Project description:Co-ordinated regulation of endometrial gene expression is essential for successful pregnancy establishment. A non-receptive uterine environment may be a key contributor to pregnancy loss, as the majority of pregnancy losses occur prior to embryo implantation. DNA methylation has been highlighted as a potential contributor in regulating early pregnancy events in the uterus. It was hypothesized that DNA methylation regulates expression of key genes in the uterus during pregnancy. To gain support for this hypothesis the correlation between DNA methylation and gene expression was tested. Endometrial samples from fertile and sub-fertile dairy cow strains were obtained at day 17 of pregnancy or the reproductive cycle. Microarrays were used to characterize genome-wide DNA methylation profiles and data compared with transcription profiles which have been previously reported. 39% of DNA methylation probes assayed mapped to RefSeq genes with transcription measurements. The 1,000 most significant correlations were used for subsequent analysis. Of these, 52% percent were negatively correlated with gene expression. When this gene list was compared with previously reported gene expression studies on the same tissues, 42% were differentially expressed when comparing pregnant and cycling animals and 11% were differentially expressed comparing pregnant fertile and sub-fertile animals. DNA methylation status was correlated with gene expression in several pathways implicated in early pregnancy events. Although these data do not provide direct evidence of a causative association between DNA methylation and gene expression, this study provides critical support for an effect of DNA methylation in early pregnancy events and highlights candidate genes for future studies. The estrous cycles of 24 lactating dairy cows were synchronized (at 58.8 (SEM 3.77) and 60.2 (SEM 1.51) days post calving in dairy cows of sub-fertile and fertile strains, respectively) and 14 received a single embryo transferred on day 7 of the estrous cycle. Animals were slaughtered at day 17 of the reproductive cycle and endometrial tissues (both caruncular and intercaruncular) were sampled. Selection criteria for the study included strain and calving date, and health postcalving was an exclusion criterion (cows with severe uterine infections or mastitis were excluded before being enrolled in the embryo transfer round). Cows in each strain were matched for calving number and age. A total of 10 cycling and 12 pregnant animals enrolled in the study were utilized, due to the associated costs of slaughtering the cows. These animals represented fertile (six pregnant and five cycling Holstein-Friesian cows with New Zealand ancestry/M-bM-^IM-$30% North American genetics, n=11, NZ) and sub-fertile (six pregnant and five cycling Holstein-Friesian cows with >87% North American ancestry, n=11, NA) phenotypes of Holstein-Friesian dairy cows
Project description:The regulation of endometrial inflammation has important consequences for the resumption of bovine fertility post-partum. All cows experience bacterial influx into the uterus after calving; however a significant proportion fail to clear infection leading to the development of cytological endometritis (CE) and compromised fertility. We hypothesised that early immunological changes could not only act as potential prognostic biomarkers for the subsequent development of disease but also shed light on the pathogenesis of endometritis in the post-partum dairy cow. Here, next-generation sequencing from endometrial biopsies taken at 7 days post-partum (DPP) identified significant expression of inflammatory genes in all cows. Despite the common inflammatory profile and enrichment of the Toll-like receptor, NF?B and TNF signalling pathways, 73 genes and 31 miRNAs differentiated between healthy cows (HC, n=9) and cows which subsequently developed CE at 7 DPP (n=6, FDR<0.1). In healthy cows, 4197 differentially expressed genes between 7 and 21 DPP whereas only 31 genes were differentially expressed in samples from cows with CE. At 21 DPP, a further 1167 genes were differentially expressed between HC cows and cows diagnosed with CE (FDR<0.1). These changes in host gene expression reflected culture-independent microbiological analysis which showed significant differences in uterine bacterial profiles between groups. Inflammatory activity was not confined to the uterus; decreased circulating granulocytes and increased Acute Phase Protein (SAA and HP) plasma expression levels were detected at 7 DPP in cows that developed CE. In conclusion, our data suggests that the major inflammatory cascade activated early post-partum is resolved thereby restoring homeostasis in healthy cows by 21 DPP, but this transition fails to occur in cows which develop CE. Despite a common inflammatory profile, differential expression of specific immune genes may identify cows at risk of prolonged inflammation and the development of CE post-partum. Sixteen Holstein Friesian cows, of mixed parity, within the same university dairy herd were sampled 7 and 21 days postpartum (DPP) in the morning after milking, over an eight week period.
