Analysis of genetic variation contributing to measured speed in Thoroughbreds identifies genomic regions involved in the transcriptional response to exercise.

Despite strong selection for athletic traits in Thoroughbred horses, there is marked variation in speed and aptitude for racing performance within the breed. Using global positioning system monitoring during exercise training, we measured speed variables and temporal changes in speed with age to derive phenotypes for GWAS. The aim of the study was to test the hypothesis that genetic variation contributes to variation in end-point physiological traits, in this case galloping speed measured during field exercise tests. Standardisation of field-measured phenotypes was attempted by assessing horses exercised on the same gallop track and managed under similar conditions by a single trainer. PCA of six key speed indices captured 73.9% of the variation with principal component 1 (PC1). Verifying the utility of the phenotype, we observed that PC1 (median) in 2-year-old horses was significantly different among elite, non-elite and unraced horses (P < 0.001) and the temporal change with age in PC1 varied among horses with different myostatin (MSTN) g.66493737C>T SNP genotypes. A GWAS for PC1 in 2-year-old horses (n = 122) identified four SNPs reaching the suggestive threshold for association (P < 4.80 × 10-5 ), defining a 1.09 Mb candidate region on ECA8 containing the myosin XVIIIB (MYO18B) gene. In a GWAS for temporal change in PC1 with age (n = 168), five SNPs reached the suggestive threshold for association and defined candidate regions on ECA2 and ECA11. Both regions contained genes that are significantly differentially expressed in equine skeletal muscle in response to acute exercise and training stimuli, including MYO18A. As MYO18A plays a regulatory role in the skeletal muscle response to exercise, the identified genomic variation proximal to the myosin family genes may be important for the regulation of the response to exercise and training.

Genetic contributions to precocity traits in racing Thoroughbreds.

Adaptation to early training and racing (i.e. precocity), which is highly variable in racing Thoroughbreds, has implications for the selection and training of horses. We hypothesised that precocity in Thoroughbred racehorses is heritable. Age at first sprint training session (work day), age at first race and age at best race were used as phenotypes to quantify precocity. Using high-density SNP array data, additive SNP heritability (hSNP2) was estimated to be 0.17, 0.14 and 0.17 for the three traits respectively. In genome-wide association studies (GWAS) for age at first race and age at best race, a 1.98-Mb region on equine chromosome 18 (ECA18) was identified. The most significant association was with the myostatin (MSTN) g.66493737C>T SNP (P = 5.46 × 10-12 and P = 1.89 × 10-14 respectively). In addition, two SNPs on ECA1 (g.37770220G>A and g.37770305T>C) within the first intron of the serotonin receptor gene HTR7 were significantly associated with age at first race and age at best race. Although no significant associations were identified for age at first work day, the MSTN:g.66493737C>T SNP was among the top 20 SNPs in the GWAS (P = 3.98 × 10-5 ). Here we have identified variants with potential roles in early adaptation to training. Although there was an overlap in genes associated with precocity and distance aptitude (i.e. MSTN), the HTR7 variants were more strongly associated with precocity than with distance. Because HTR7 is closely related to the HTR1A gene, previously implicated in tractability in young Thoroughbreds, this suggests that behavioural traits may influence precocity.

Fertility and genomics: comparison of gene expression in contrasting reproductive tissues of female cattle.

To compare gene expression among bovine tissues, large bovine RNA-seq datasets were used, comprising 280 samples from 10 different bovine tissues (uterine endometrium, granulosa cells, theca cells, cervix, embryos, leucocytes, liver, hypothalamus, pituitary, muscle) and generating 260 Gbases of data. Twin approaches were used: an information-theoretic analysis of the existing annotated transcriptome to identify the most tissue-specific genes and a de-novo transcriptome annotation to evaluate general features of the transcription landscape. Expression was detected for 97% of the Ensembl transcriptome with at least one read in one sample and between 28% and 66% at a level of 10 tags per million (TPM) or greater in individual tissues. Over 95% of genes exhibited some level of tissue-specific gene expression. This was mostly due to different levels of expression in different tissues rather than exclusive expression in a single tissue. Less than 1% of annotated genes exhibited a highly restricted tissue-specific expression profile and approximately 2% exhibited classic housekeeping profiles. In conclusion, it is the combined effects of the variable expression of large numbers of genes (73%-93% of the genome) and the specific expression of a small number of genes (<1% of the transcriptome) that contribute to determining the outcome of the function of individual tissues.

