Bovine pre-transfer endometrium and embryo transcriptome fingerprints as predictors of pregnancy success after embryo transfer

Aberrant endometrial and embryonic gene expression is one of the causes of pregnancy failure in cattle. However, selecting cows with adequate endometrial receptivity and embryos of higher developmental competence based on the gene expression pattern has been a greater challenge. To investigate whether the pre-transfer endometrial and embryonic gene expression pattern has a direct relation with upcoming pregnancy success, a global endometrial and embryonic transcriptome analysis was carried out in endometrial and embryo biopsy samples using GeneChip® Bovine Genome Array and preimplantation specific cDNA array, respectively. For this, endometrium biopsies were taken at days 7 and 14 of the estrous cycle in Simmental heifers during the pre-transfer period. In the next cycle, in vivo produced day 7 blastocysts were transferred to the recipients at day 7 of the estrous cycle after taking 30-40% parts of the blastocyst as a biopsy for transcriptome analysis. After pregnancy diagnosis, the heifers were classified as calf delivery (receptive endometrium) and no pregnancy (non-receptive endometrium) groups. Subsequently, the endometrial biopsies (taken at days 7 and 14 of the estrous cycle) and the embryo biopsies were categorized as calf delivery or no pregnancy groups. The results revealed 1126 genes were differentially expressed between receptive and non-receptive endometrium at day 7 of the estrous cycle. These differences were accompanied by qualitative and quantitative alteration of major biological process and molecular pathways including cellular localization, post-transcriptional modification, signal transduction, apoptosis, cell cycle and immune response. However, only 14 genes were differentially expressed between receptive and non-receptive endometrium at day 14 of the estrous cycle. Furthermore, the transcriptome dynamics of receptive and non-receptive endometrium between day 7 and 14 of the estrous cycle revealed 1867 and 254 differentially expressed genes, respectively. The higher number of differentially expressed genes and functional categories between day 7 and 14 of the estrous cycle in receptive compared to non-receptive endometrium revealed the transcriptome plasticity of receptive endometrium. In addition, the gene expression profile in embryos biopsies resulted in calf delivery and those resulted in no pregnancy revealed 70 genes to be differentially expressed between the two embryo groups. Among those, 32 genes including SPAG17, PF6, UBE2D3P, DFNB31, AMD1, DTNBP1 and ARL8B were elevated in calf delivery groups and 38 genes including SGK1, GBF1, KRT8, DTX2, RNF34, ARL8B, RYBP and EEF1 were elevated in no pregnancy embryo groups. Therefore, the present study highlights the potential of pre-transfer endometrial and embryonic gene expression patterns as predictors of pregnancy success in cattle.Genexprssionsprofile von vor dem Transfer biopsierten bovinen Endometrien und Embryonen als Prädikator für den Trächtigkeitserfolg beim Rind Abweichende Genexpressionen sowohl des Embryos als auch des Endometriums sind ein Grund für verminderte Trächtigkeitsraten nach dem Embryotransfer beim Rind. Die Selektion der Embryonen einerseits und der Empfängertiere an Hand des Endometriums andererseits basierend auf den Genexpressionsmustern, stellt eine große Herausforderung dar. Um herauszufinden, ob Korrelationen zwischen der Genexpression des Endometriums vor dem Transfer und der des Embryos auf der einen Seite und einer erfolgreichen Trächtigkeit auf der anderen Seite, vorliegen, wurde eine globale Transkriptionsanalyse der Biopsien des Endometriums und des Embryos mittels des GeneChip® Bovine Genome Array und einem präimplantations spezifischen cDNA Arrays durchgeführt. Die Biopsieproben des Endometriums wurden an Tag 7 und Tag 14 des Vorzykluses der Versuchstiere (Simmental Färsen) genommen. Des Weitern wurden in vivo Blastozysten am Tag 7 gespült, eine Biopsieprobe genommen (ca. 30-40%) und die Embryonen auf die Empfängertiere übertragen. Diese Biopsieproben wurden nach der Trächtigkeitsuntersuchung den Gruppen der trächtigen Färsen (rezeptives Endometrium) und der nicht-trächtigen Färsen (nicht-rezeptives Endometrium) zugeordnet. Insgesamt wurden 1126 unterschiedlich exprimierte Gene zwischen den rezeptiven und nichtrezeptiven Endometrien an Tag 7 detektiert. Des Weiteren zeigten sich qualitative und quantitative Veränderungen in bedeutenden biologischen Prozessen und molekularen Pathways, wie in der zellulären Anordnung, posttranskriptionalen Modifikation, Signaltransduktion, Apoptose, im Zellzyklus und in der Imunantwort. Konträr zu diesen Ergebnissen waren an Tag 14 nur 14 Gene zwischen den Endometrien unterschiedlich reguliert. Im Bereich der Transkriptionsdynamik waren zwischen den Gruppen an Tag 7 1867 Gene und am Tag 14 254 Gene unterschiedlich exprimiert. Diese relativ große Anzahl unterschiedlich regulierter Gene zeigt die Transkriptomplastizität des rezeptiven Endometriums. Zwischen den Biopsieproben der Embryonen, die zu einer Trächtigkeit führten und denen die keine Trächtigkeit induzierten zeigten 70 Gene eine unterschiedliche Expression. In der ersten Gruppe wurden 32 Gene, darunter SPAG17, PF6, UBE2D3P, DFNB31, AMD1, DTNBP1 und ARL8B expremiert und in der zweiten Gruppe 38 Gene darunter SGK1, GBF1, KRT8, DTX2, RNF34, ARL8B, RYBP und EEF1. Die vorliegende Arbeit zeigt demzufolge, dass das Genexpressionsprofil des Endometriums und der Embryonen vor dem Transfer als Vorhersage für den Trächtigkeitserfolg dienen kann

