C, 32 genes were differentially expressed. Differentially expressed genes were involved in the immune response and cellular motility in RA and RO groups, respectively, compared to C group. The largest difference between groups was observed in the comparison between RA and RO heifers, with 1094 genes shown to be significantly differentially expressed (SDE). Pathway analysis showed that these SDE genes were associated with 6 canonical pathways (P < 0.01), of which neuroactive ligand-receptor interaction was the most significant. Within the comparisons the main over-represented pathway functions were immune response including neuroprotection (CXCL10, Q1KLR3, IFIH1, IL1 and IL8; RA v C and RA v RO); energy homeostasis (AgRP and NPY; RA v RO); cell motility (CADH1, DSP and TSP4; RO v C) and prevention of GnRH release (NTSR1 IL1α, IL1β, NPY and PACA; RA v RO). Conclusions This information will assist in understanding the genomic factors regulating the influence of diet restriction on fertility and may assist in optimising nutritional and management systems for the improvement in reproductive performance.
    • Establishment of a bovine/Quercus silvopastoral experiment in lowland Ireland.

      Short, Ian; McAdam, J.; Culleton, Noel; Douglas, Gerry C. (CAB International, 2005)
      A silvopastoral experiment was established at Teagasc in County Wexford, Ireland, in 2002 with oak (Quercus robur L.) in an alley design and bovines. The experiment includes some treatments with trees produced with an enhanced root system (RPM). The treatments are: (1) control pasture plots; (2) RPM agroforestry (400 stems/ha); (3) conventional agroforestry (400 stems/ha); (4) RPM forestry (6600stems/ha; and (5) conventional forestry (6600 stems/ha). The trees were successfully established and cattle were successfully managed in combination with the trees. In the first year, height growth of bare-root oaks was significantly greater in the forestry treatment compared to the agroforestry treatment and, overall, RPM oaks were taller than bare-root plants. Among the RPM trees, the agroforestry system resulted in a greater stem diameter than those in the forestry plots. Height increment was greater for RPM trees than for bare-root trees.
    • Investigation of the presence and activity of the innate immune component, Complement, in bovine milk

      Maye, Susan (2016-04)
      The occurrence of Complement in human milk indicates the presence of innate immune components of maternal origin and their putative defensive role in the neonatal gut. In order to ascertain the status of Complement in bovine milk, it was necessary to validate the suitability of the Complement-sensitive bacterial sequestration assay used to monitor human milk. The relative bacteriostatic effects, expressed as a differential between the initial and final assay counts following inhibition of the E. coli O111 marker strain were, 6.20 and 6.06 log CFU/ml for raw bovine and human milks, respectively. Lower levels of Complement activity measured in pasteurised and low-fat milks were observed during the course of heat-inactivation studies, while gravity separation of cream over a 24 h period confirmed a greater degree of attachment by Complement to the rising cream layer. The E. coli O111 sequestration assay strain had restricted growth of 7.5 and 8.2 CFU/ml in the higher and lower gravity-separated fractions, respectively. An animal health effect was observed, with Complement appearing to be more active in milk samples from cows with a higher somatic cell counts (SCC). Inducing sub-clinical mastitis in the healthy quarter of a lactating cow confirmed that increased Complement activity effect was local to the affected quarter and not manifested systemically. Complement activity levels varied in the milks of 7 commercially important prominent dairy breeds and crossbreeds surveyed. Both the Norwegian Red and Kerry breed cows stood out as having higher Complement activity (6.25 and 6.92 CFU/ml, respectively); these two breeds are known to have stronger immunity and a better capacity to resist mastitic infection. The findings of this study have implications for both milk production and processing disciplines. In order to retain the antimicrobial efficacy associated with xi Complement in raw bovine milk, milder process treatments than hitherto practised in industry should be adopted.
    • Next Generation Sequencing Reveals the Expression of a Unique miRNA Profile in Response to a Gram-Positive Bacterial Infection