N-glycan profiling of bovine follicular fluid at key dominant follicle developmental stages.

Follicular fluid (FF), an important microenvironment for the development of oocytes, contains many proteins that are glycosylated with N-linked glycans. This study aimed i) to present an initial analysis of the N-linked glycan profile of bovine FF using hydrophilic interaction liquid chromatography, anion exchange chromatography, high performance liquid chromatography (HPLC)-based separations and subsequent liquid chromatography-mass spectrometry/mass spectrometry analysis; ii) to determine differences in the N-glycan profile between FF from dominant and subordinate follicles from dairy heifers and lactating dairy cows and iii) to identify alterations in the N-glycan profile of FF during preovulatory follicle development using newly selected, differentiated (preovulatory) and luteinised dominant follicles from dairy heifers and lactating cows. We found that the majority of glycans on bovine FF are based on biantennary hypersialylated structures, where the glycans are sialylated on both the galactose and N-acetylglucosamine terminal sugars. A comparison of FF N-glycans from cows and heifers indicated higher levels of nonsialylated glycans with a lower proportion of sialylated glycans in cows than in heifers. Overall, as the follicle develops from Selection, Differentiation and Luteinisation in both cows and heifers, there is an overall decrease in sialylated structures on FF N-glycans.

Expression of microRNAs and their target genes and pathways associated with ovarian follicle development in cattle.

Development of ovarian follicles is controlled at the molecular level by several gene products whose precise expression leads to regression or ovulation of follicles. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression through sequence-specific base pairing with target messenger RNAs (mRNAs) causing translation repression or mRNA degradation. The aim of this study was to identify miRNAs expressed in theca and/or granulosa layers and their putative target genes/pathways that are involved in bovine ovarian follicle development. By using miRCURY microarray (Exiqon) we identified 14 and 49 differentially expressed miRNAs (P < 0.01) between dominant and subordinate follicles in theca and granulosa cells, respectively. The expression levels of four selected miRNAs were confirmed by qRT-PCR. To identify target prediction and pathways of differentially expressed miRNAs we used Union of Genes option in DIANA miRPath v.2.0 software. The predicted targets for these miRNAs were enriched for pathways involving oocyte meiosis, Wnt, TGF-beta, ErbB, insulin, P13K-Akt, and MAPK signaling pathways. This study identified differentially expressed miRNAs in the theca and granulosa cells of dominant and subordinate follicles and implicates them in having important roles in regulating known molecular pathways that determine the fate of ovarian follicle development.

Temporal regulation of fibroblast growth factors and their receptors in the endometrium and conceptus during the pre-implantation period of pregnancy in cattle.

We hypothesised that the expression pattern of members of the fibroblast growth factor (FGF) family would be altered in the endometrium as the oestrous cycle/early pregnancy progressed associated with changes in the expression pattern of their receptors in the developing embryo/conceptus. Expression of FGF1 and FGF10 transcript variants 1 and 2 increased significantly as the oestrous cycle/early pregnancy progressed. Neither progesterone (P4) supplementation nor pregnancy status significantly affected the expression of any of the FGF ligands studied. However, there was a significant interaction between day, pregnancy and P4 status on FGF2 expression (P<0.05) and a significant interaction between P4 status and day on FGF10_tv2 expression. FGF10 protein was localised in the luminal and glandular epithelium as well as the stroma but was not detected in the myometrium. By RNA sequencing, the expression of FGF ligands in the developing embryo/conceptus was found to be minimal. The expression of FGF receptor 1 (FGFR1), FGFR2, FGFR3, FGFR4, FGFRL1 and FRS3 was significantly affected by the stage of conceptus development. Interestingly, the expression of FGFR1 and FGFR4 was higher during early embryo development (days 7-13, P<0.05) but decreased on day 16 (P<0.05) while FGFR2 (P<0.001) expression was similar from day 7 through to day 13, with a significant increase by day 16 (P<0.05) that was maintained until day 19 (P>0.05). In conclusion, these data demonstrate that FGF ligands are primarily expressed by the endometrium and their modulation throughout the luteal phase of the oestrous cycle/early pregnancy are associated with alterations in the expression of their receptors in the embryo/conceptus.

