Web-based Tools for the Analysis of DNA Microarrays

End of project reportDNA microarrays are widely used for gene expression profiling. Raw data resulting from microarray experiments, however, tends to be very noisy and there are many sources of technical variation and bias. This raw data needs to be quality assessed and interactively preprocessed to minimise variation before statistical analysis in order to achieve meaningful result. Therefore microarray analysis requires a combination of visualisation and statistical tools, which vary depending on what microarray platform or experimental design is used.Bioconductor is an existing open source software project that attempts to facilitate analysis of genomic data. It is a collection of packages for the statistical programming language R. Bioconductor is particularly useful in analyzing microarray experiments. The problem is that the R programming language’s command line interface is intimidating to many users who do not have a strong background in computing. This often leads to a situation where biologists will resort to using commercial software which often uses antiquated and much less effective statistical techniques, as well as being expensively priced. This project aims to bridge this gap by providing a user friendly web-based interface to the cutting edge statistical techniques of Bioconductor

Studies relating to protein expression in the uterus of the cow

End of project reportEmbryo loss is a major cause of reproductive wastage in the cow. The majority of embryo loss occurs in the first 16 days after fertilisation when the embryo is critically dependent on the maternal uterine environment for survival. Despite the central role of uterine fluid in the normal growth and development of the embryo, there is limited information on the protein composition of these fluids. The main objectives of the studies in this thesis were to examine the protein composition of the bovine uterus during the oestrous cycle and to examine the relationship between the concentration of systemic progesterone and uterine protein expression in the cow. In the first study, the concentration of retinol-binding protein (RBP) in the bovine uterus was found to vary across the cycle and was 5-15-fold higher (P0.05) of the cycle, which was surprising given that previous studies have indicated that uterine RBP gene expression was positively associated with the concentration of systemic progesterone. In the second study, IGF binding protein 2 (IGFBP-2), IGFBP-3, IGFBP-4 and IGFBP-5 were identified in uterine fluid on days 3, 7, 11 and 15 of the oestrous cycle. There was a local effect on the concentration of IGFBPs where the concentration was greater on the ipsilateral side than that on the contralateral side for IGFBP-2 (P<0.05), 3 (P<0.01) and 5 (P<0.01) on day 15. This difference is a further indication of a local controlling mechanism regulating proteins between the uterine horns. Similar to RBP expression this study could find no significant relationship between the concentration of systemic progesterone and IGFBP concentrations on Day 7 of the oestrous cycle. In the third study, changes in the global pattern of uterine proteins between Days 3 and Day 15 of the oestrous cycle were examined using two-dimensional electrophoresis (2-DE). Six proteins were found to be upregulated on Day 15 compared to Day 3. Three proteins of these were identified as aldose reductase, plakoglobin and heat shock protein 27 while the other three proteins were identified as bovine serum albumin. Aldose reductase, an enzyme directly involved in the production of sorbitol and indirectly of fructose, was 10-fold higher (P<0.0001) on Day 15 compared to Day 3. Plakoglobin (Pg) was upregulated 2.3-fold (P<0.0001) on Day 15 compared to Day 3. Pg is a component of cellular junctions and its up-regulation may have a role in the uterine glandular epithelium. Heat shock protein 27 (Hsp27) was higher on Day 15 than on Day 3 (P<0.01) and Hsp27 was 1.4-fold higher in the ipsilateral compared to the contralateral uterine horn (P<0.01). Hsp27 may be secreted in response to potential stresses in the uterus or act as a molecular chaperone. On Day 7 there was no difference (P<0.05) in the pattern of proteins secreted between cows with low (2.7±0.10ng/ml) and high (4.8±0.13 ng/ml) concentrations of systemic progesterone on Day 7. The results of these studies have shown that dramatic changes occur in protein expression across the bovine oestrous cycle. Additionally, it emphasises the need for gene studies to be followed with protein studies as an adjunct or complementary tool. Proteins have a wide range of essential roles in the uterus and together these studies provide novel information on protein expression in the uterus of the cow