Project description:The regulation of endometrial inflammation has important consequences for the resumption of bovine fertility post-partum. All cows experience bacterial influx into the uterus after calving; however a significant proportion fail to clear infection leading to the development of cytological endometritis (CE) and compromised fertility. We hypothesised that early immunological changes could not only act as potential prognostic biomarkers for the subsequent development of disease but also shed light on the pathogenesis of endometritis in the post-partum dairy cow. Here, next-generation sequencing from endometrial biopsies taken at 7 days post-partum (DPP) identified significant expression of inflammatory genes in all cows. Despite the common inflammatory profile and enrichment of the Toll-like receptor, NFκB and TNF signalling pathways, 73 genes and 31 miRNAs differentiated between healthy cows (HC, n=9) and cows which subsequently developed CE at 7 DPP (n=6, FDR<0.1). In healthy cows, 4197 differentially expressed genes between 7 and 21 DPP whereas only 31 genes were differentially expressed in samples from cows with CE. At 21 DPP, a further 1167 genes were differentially expressed between HC cows and cows diagnosed with CE (FDR<0.1). These changes in host gene expression reflected culture-independent microbiological analysis which showed significant differences in uterine bacterial profiles between groups. Inflammatory activity was not confined to the uterus; decreased circulating granulocytes and increased Acute Phase Protein (SAA and HP) plasma expression levels were detected at 7 DPP in cows that developed CE. In conclusion, our data suggests that the major inflammatory cascade activated early post-partum is resolved thereby restoring homeostasis in healthy cows by 21 DPP, but this transition fails to occur in cows which develop CE. Despite a common inflammatory profile, differential expression of specific immune genes may identify cows at risk of prolonged inflammation and the development of CE post-partum. Sixteen Holstein Friesian cows, of mixed parity, within the same university dairy herd were sampled 7 and 21 days postpartum (DPP) in the morning after milking, over an eight week period.
Project description:The experiment is part of a study aimed at identifying and studying genes that contribute to differences in oestrous behaviour expression and fertility levels of dairy cows. Samples from 4 brain areas (dorsal hypothalamus, ventral hypothalamus, amygdala and hippocampus) and the anterior pituitary were collected from 28 primiparous Holstein Friesian cows, 14 of which were sacrificed at start of oestrus and 14 at mid of oestrous cycle. Differential gene expression between the 2 phases of oestrous cycle as well as the association of gene expression patterns with the level of oestrous behaviour expression are studied.
Project description:The aim of this study was to determine the effects of linseed dietary supplementation on gene expression in the mammary gland of grazing dairy cows. Milk composition and gene expression in the mammary gland tissue were evaluated in dairy cows supplemented with linseed. The linseed supplementation improves the health and nutrition quality aspects of dairy milk, but also affects the gene networks expression signature associated with cellular growth and proliferation, cell-death, signalling, nutrient metabolism, and immune response, and in turn, the mammary gland integrity and health. The experiment was carried out in a complete randomized blocked designed structure comprising 14 Holstein-Friesian cows (6 second parity, 2 third parity and 6 older cows), selected from a 550-cow herd. Cows were paired in 7 blocks on the basis of similarity in parity (second parity, third parity and older cows), expected date of calving, and milk performance in the previous lactation (in order of priority). Cows within each block were randomly allocated to one of two treatment groups, “Omega” or “Control”. The dietary Omega treatment consisted of a basal diet supplemented with a concentrate-mixture including linseed on a dry matter (DM) basis, whereas cows in treatment group Control were supplemented with a concentrate mixture without linseed. Linseed was chosen because it is rich in c9,c12,c15-18:3 (ALA). Concentrate mixtures were fed with a concentrate dispenser. Experimental treatments started 3 weeks before the expected calving date (wk -3) and lasted until 6 weeks after calving (wk 6).
Project description:The ruminant liver has multiple roles in the dairy cow and many of these are crucial in nutrient supply during lactation. Reduced feed intake alters the expression of many genes and pathways in the liver, inducing a period of negative energy balance. Once-daily milking is a management strategy to reduce the effects of periods of negative energy balance so the objective of this study was to determine if once-daily milking altered hepatic gene transcription during a period of negative energy balance induce by caloric restriction. Multiparous Holstein-Friesian and Holstein-Friesian x Jersey cows (n = 120) were grazed on pasture and milked twice daily (2X) from calving until 34 ± 6 days in milk (mean ± standard deviation). Cows were then allocated to one of four treatments in a 2 x 2 factorial arrangement. Treatments consisted of two milking frequencies (2X or once daily; 1X) and two feeding levels for three weeks: adequately fed (AF), consuming 14.3 kg dry matter intake/cow per d, or underfed (UF) consuming 8.3 kg dry matter intake /cow per d. After the treatment period, all cows were fed to target grazing residuals ? 1600 kg DM/cow per d and milked 2X for 20 wk. Liver tissue was collected from 12 cows per treatment by subcutaneous biopsy at 3 wk relative to treatment start, RNA extracted and transcript abundance of genes quantified.