Physiological status alters immunological regulation of bovine follicle differentiation in dairy cattle.

Lactation in dairy cattle is associated with a multitude of endocrine, metabolic and immunological changes that not only influence animal health, but also affect fertility, and in particular ovarian function. We have previously generated a global transcriptomic profile of bovine follicular tissue using RNA sequencing. This study aimed to: identify key immune-related transcriptional changes that occur during follicle differentiation and luteinisation using ingenuity pathway analysis (IPA); and determine if a compromised model of stress (i.e. lactation) influences the temporal expression of these genes. Ovarian follicular tissue from Holstein-Friesian non-lactating heifers (n=17) and lactating cows (n=16) was compared at three stages of preovulatory follicle development: (A) the newly selected dominant follicle in the luteal phase (Selection); (B) the follicular phase before the LH surge (Differentiation), and (C) the preovulatory phase after the LH surge (Luteinisation). IPA revealed an over-representation of immune-related pathways in theca compared with granulosa cells during differentiation; these were related to leucocyte extravasation and chemotaxis. Conversely, luteinisation was characterised by over-representation of immune-related pathways in granulosa compared with theca cells; these were related to inflammation and innate immune response. Notably, comparison of follicles from heifers and lactating cows revealed a large number of differentially expressed genes associated with immune cell subpopulations and chemotaxis. In conclusion, identification of immune-related canonical pathways during follicle development supports the hypothesis that ovulation is an inflammatory event. This process is influenced by the physiological status of lactation and likely contributes to compromised peri-ovulatory follicle function by impairing the inflammatory process of ovulation.

Evidence for an early endometrial response to pregnancy in cattle: both dependent upon and independent of interferon tau.

The aims of this study were to 1) identify the earliest transcriptional response of the bovine endometrium to the presence of the conceptus (using RNAseq), 2) investigate if these genes are regulated by interferon tau (IFNT) in vivo, and 3) determine if they are predictive of the pregnancy status of postpartum dairy cows. RNAseq identified 459 differentially expressed genes (DEGs) between pregnant and cyclic endometria on day 16. Quantitative real-time PCR analysis of selected genes revealed PARP12, ZNFX1, HERC6, IFI16, RNF213, and DDX58 expression increased in pregnant compared with cyclic endometria on day 16 and were directly upregulated by intrauterine infusion of IFNT in vivo for 2 h (P < 0.05). On day 13 following estrous endometrial expression of nine genes increased [ARHGAP1, MGC127874, LIMS2, TBC1D1, FBXL7, C25H16orf71, LOC507810, ZSWIM4, and one novel gene (ENSBTAT00000050193)] and seven genes decreased (SERBP1, SRGAP2, AL7A1, TBK1, F2RL2, MGC128929, and WBSCR17; P < 0.05) in pregnant compared with cyclic heifers. Of these DEGs, significant differences in expression between pregnant and cyclic endometria were maintained on day 16 for F2RL2, LIMS2, LOC507810, MGC127874, TBC1D1, WBSCR17, and ZSWIM4 (P < 0.05) both their expression was not directly regulated by IFNT in vivo. Analysis of the expression of selected interferon-stimulated genes in blood samples from postpartum dairy cows revealed a significant increase (P < 0.05) in expression of ZXFX1, PARP12, SAMD9, and HERC6 on day 18 following artificial insemination in cows subsequently confirmed pregnant compared with cyclic controls. In conclusion, RNAseq identified a number of novel pregnancy-associated genes in the endometrium of cattle during early pregnancy that are not regulated by IFNT in vivo. In addition, a number of genes that are directly regulated by short term exposure to IFNT in vivo are differentially expressed on day 18 following estrus detection in the blood of postpartum dairy cows depending on their pregnancy status.

