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

      Moore, Stephen; Ericsson, Aaron C; Behura, Susanta K; Lamberson, William R; Evans, Timothy J; McCabe, Matthew S; Poock, Scott E; Lucy, Matthew C; European Union; USDA Animal Health Formula Funds; et al. (Biomed Central, 2019-05-22)
      Background Fertility in dairy cows depends on ovarian cyclicity and on uterine involution. Ovarian cyclicity and uterine involution are delayed when there is uterine dysbiosis (overgrowth of pathogenic bacteria). Fertility in dairy cows may involve a mechanism through which the uterine microbiota affects ovarian cyclicity as well as the transcriptome of the endometrium within the involuting uterus. The hypothesis was that the transcriptome of the endometrium in postpartum cows would be associated with the cyclicity status of the cow as well as the microbiota during uterine involution. The endometrium of first lactation dairy cows was sampled at 1, 5, and 9 weeks postpartum. All cows were allowed to return to cyclicity without intervention until week 5 and treated with an ovulation synchronization protocol so that sampling at week 9 was on day 13 of the estrous cycle. The endometrial microbiota was measured by 16S rRNA gene sequencing and principal component analysis. The endometrial transcriptome was measured by mRNA sequencing, differential gene expression analysis, and Ingenuity Pathway Analysis. Results The endometrial microbiota changed from week 1 to week 5 but the week 5 and week 9 microbiota were similar. The endometrial transcriptome differed for cows that were either cycling or not cycling at week 5 and cyclicity status depended in part on the endometrial microbiota. Compared with cows cycling at week 5, there were large changes in the transcriptome of cows that progressed from non-cycling at week 5 to cycling at week 9. There was evidence for concurrent and longer-term associations between the endometrial microbiota and transcriptome. The week 1 endometrial microbiota had the greatest effect on the subsequent endometrial transcriptome and this effect was greatest at week 5 and diminished by week 9. Conclusions The cumulative response of the endometrial transcriptome to the microbiota represented the combination of past microbial exposure and current microbial exposure. The endometrial transcriptome in postpartum cows, therefore, depended on the immediate and longer-term effects of the uterine microbiota that acted directly on the uterus. There may also be an indirect mechanism through which the microbiome affects the transcriptome through the restoration of ovarian cyclicity postpartum.
    • A differential interplay between the expression of Th1/Th2/Treg related cytokine genes in Teladorsagia circumcincta infected DRB1*1101 carrier lambs

      Hassan, Musa; Hanrahan, James P; Good, Barbara; Mulcahy, Grace; Sweeney, Torres; Teagasc Walsh Fellowship Programme (Biomed Central, 2011-03-08)
      Substantial debate exists on whether the immune response between sheep resistant and susceptible to gastrointestinal nematodes can be differentiated into a Th1 and Th2 phenotype. The present study addresses the hypothesis that variation in resistance to Teladorsagia circumcincta between DRB1*1101 (associated with reduced faecal egg count and worm burden) carriers and non-carriers is due to a differential interplay in the expression of Th1/Th2 and regulatory T (Treg) related cytokine genes. Lambs from each genotype were either slaughtered at day 0 (un-infected control) or infected with 3 × 104 Teladorsagia circumcincta L3 and slaughtered at 3, 7, 21, and 35 days later. Lambs carrying the DRB1*1101 allele had a significantly lower worm burden (P < 0.05) compared to the non-carriers. Abomasal mucosal cytokine gene expression was evaluated by quantitative real-time PCR and comparison made for time and genotype effects. The response generated varied through the course of infection and was affected by genotype. DRB1*1101 carriers had an up-regulated expression of the Th1-related cytokine genes (IL-1β, TNFα, and IFN-γ) at day 3, but this was replaced by an up-regulated expression of Th2-related cytokine genes (IL-10 and IL-13) and Treg-related cytokine genes (IL-2RA-CD25, TGFα, TGFβ, Arg2, MIF and FOXP3) by day 7. Conversely, in the non-carriers these changes in gene expression were delayed until days 7 and 21 post infection (pi), respectively. It is concluded that resistance to Teladorsagia circumcincta in animals carrying the DRB1*1101 allele is influenced by an earlier interplay between Th1, Th2 and T regulatory immune response genes.
    • 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.
    • 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.
    • Global endometrial transcriptomic profiling: transient immune activation precedes tissue proliferation and repair in healthy beef cows