      Lawless, Nathan; Foroushani, Amir B. K.; McCabe, Matthew S.; O'Farrelly, Cliona; Lynn, David J. (PLOS, 2013-01-25)
      MicroRNAs (miRNAs) are short, non-coding RNAs, which post-transcriptionally regulate gene expression and are proposed to play a key role in the regulation of innate and adaptive immunity. Here, we report a next generation sequencing (NGS) approach profiling the expression of miRNAs in primary bovine mammary epithelial cells (BMEs) at 1, 2, 4 and 6 hours post-infection with Streptococcus uberis, a causative agent of bovine mastitis. Analysing over 450 million sequencing reads, we found that 20% of the approximately 1,300 currently known bovine miRNAs are expressed in unchallenged BMEs. We also identified the expression of more than 20 potentially novel bovine miRNAs. There is, however, a significant dynamic range in the expression of known miRNAs. The top 10 highly expressed miRNAs account for >80% of all aligned reads, with the remaining miRNAs showing much lower expression. Twenty-one miRNAs were identified as significantly differentially expressed post-infection with S. uberis. Several of these miRNAs have characterised roles in the immune systems of other species. This miRNA response to the Gram-positive S. uberis is markedly different, however, to lipopolysaccharide (LPS) induced miRNA expression. Of 145 miRNAs identified in the literature as being LPS responsive, only 9 were also differentially expressed in response to S. uberis. Computational analysis has also revealed that the predicted target genes of miRNAs, which are down-regulated in BMEs following S. uberis infection, are statistically enriched for roles in innate immunity. This suggests that miRNAs, which potentially act as central regulators of gene expression responses to a Gram-positive bacterial infection, may significantly regulate the sentinel capacity of mammary epithelial cells to mobilise the innate immune system.
    • 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 (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.
    • RNA-seq analysis of differential gene expression in liver from lactating dairy cows divergent in negative energy balance

      McCabe, Matthew; Waters, Sinead M.; Morris, Dermot G.; Kenny, David J; Lynn, David J; Creevey, Christopher J. (Biomed Central, 2012-05-20)
      Background: The liver is central to most economically important metabolic processes in cattle. However, the changes in expression of genes that drive these processes remain incompletely characterised. RNA-seq is the new gold standard for whole transcriptome analysis but so far there are no reports of its application to analysis of differential gene expression in cattle liver. We used RNA-seq to study differences in expression profiles of hepatic genes and their associated pathways in individual cattle in either mild negative energy balance (MNEB) or severe negative energy balance (SNEB). NEB is an imbalance between energy intake and energy requirements for lactation and body maintenance. This aberrant metabolic state affects high-yielding dairy cows after calving and is of considerable economic importance because of its negative impact on fertility and health in dairy herds. Analysis of changes in hepatic gene expression in SNEB animals will increase our understanding of NEB and contribute to the development of strategies to circumvent it. Results: RNA-seq analysis was carried out on total RNA from liver from early post partum Holstein Friesian cows in MNEB (n = 5) and SNEB (n = 6). 12,833 genes were deemed to be expressed (>4 reads per gene per animal), 413 of which were shown to be statistically significantly differentially expressed (SDE) at a false discovery rate (FDR) of 0.1% and 200 of which were SDE (FDR of 0.1%) with a ≥2-fold change between MNEB and SNEB animals. GOseq/KEGG pathway analysis showed that SDE genes with ≥2- fold change were associated (P <0.05) with 9 KEGG pathways. Seven of these pathways were related to fatty acid metabolism and unexpectedly included ‘Steroid hormone biosynthesis’, a process which mainly occurs in the reproductive organs rather than the liver. Conclusions: RNA-seq analysis showed that the major changes at the level of transcription in the liver of SNEB cows were related to fat metabolism. 'Steroid hormone biosynthesis', a process that normally occurs in reproductive tissue, was significantly associated with changes in gene expression in the liver of SNEB cows. Changes in gene expression were found in this pathway that have not been previously been identified in SNEB cows.
    • Understanding and using somatic cell counts to improve milk quality

      Ruegg, P.L.; Pantoja, J.C.F. (Teagasc (Agriculture and Food Development Authority), Ireland, 2013)
      The production of high quality milk is a requirement to sustain a profitable dairy industry and somatic cell count (SCC) values are routinely used to identify subclinical mastitis and define quality standards. The objective of this paper is to review the use of SCC as a diagnostic tool for subclinical mastitis in order to improve milk quality on dairy farms. Mastitis is detected based on inflammation subsequent to intramammary infection (IMI) by pathogenic organisms. Individual cow SCC values are used to detect the inflammation that results from IMI and are necessary to define the prevalence and incidence of subclinical IMI. A threshold of <200,000 cells/mL is considered to be of the most practical value used to define a mammary quarter as healthy. The development of IMI is the most significant factor that influences milk SCC and assessment of monthly values to determine newly and chronically increased SCC can be highly diagnostic for resolving problems with increased bulk tank SCC. Methods to reduce the development of new IMI are well known and adoption of best management practices for milking and herd management have consistently been shown to result in reductions in bulk tank SCC. Implementation of mastitis control programmes can be improved by focusing on three practical recommendations: 1) Farmers should work with their advisors to develop an annual udder health plan that includes clear goals for milk quality. 2) The annual udder health plan should emphasise prevention of new IMI. 3) Farmers must identify and manage chronically infected cows. Proactive management of IMI can be extremely effective in helping farmers produce milk that meets industry standards for milk quality.