Effect of the metabolic environment at key stages of follicle development in cattle: focus on steroid biosynthesis.

Cellular mechanisms that contribute to low estradiol concentrations produced by the preovulatory ovarian follicle in cattle with a compromised metabolic status are largely unknown. To gain insight into the main metabolic mechanisms affecting preovulatory follicle function, two different animal models were used. Experiment 1 compared Holstein-Friesian nonlactating heifers (n = 17) and lactating cows (n = 16) at three stages of preovulatory follicle development: 1) newly selected dominant follicle in the luteal phase (Selection), 2) follicular phase before the LH surge (Differentiation), and 3) preovulatory phase after the LH surge (Luteinization). Experiment 2 compared newly selected dominant follicles in the luteal phase in beef heifers fed a diet of 1.2 times maintenance (M, n = 8) or 0.4 M (n = 11). Lactating cows and 0.4 M beef heifers had higher concentrations of ß-hydroxybutyrate, and lower concentrations of glucose, insulin, and IGF-I compared with dairy heifers and 1.2 M beef heifers, respectively. In lactating cows this altered metabolic environment was associated with reduced dominant follicle estradiol and progesterone synthesis during Differentiation and Luteinization, respectively, and in 0.4 M beef heifers with reduced dominant follicle estradiol synthesis. Using a combination of RNA sequencing, Ingenuity Pathway Analysis, and qRT-PCR validation, we identified several important molecular markers involved in steroid biosynthesis, such as the expression of steroidogenic acute regulatory protein (STAR) within developing dominant follicles, to be downregulated by the catabolic state. Based on this, we propose that the adverse metabolic environment caused by lactation or nutritional restriction decreases preovulatory follicle function mainly by affecting cholesterol transport into the mitochondria to initiate steroidogenesis.

Conceptus-induced changes in the endometrial transcriptome: how soon does the cow know she is pregnant?

This study sought to determine the earliest response of the bovine uterine endometrium to the presence of the conceptus at key developmental stages of early pregnancy. There were no detectable differences in gene expression in endometria from pregnant and cyclic heifers on Days 5, 7, and 13 postestrus, but the expression of 764 genes was altered due to the presence of the conceptus at maternal recognition of pregnancy (Day 16). Of these 514 genes, MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD9, EIF4E, and IFIT2 increased to the greatest extent in pregnant endometria (>8-fold log2 fold change increase). The expression of OXTR, Bt.643 (unofficial symbol), and KCNMA1 was reduced the most, but short-term treatment with recombinant ovine interferon tau (IFNT) in vitro or in vivo did not alter their expression. In vivo intrauterine infusion of IFNT induced the expression of EIF4E, IFIT2, IFI44, ISG20, MX2, RSAD2, SAMD9, and USP18. These results revealed for the first time that changes that occur in the endometrial transcriptome are independent of the presence of a conceptus until pregnancy recognition. The differentially expressed genes (including MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD, and EIF4E) are a consequence of IFNT production by the conceptus. The identified genes represent known and novel early markers of conceptus development and/or return to cyclicity and may be useful to identify the earliest stage at which the endometrial response to the conceptus is detectable.

Clustal W and Clustal X version 2.0.

SUMMARY: The Clustal W and Clustal X multiple sequence alignment programs have been completely rewritten in C++. This will facilitate the further development of the alignment algorithms in the future and has allowed proper porting of the programs to the latest versions of Linux, Macintosh and Windows operating systems. AVAILABILITY: The programs can be run on-line from the EBI web server: http://www.ebi.ac.uk/tools/clustalw2. The source code and executables for Windows, Linux and Macintosh computers are available from the EBI ftp site ftp://ftp.ebi.ac.uk/pub/software/clustalw2/

Prescribers prefer people: The sources of information used by doctors for prescribing suggest that the medium is more important than the message.