      Foley, Cathriona; Chapwanya, Aspinas; Creevey, Christopher J.; Narciandi, Fernando; Morris, Derek W.; Kenny, Elaine; Cormican, Paul; Callanan, John J; O'Farrelly, Cliona; Meade, Kieran G (Biomed Central, 2012-09-18)
      Background: All cows experience bacterial contamination and tissue injury in the uterus postpartum, instigating a local inflammatory immune response. However mechanisms that control inflammation and achieve a physiologically functioning endometrium, while avoiding disease in the postpartum cow are not succinctly defined. This study aimed to identify novel candidate genes indicative of inflammation resolution during involution in healthy beef cows. Previous histological analysis of the endometrium revealed elevated inflammation 15 days postpartum (DPP) which was significantly decreased by 30 DPP. The current study generated a genome-wide transcriptomic profile of endometrial biopsies from these cows at both time points using mRNA-Seq. The pathway analysis tool GoSeq identified KEGG pathways enriched by significantly differentially expressed genes at both time points. Novel candidate genes associated with inflammatory resolution were subsequently validated in additional postpartum animals using quantitative real-time PCR (qRT-PCR). Results: mRNA-Seq revealed 1,107 significantly differentially expressed genes, 73 of which were increased 15 DPP and 1,034 were increased 30 DPP. Early postpartum, enriched immune pathways (adjusted P < 0.1) included the T cell receptor signalling pathway, graft-versus-host disease and cytokine-cytokine receptor interaction pathways. However 30 DPP, where the majority of genes were differentially expressed, the enrichment (adjusted P < 0.1) of tissue repair and proliferative activity pathways was observed. Nineteen candidate genes selected from mRNA-Seq results, were independently assessed by qRT-PCR in additional postpartum cows (5 animals) at both time points. SAA1/2, GATA2, IGF1, SHC2, and SERPINA14 genes were significantly elevated 30 DPP and are functionally associated with tissue repair and the restoration of uterine homeostasis postpartum. Conclusions: The results of this study reveal an early activation of the immune response which undergoes a temporal functional change toward tissue proliferation and regeneration during endometrial involution in healthy postpartum cows. These molecular changes mirror the activation and resolution of endometrial inflammation during involution previously classified by the degree of neutrophil infiltration. SAA1/2, GATA2, IGF1, SHC2, and SERPINA14 genes may become potential markers for resolution of endometrial inflammation in the postpartum cow.
    • RNA-seq of muscle from pigs divergent in feed efficiency and product quality identifies differences in immune response, growth, and macronutrient and connective tissue metabolism

      Horodyska, Justyna; Wimmers, Klaus; Reyer, Henry; Trakooljul, Nares; Mullen, Anne Maria; Lawlor, Peadar G; Hamill, Ruth M; European Union; 311794 (Biomed Central, 2018-11-01)
      Background Feed efficiency (FE) is an indicator of efficiency in converting energy and nutrients from feed into a tissue that is of major environmental and economic significance. The molecular mechanisms contributing to differences in FE are not fully elucidated, therefore the objective of this study was to profile the porcine Longissimus thoracis et lumborum (LTL) muscle transcriptome, examine the product quality from pigs divergent in FE and investigate the functional networks underpinning the potential relationship between product quality and FE. Results RNA-Seq (n = 16) and product quality (n = 40) analysis were carried out in the LTL of pigs differing in FE status. A total of 272 annotated genes were differentially expressed with a P < 0.01. Functional annotation revealed a number of biological events related to immune response, growth, carbohydrate & lipid metabolism and connective tissue indicating that these might be the key mechanisms governing differences in FE. Five most significant bio-functions altered in FE groups were ‘haematological system development & function’, ‘lymphoid tissue structure & development’, ‘tissue morphology’, ‘cellular movement’ and ‘immune cell trafficking’. Top significant canonical pathways represented among the differentially expressed genes included ‘IL-8 signalling’, ‘leukocyte extravasation signalling, ‘sphingosine-1-phosphate signalling’, ‘PKCθ signalling in T lymphocytes’ and ‘fMLP signalling in neutrophils’. A minor impairment in the quality of meat, in relation to texture and water-holding capacity, produced by high-FE pigs was observed. High-FE pigs also had reduced intramuscular fat content and improved nutritional profile in terms of fatty acid composition. Conclusions Ontology analysis revealed enhanced activity of adaptive immunity and phagocytes in high-FE pigs suggesting more efficient conserving of resources, which can be utilised for other important biological processes. Shifts in carbohydrate conversion into glucose in FE-divergent muscle may underpin the divergent evolution of pH profile in meat from the FE-groups. Moreover, altered amino acid metabolism and increased mobilisation & flux of calcium may influence growth in FE-divergent muscle. Furthermore, decreased degradation of fibroblasts in FE-divergent muscle could impact on collagen turnover and alter tenderness of meat, whilst enhanced lipid degradation in high-FE pigs may potentially underlie a more efficient fat metabolism in these animals.
    • SNP variation in the promoter of the PRKAG3 gene and association with meat quality traits in pig