Alterations in hepatic miRNA expression during negative energy balance in postpartum dairy cattle

peer-reviewedBackground Negative energy balance (NEB), an altered metabolic state, occurs in early postpartum dairy cattle when energy demands to support lactation exceed energy intake. During NEB the liver undergoes oxidative stress and increased breakdown of fatty acids accompanied by changes in gene expression. It is now known that micro RNAs (miRNA) can have a role in mediating such alterations in gene expression through repression or degradation of target mRNAs. miRNA expression is known to be altered by metabolism and environmental factors and miRNAs are implicated in expression modulation of metabolism related genes. Results miRNA expression was profiled in the liver of moderate yielding dairy cattle under severe NEB (SNEB) and mild NEB (MNEB) using the Affymetrix Gene Chip miRNA_2.0 array with 679 probe sets for Bos-taurus miRNAs. Ten miRNAs were found to be differentially expressed using the ‘samr’ statistical package (delta = 0.6) at a q-value FDR of < 12%. Five miRNAs including miR-17-5p, miR-31, miR-140, miR-1281 and miR-2885 were validated using RT-qPCR, to be up-regulated under SNEB. Liver diseases associated with these miRNAs include non-alcoholic fatty liver (NAFLD) and hepatocellular carcinoma (HCC). miR-140 and miR-17-5p are known to show differential expression under oxidative stress. A total of 32 down-regulated putative target genes were also identified among 418 differentially expressed hepatic genes previously reported for the same animal model. Among these, GPR37 (G protein-coupled receptor 37), HEYL (hairy/enhancer-of-split related with YRPW motif-like), DNJA1, CD14 (Cluster of differentiation 14) and GNS (glucosamine (N-acetyl)-6-sulfatase) are known to be associated with hepatic metabolic disorders. In addition miR-140 and miR-2885 have binding sites on the most down-regulated of these genes, FADS2 (Fatty acid desaturase 2) which encodes an enzyme critical in lipid biosynthesis. Furthermore, HNF3-gamma (Hepatocyte nuclear factor 3-gamma), a hepatic transcription factor (TF) that is involved in IGF-1 expression regulation and maintenance of glucose homeostasis is a putative target of miR-31. Conclusions This study shows that SNEB affects liver miRNA expression and these miRNAs have putative targets in hepatic genes down-regulated under this condition. This study highlights the potential role of miRNAs in transcription regulation of hepatic gene expression during SNEB in dairy cattle. Background Negative energy balance (NEB), an altered metabolic state, occurs in early postpartum dairy cattle when energy demands to support lactation exceed energy intake. During NEB the liver undergoes oxidative stress and increased breakdown of fatty acids accompanied by changes in gene expression. It is now known that micro RNAs (miRNA) can have a role in mediating such alterations in gene expression through repression or degradation of target mRNAs. miRNA expression is known to be altered by metabolism and environmental factors and miRNAs are implicated in expression modulation of metabolism related genes. Results miRNA expression was profiled in the liver of moderate yielding dairy cattle under severe NEB (SNEB) and mild NEB (MNEB) using the Affymetrix Gene Chip miRNA_2.0 array with 679 probe sets for Bos-taurus miRNAs. Ten miRNAs were found to be differentially expressed using the ‘samr’ statistical package (delta = 0.6) at a q-value FDR of < 12%. Five miRNAs including miR-17-5p, miR-31, miR-140, miR-1281 and miR-2885 were validated using RT-qPCR, to be up-regulated under SNEB. Liver diseases associated with these miRNAs include non-alcoholic fatty liver (NAFLD) and hepatocellular carcinoma (HCC). miR-140 and miR-17-5p are known to show differential expression under oxidative stress. A total of 32 down-regulated putative target genes were also identified among 418 differentially expressed hepatic genes previously reported for the same animal model. Among these, GPR37 (G protein-coupled receptor 37), HEYL (hairy/enhancer-of-split related with YRPW motif-like), DNJA1, CD14 (Cluster of differentiation 14) and GNS (glucosamine (N-acetyl)-6-sulfatase) are known to be associated with hepatic metabolic disorders. In addition miR-140 and miR-2885 have binding sites on the most down-regulated of these genes, FADS2 (Fatty acid desaturase 2) which encodes an enzyme critical in lipid biosynthesis. Furthermore, HNF3-gamma (Hepatocyte nuclear factor 3-gamma), a hepatic transcription factor (TF) that is involved in IGF-1 expression regulation and maintenance of glucose homeostasis is a putative target of miR-31. Conclusions This study shows that SNEB affects liver miRNA expression and these miRNAs have putative targets in hepatic genes down-regulated under this condition. This study highlights the potential role of miRNAs in transcription regulation of hepatic gene expression during SNEB in dairy cattle