AIMS: The sources of prescribing information are legion but there is little knowledge about which are actually used in practice by doctors when prescribing. The aims of this study were to determine the sources of prescribing information considered important by doctors, establish which were used in practice, and investigate if hospital and primary care physicians differed in their use of the sources. METHODS: Two hundred general practitioners (GPs) and 230 hospital doctors were asked to rate information sources in terms of their importance for prescribing 'old' and 'new' drugs, and then to name the source from which information about the last new drug prescribed was actually derived. RESULTS: Among 108 GPs, the Drugs and Therapeutics Bulletin and medical journal articles were most frequently rated as important for information on both old and new drugs while pharmaceutical representatives and hospital/consultant recommendations were more important for information on new drugs, as opposed to old. In practice, information on the last new drug prescribed was derived from pharmaceutical representatives in 42% of cases and hospital/consultant recommendations in 36%, with other sources used infrequently. Among 118 hospital doctors, the British National Formulary (BNF) and senior colleagues were of greatest theoretical importance. In practice, information on the last new drug prescribed was derived from a broad range of sources: colleagues, 29%; pharmaceutical representatives, 18%; hospital clinical meetings, 15%; journal articles, 13%; lectures, 10%. GPs and hospital doctors differed significantly in their use of pharmaceutical representatives (42% vs 18%) and colleagues (7% vs 29%) as sources of prescribing information (P < 0.0001 for both). CONCLUSIONS: The sources most frequently rated important in theory were not those most used in practice, especially among GPs. Both groups under-estimated the importance of pharmaceutical representatives. Most importantly, the sources of greatest practical importance were those involving the transfer of information through the medium of personal contact.

Current problems with non-specific COX inhibitors.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used and effective treatments for pain and inflammation. They have a substantial toxicity profile with side effects mainly affecting the gastrointestinal tract, heart and kidneys. Although they comprise a chemically diverse group of drugs, NSAIDs are unified by a common mode of action the ability to inhibit the enzyme cyclo-oxygenase (COX). This also accounts for much of their toxicity. The enzyme exists in at least 2 isoforms. COX-1 generates prostaglandins with physiological functions, COX-2 is induced by inflammation and its physiologic functions are unclear at present. Conventional NSAIDs, like diclofenac, ibuprofen, and naproxen, are non-selective COX inhibitors, blocking the production of both physiologic and inflammatory prostaglandins. In this chapter, we describe the main predictable gastrointestinal, cardiac and renal toxicities that can be explained by such blockade and review the supporting clinical and epidemiological evidence. In the gastrointestinal tract, the side effects associated with conventional NSAIDs are both local and systemic, and include ulceration, bleeding, perforation, and obstruction. The upper gastrointestinal tract is more commonly affected than the lower. The cardiac and renal side effects are most likely to occur in patients with existing heart or kidney disease, where prostaglandins play an essential role in maintaining the vasoconstrictor/dilator balance necessary for homeostasis. The patients at highest risk of toxicity are the elderly, those with a prior history of ulceration or bleeding, and those with a history of cardiac disease. Among such patients, the decision to prescribe NSAIDs requires careful consideration of the potential benefits and harms.

COX-2 inhibitors. What is their place?

BACKGROUND: Nonsteroid anti-inflammatory drugs (NSAIDs) are widely used in Australia for the treatment of arthritis and the relief of pain. However, side effects, particularly those affecting the gastrointestinal tract, are of considerable cost to the individual and the community. OBJECTIVE: To examine the role of selective cyclo-oxygenase-2 (COX-2) inhibitors in the anti-inflammatory armamentarium. DISCUSSION: COX-2 inhibitors are of similar clinical efficacy to nonselective NSAIDs. They cause significantly fewer endoscopic ulcers, but these lesions are usually clinically asymptomatic. Dyspepsia rates are 1-2% lower. The frequency of serious gastrointestinal complications (symptomatic ulceration, bleeding, perforation) appears to be reduced by 0.5-1.0%. As these problems occur infrequently, routine use of COX-2 inhibitors would not be cost effective. Although no data are yet available, patients at high risk of NSAID induced gastrointestinal complications may be the group for whom COX-2 inhibitors will provide most clinical benefit.

Growth in use of statins after trials is not targeted to most appropriate patients.