Project description:Liver plays a profound role in the acute phase response (APR) observed in the early phase of acute bovine mastitis caused by Escherichia coli (E. coli). To gain an insight into the genes and pathways involved in hepatic APR of dairy cows we performed a global gene expression analysis of liver tissue sampled at different time points before and after intra-mammary (IM) exposure to E. coli lipopolysaccharide (LPS) treatment. Experiment Overall Design: Eight healthy, high yielding Holstein-Friesian dairy cows in their first lactation (9 to 12 weeks after calving) were chosen for this study. At time 0 the right front quarter was infused with 200 μg E. coli LPS dissolved in 10 ml 0.9% NaCl solution, the left front quarter serving as control was infused with 10 ml 0.9% NaCl solution. Liver biopsies were taken at â22, 3, 6, 9, 12 and 48 hours relative to LPS infusion in 4 cows, and also at â22, 9 and 48 hours in the remaining 4 cows. RNA from liver biopsies was isolated and biotin labeled cRNA was loaded onto the Affymetric GeneChip Bovine Genome Array. A control study using cows infused with 0.9% NaCl showed that there was no effect of taking the biopsy, neither in the clinical measurement nor in the expression of a selected subset of genes. Therefore, only samples taken from the LPS treated cows were measured for the gene expression using microarrays.
Project description:This study aimed at evaluating the global gene expression of blood-derived neutrophils from periparturient cows. Blood was collected from Holstein Friesian periparturient cows (N=3) at −14 d relative to expected calving date and 7 d relative to actual calving date. Neutrophils were isolated and subsequently used for transcriptional profiling using the Agilent bovine (v2) 4 × 44 K array. Calculation of fold change in gene expression and pathway analysis was conducted using the GeneSpring GX software 13.0. Periparturient period impacted global gene expression and resulted in 249 genes that were differentially expressed (FC≥2, p<0.05.); 162 were upregulated post-calving 87 of these were downregulated. Genes that code for proinflammatory receptors (CD58, GLRX3), chemokines (CMKLR1), and transcriptional regulation (MTA) were upregulated. Concurrently, genes that code for cellular adhesion and migration (ADRM1 and THY1), and immune induction (CATHL2) and homeostasis were downregulated gene. Pathway analysis revealed that 118 pathways are affected in bovine neutrophils during the periparturient period (p<0.05). These pathways included the Wnt signaling, one carbon Metabolism, TLR, inflammation response, Oxidative Stress, T-Cell Receptor signaling, adipogenesis, and MAPK Signaling Pathways.
Project description:Pregnancy induces changes in the transcriptome of the bovine endometrium from 15 days after insemination. However, pregnancy is less likely to occur if cows had a postpartum bacterial infection of the uterus. We hypothesized that uterine bacterial infection alters the endometrial transcriptomic signature of pregnancy. To examine the endometrial transcriptomic signature of pregnancy, cows were inseminated 130 days after intrauterine infusion of pathogenic bacteria and endometrium was collected 16 days later for RNA sequencing. We found 171 pregnancy regulated genes in cows 146 days after bacterial infection. When comparing our findings with three previous studies that described the endometrial transcriptomic signature of pregnancy in healthy cows, 24 genes were consistently differentially expressed in pregnancy, including MX1, MX2 and STAT1. However, 12 pregnancy regulated genes were only found in the endometrium of healthy cows, including ISG15 and TRANK1. Furthermore, 28 pregnancy regulated genes were only found in the endometrium of cows following bacterial infection and these were associated with altered iNOS, TLR, and IL-7 canonical signaling pathways. Although 94 predicted upstream regulators were conserved amongst the studies, 14 were found only in the endometrium of pregnant healthy cows, and 5 were found only in cows following bacterial infection, including AIRE, NFKBIA, and DUSP1. In conclusion, there were both consistent and discordant features of the endometrial transcriptomic signature of pregnancy 146 days after intrauterine bacterial infusion. These findings imply that there is an essential transcriptomic signature of pregnancy, but that infection induces long term changes in the endometrium that affect the transcriptomic response to pregnancy.