The miRNAome of the postpartum dairy cow liver in negative energy balance

peer-reviewedBackground: Negative energy balance (NEB) is an altered metabolic state in high yielding cows that occurs during the first few weeks postpartum when energy demands for lactation and maintenance exceed the energy supply from dietary intake. NEB can, in turn, lead to metabolic disorders and to reduced fertility. Alterations in the expression of more than 700 hepatic genes have previously been reported in a study of NEB in postpartum dairy cows. miRNAs (microRNA) are known to mediate many alterations in gene expression post transcriptionally. To study the hepatic miRNA content of postpartum dairy cows, including their overall abundance and differential expression, in mild NEB (MNEB) and severe NEB (SNEB), short read RNA sequencing was carried out. To identify putative targets of differentially expressed miRNAs among differentially expressed hepatic genes reported previously in dairy cows in SNEB computational target identification was employed. Results: Our results indicate that the dairy cow liver expresses 53 miRNAs at a lower threshold of 10 reads per million. Of these, 10 miRNAs accounted for greater than 95% of the miRNAome (miRNA content). Of the highly expressed miRNAs, miR-122 constitutes 75% followed by miR-192 and miR-3596. Five out of thirteen let-7 miRNA family members are also among the highly expressed miRNAs. miR-143, down-regulated in SNEB, was found to have 4 putative up-regulated gene targets associated with SNEB including LRP2 (low density lipoprotein receptor-related protein 2), involved in lipid metabolism and up-regulated in SNEB. Conclusions: This is the first liver miRNA-seq profiling study of moderate yielding dairy cows in the early postpartum period. Tissue specific miR-122 and liver enriched miR-192 are two of the most abundant miRNAs in the postpartum dairy cow liver. miR-143 is significantly down-regulated in SNEB and putative targets of miRNA-143 which are up-regulated in SNEB, include a gene involved in lipid metabolism.Teagasc Walsh Fellowship Programm

Biotechnology in Cattle Reproduction.

End of Project ReportTeagasc acknowledges support from the European Union 3rd and 4th Framework Programmes (CT-92-0163 and CT-95-0190)Over the next decade the Irish agri-food industry will have to compete in a rapidly changing world environment arising from increased competitiveness, decreased world market prices and increased consumer demands for higher quality, healthier and safer food. To become competitive in this environment the scale and efficiency of production at both farm and factory level will have to increase significantly and this must be achieved with due regard for the protection of the environment and the welfare of animals. New technologies will be needed to achieve this. Biotechnology will be central to the development of these new technologies. This project has been concerned with the identification and evaluation of biotechnology developments that have the potential to increase reproductive efficiency in cattle. This includes a range of technologies relating to the in vitro production, manipulation, cryopreservation and transfer of cattle embryos. The potential of other emerging technologies such as embryo and sperm sexing, cloning and biopharming or the production of commercially desirable proteins in cows milk are also addressed in this report.European Unoi

Viability of in vitro produced cattle embryos.

End of Project ReportEmbryo transfer is being increasingly used in the cattle industry. As well as direct embryo transfers, many embryo-based biotechnologies have the potential to improve cattle production efficiency through enhanced breeding strategies, by facilitating the introduction of desirable traits such as disease resistance and through the production of desirable medical or pharmaceutical products in the milk. These biotechnologies are, however, dependent on a supply of viable in vitro produced (IVP) embryos. While the in vitro fertilization rate is high (80%) in cattle, only about 30 transferable embryos, or blastocysts, are produced from every 100 fertilized oocytes. A major factor affecting the viability of IVP embryos is their failure, in a high proportion of cases, to undergo normal development to the blastocyst stage in the manner of in vivo embryos. The major problem relates to a failure of the cells of IVP embryos to form a compact cell mass when they are 5 - 6 days old. This ultimately leads to developmental problems and compromised viability. Cell compaction is recognized as a critical event in early embryo development and has been associated with marked changes in protein synthesis and phosphorylation in the embryos of some species. This report is the first, to our knowledge, to describe the rate and pattern of protein synthesis and phosphorylation before, during and after compaction in both in vivo and in IVP cattle embryos. The main results are summarised below.European Union 4th Framework Programmes (Contract CT-95-0032

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

peer-reviewedBackground 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.Teagasc Walsh Fellowship Programm

Transcriptome analysis of porcine M. semimembranosus divergent in intramuscular fat as a consequence of dietary protein restriction