      Ryan, Marion T; Hamill, Ruth M; O'Halloran, Aisling M; Davey, Grace C; McBryan, Jean; Mullen, Anne Maria; McGee, Chris; Gispert, Marina; Southwood, Olwen I; Sweeney, Torres; et al. (Biomed Central, 25/07/2012)
      Background: The PRKAG3 gene encodes the γ3 subunit of adenosine monophosphate activated protein kinase (AMPK), a protein that plays a key role in energy metabolism in skeletal muscle. Non-synonymous single nucleotide polymorphisms (SNPs) in this gene such as I199V are associated with important pork quality traits. The objective of this study was to investigate the relationship between gene expression of the PRKAG3 gene, SNP variation in the PRKAG3 promoter and meat quality phenotypes in pork. Results: PRKAG3 gene expression was found to correlate with a number of traits relating to glycolytic potential (GP) and intramuscular fat (IMF) in three phenotypically diverse F1 crosses comprising of 31 Large White, 23 Duroc and 32 Pietrain sire breeds. The majority of associations were observed in the Large White cross. There was a significant association between genotype at the g.-311A>G locus and PRKAG3 gene expression in the Large White cross. In the same population, ten novel SNPs were identified within a 1.3 kb region spanning the promoter and from this three major haplotypes were inferred. Two tagging SNPs (g.-995A>G and g.-311A>G) characterised the haplotypes within the promoter region being studied. These two SNPs were subsequently genotyped in larger populations consisting of Large White (n = 98), Duroc (n = 99) and Pietrain (n = 98) purebreds. Four major haplotypes including promoter SNP’s g.-995A>G and g.-311A>G and I199V were inferred. In the Large White breed, HAP1 was associated with IMF% in the M. longissmus thoracis et lumborum (LTL) and driploss%. HAP2 was associated with IMFL% GP-influenced traits pH at 24 hr in LTL (pHULT), pH at 45 min in LTL (pH45LT) and pH at 45 min in the M. semimembranosus muscle (pH45SM). HAP3 was associated with driploss%, pHULT pH45LT and b* Minolta. In the Duroc breed, associations were observed between HAP1 and driploss% and pHUSM. No associations were observed with the remaining haplotypes (HAP2, HAP3 and HAP4) in the Duroc breed. The Pietrain breed was monomorphic in the promoter region. The I199V locus was associated with several GP-influenced traits across all three breeds and IMF% in the Large White and Pietrain breed. No significant difference in promoter function was observed for the three main promoter haplotypes when tested in vitro. Conclusion: Gene expression levels of the porcine PRKAG3 are associated with meat quality phenotypes relating to glycolytic potential and IMF% in the Large White breed, while SNP variation in the promoter region of the gene is associated with PRKAG3 gene expression and meat quality phenotypes.
    • Transcriptomics of liver and muscle in Holstein cows genetically divergent for fertility highlight differences in nutrient partitioning and inflammation processes

      Moran, Bruce; Cummins, Sean B; Creevey, Christopher J.; Butler, Stephen T. (Biomed Central, 2016-08-11)
      Background The transition between pregnancy and lactation is a major physiological change for dairy cows. Complex systemic and local processes involving regulation of energy balance, galactopoiesis, utilisation of body reserves, insulin resistance, resumption of oestrous cyclicity and involution of the uterus can affect animal productivity and hence farm profitability. Here we used an established Holstein dairy cow model of fertility that displayed genetic and phenotypic divergence in calving interval. Cows had similar genetic merit for milk production traits, but either very good genetic merit for fertility traits (‘Fert+’; n = 8) or very poor genetic merit for fertility traits (‘Fert-’; n = 8). We used RNA sequencing to investigate gene expression profiles in both liver and muscle tissue biopsies at three distinct time-points: late pregnancy, early lactation and mid lactation (-18, 1 and 147 days relative to parturition, respectively). Results We found 807 and 815 unique genes to be differentially expressed in at least one time-point in liver and muscle respectively, of which 79 % and 83 % were only found in a single time-point; 40 and 41 genes were found differentially expressed at every time-point indicating possible systemic or chronic dysregulation. Functional annotation of all differentially expressed genes highlighted two physiological processes that were impacted at every time-point in the study, These were immune and inflammation, and metabolic, lipid and carbohydrate-binding. Conclusion These pathways have previously been identified by other researchers. We show that several specific genes which are differentially regulated, including IGF-1, might impact dairy fertility. We postulate that an increased burden of reactive oxidation species, coupled with a chronic inflammatory state, might reduce dairy cow fertility in our model.