OBJECTIVE: To determine whether growth in the use of lipid-lowering drugs after publication of studies in the primary and secondary prevention of coronary heart disease is in the population in which benefit was established, particularly middle-aged men. METHODS: We performed a series of pharmacoepidemiologic surveys of community prescribing in Ireland over 4 years. RESULTS: Nationally, the use of lipid-lowering drugs (92% statins) increased approximately fourfold from 1994 to 1998. In the Eastern Health Board region, the number of monthly recipients increased from 447 in April 1994 to 3530 in March 1998. Although use increased steadily after publication of Scandinavian Simvastatin Survival Study (4S) and West of Scotland Coronary Prevention Study (WOSCOPS) in 1994 and 1995, respectively, this occurred to a greater extent in women. However, after the Cholesterol and Recurrent Events (CARE) study in 1996 and subsequent recommendations that targeted statin use, particularly in men from 35 to 69 years old, there was a relatively greater increase in that population but, at 2.3%, it was well short of the target population of 5.8%. More women than men older than 65 years are receiving statins. The 10-mg dosage (a fourth or half that used in studies) is the most frequently dispensed. CONCLUSION: The use of statins, although rising rapidly, is below targets and was initially not directed at the population likely to benefit most or in the recommended dosage. Consequently, the benefits projected from clinical trials may not be seen in clinical practice.

Anti-depressants in primary care: analysis of treatment discontinuations.

It is well known that adherence to anti-depressant therapy is often poor, but the literature describes little in the way of systematic analyses to determine co-relation between treatment discontinuation and possible contributing factors. We used a community dispensing database to review anti-depressant prescribing patterns and continuity of therapy over a period of 10 months among a population of community-based general practice patients. Some 109,228 anti-depressant prescriptions were dispensed to 24,073 patients, of whom 37.5% collected a single prescription only. Tricyclic anti-depressant prescribing declined significantly during the observation period (from 70% of prescriptions in month 1 to 66% in month 10) while that of selective serotonin reuptake inhibitors (SSRIs) increased (23% in month 1, 28% in month 10) ( p < 0.0001). Some 27% of those on tricyclics were prescribed <50% of the defined daily dose (DDD) compared with 2% of those on SSRIs. Among patients new to therapy who collected >1 prescription, adherence was poor and declined over time. The factors that influenced the extent to which patients failed to adhere to therapy included dosage level (% DDD) and age ( p <0.0001 for both), but not drug class or sex. The findings suggest that low dosage is a contributory factor in treatment discontinuation, and that contrary to common perception, SSRIs are not necessarily associated with better adherence to therapy than tricyclics. Copyright (c) 2000 John Wiley & Sons, Ltd.

The effects of information framing on the practices of physicians.

OBJECTIVE: The presentation format of clinical trial results, or the "frame," may influence perceptions about the worth of a treatment. The extent and consistency of that influence are unclear. We undertook a systematic review of the published literature on the effects of information framing on the practices of physicians. DESIGN: Relevant articles were retrieved using bibliographic and electronic searches. Information was extracted from each in relation to study design, frame type, parameter assessed, assessment scale, clinical setting, intervention, results, and factors modifying the frame effect. MAIN RESULTS: Twelve articles reported randomized trials investigating the effect of framing on doctors' opinions or intended practices. Methodological shortcomings were numerous. Seven papers investigated the effect of presenting clinical trial results in terms of relative risk reduction, or absolute risk reductions or the number needing to be treated; gain/loss (positive/negative) terms were used in four papers; verbal/numeric terms in one. In simple clinical scenarios, results expressed in relative risk reduction or gain terms were viewed most positively by doctors. Factors that reduced the impact of framing included the risk of causing harm, preexisting prejudices about treatments, the type of decision, the therapeutic yield, clinical experience, and costs. No study investigated the effect of framing on actual clinical practice. CONCLUSIONS: While a framing effect may exist, particularly when results are presented in terms of proportional or absolute measures of gain or loss, it appears highly susceptible to modification, and even neutralization, by other factors that influence doctors' decision making. Its effects on actual clinical practice are unknown.