peer-reviewedBackground: Intramuscular fat (IMF) content is positively correlated with aspects of pork palatability, including flavour, juiciness and overall acceptability. The ratio of energy to protein in the finishing diet of growing pigs can impact on IMF content with consequences for pork quality. The objective of this study was to compare gene expression profiles of Musculus semimembranosus (SM) of animals divergent for IMF as a consequence of protein dietary restriction in an isocaloric diet. The animal model was derived through the imposition of low or high protein diets during the finisher stage in Duroc gilts. RNA was extracted from post mortem SM tissue, processed and hybridised to Affymetrix porcine GeneChip® arrays. Results: IMF content of SM muscle was increased on the low protein diet (3.60 ± 0.38% versus 1.92 ± 0.35%). Backfat depth was also greater in animals on the low protein diet, and average daily gain and feed conversion ratio were lower, but muscle depth, protein content and moisture content were not affected. A total of 542 annotated genes were differentially expressed (DE) between animals on low and high protein diets, with 351 down-regulated and 191 up-regulated on the low protein diet. Transcript differences were validated for a subset of DE genes by qPCR. Alterations in functions related to cell cycle, muscle growth, extracellular matrix organisation, collagen development, lipogenesis and lipolysis, were observed. Expression of adipokines including LEP, TNFα and HIF1α were increased and the hypoxic stress response was induced. Many of the identified transcriptomic responses have also been observed in genetic and fetal programming models of differential IMF accumulation, indicating they may be robust biological indicators of IMF content. Conclusion: An extensive perturbation of overall energy metabolism in muscle occurs in response to protein restriction. A low protein diet can modulate IMF content of the SM by altering gene pathways involved in lipid biosynthesis and degradation; however this nutritional challenge negatively impacts protein synthesis pathways, with potential consequences for growth.Department of Agriculture, Food and the Marine, Ireland - Food Institutional Research Measur

Control of Ovulation Rate in Beef Cattle.

End of Project ReportUnder intensive production systems, the greatest potential for effecting increases in production and economic efficiency in the beef cow herd lies in the possibility of increasing the frequency of twin births. Embryo transfer is technically a successful method of inducing twin births in cattle. While an embryo transfer approach is too costly to allow commercial twinning, it has been used to show that ovulation rate and not uterine capacity is the limiting factor in increasing the reproductive rate of the cow. While ovulation of one or more viable oocytes is central to normal reproduction, knowledge of the control of ovulation and of folliculogenesis on which ovulation depends, is limited. In spite of the fact that many follicles are subjected to the same endogenous hormonal environment and theoretically should all be capable of ovulating, only a tiny proportion do. While gonadotrophic hormones play a central role in ovarian follicle development and ovulation, their action at the ovarian level seems to be controlled by intra-ovarian factors. This intra-ovarian control of ovulation is thought to be exerted partly by the hormone inhibin and partly by other, as yet, unidentified compounds in follicular fluid. This project focused on identification and isolation of ovarian compounds involved in the control of ovulation rate, followed by immunisation against these compounds in order to study the effect on ovulation and the twin calving rates. The main results are summarised here and detailed results have been published in the papers listed at the end of this report

Effects of negative energy balance on liver gene and protein expression during the early postpartum period and its impacts on dairy cow fertility

End of project reportNegative energy balance (NEB) is a severe metabolic affecting high yielding dairy cows early post partum with both concurrent and latent negative effects on cow fertility as well as on milk production and cow health. The seasonal nature of Irish dairy production necessitates high cow fertility and a compact spring calving pattern in order to maximise grass utilisation. Poor dairy cow reproductive performance currently costs the Irish cattle industry in excess of €400 million annually. High milk yields have been associated with lower reproductive efficiency, and it has been suggested that this effect is probably mediated through its effects on the energy balance of the cow during lactation. The modern high genetic merit dairy cow prioritises nutrient supply towards milk production in early lactation and this demand takes precedence over the provision of optimal conditions for reproduction. In this study we used the bovine Affymetrix 23,000 gene microarray, which contains the most comprehensive set of bovine genes to be assembled and provides a means of investigating the modifying influences of energy balance on liver gene expression. Cows in severe negative energy balance (SNEB) in early lactation showed altered hepatic gene expression in metabolic processes as well as a down regulation of the insulin-like growth factor (IGF) system, where insulin like growth factor-1 (IGF-1), growth hormone receptor variant 1A (GHR1A) and insulin-like growth factor binding protein-acid labile subunit (IGFBP-ALS) were down regulated compared to the cows in the moderate negative energy balance MNEB group, consistent with a five-fold reduction in systemic concentrations of IGF1 in the SNEB group.Cows in SNEB showed elevated expression of key genes involved in the inflammatory response such as interleukin-8 (IL-8). There was a down regulation of genes involved in cellular growth in SNEB cows and moreover a negative regulator of cellular proliferation (HGFIN) was up regulated in SNEB cows, which is likely to compromise adaptation and recovery from NEB. The puma method of analysis revealed that 417 genes were differentially regulated by EB (P<0.05), of these genes 190 were up-regulated while 227 were down-regulated, with 405 genes having known biological functions. From Ingenuity Pathway Analysis (IPA), lipid catabolism was found to be the process most affected by differences in EB status