Machine-learning methods applied to integrated transcriptomic data from bovine blastocysts and elongating conceptuses to identify genes predictive of embryonic competence

Early pregnancy loss markedly impacts reproductive efficiency in cattle. The objectives were to model a biologically relevant gene signature predicting embryonic competence for survival after integrating transcriptomic data from blastocysts and elongating conceptuses with different developmental capacities and to validate the potential biomarkers with independent embryonic data sets through the application of machine-learning algorithms. First, two data sets from in vivo-produced blastocysts competent or not to sustain a pregnancy were integrated with a data set from long and short day-15 conceptuses. A statistical contrast determined differentially expressed genes (DEG) increasing in expression from a competent blastocyst to a long conceptus and vice versa; these were enriched for KEGG pathways related to glycolysis/gluconeogenesis and RNA processing, respectively. Next, the most discriminative DEG between blastocysts that resulted or did not in pregnancy were selected by linear discriminant analysis. These eight putative biomarker genes were validated by modeling their expression in competent or noncompetent blastocysts through Bayesian logistic regression or neural networks and predicting embryo developmental fate in four external data sets consisting of in vitro-produced blastocysts (i) competent or not, or (ii) exposed or not to detrimental conditions during culture, and elongated conceptuses (iii) of different length, or (iv) developed in the uteri of high- or subfertile heifers. Predictions for each data set were more than 85% accurate, suggesting that these genes play a key role in embryo development and pregnancy establishment. In conclusion, this study integrated transcriptomic data from seven independent experiments to identify a small set of genes capable of predicting embryonic competence for survival

Endometrial DNA methylation signatures during the time of breeding in relation to the pregnancy outcome in postpartum dairy cows fed a control diet or supplemented with rumen-protected methionine

Post calving metabolic stress reduces the fertility of high producing dairy cows possibly by altering the expression of genes in the maternal environment via epigenetic modifications. Therefore, this study was conducted to identify endometrial DNA methylation marks that can be associated with pregnancy outcomes in postpartum cows at the time of breeding. For this, twelve days post-calving, cows were either offered a control diet or supplemented daily with rumen-protected methionine. Cows showing heat 50–64 days postpartum were artificially inseminated. Endometrial cytobrush samples were collected 4–8 h after artificial insemination and classified based on the pregnancy out comes as those derived from cows that resulted in pregnancy or resulted in no pregnancy. The DNAs isolated from endometrial samples were then subject to reduced representative bisulfite sequencing for DNA methylation analysis. Results showed that in the control diet group, 1,958 differentially methylated CpG sites (DMCGs) were identified between cows that resulted in pregnancy and those that resulted in no pregnancy of which 890 DMCGs were located on chr 27: 6217254–6225600 bp. A total of 537 DMCGs were overlapped with 313 annotated genes that were involved in various pathways including signal transduction, signalling by GPCR, aldosterone synthesis and secretion. Likewise, in methionine supplemented group, 3,430 CpG sites were differentially methylated between the two cow groups of which 18.7% were located on Chr27: 6217254–6225600 bp. A total of 1,781 DMCGS were overlapped with 890 genes which involved in developmental and signalling related pathways including WNT-signalling, focal adhesion and ECM receptor interaction. Interestingly, 149 genes involved in signal transduction, axon guidance and non-integrin membrane-ECM interactions were differentially methylated between the two cow groups irrespective of their feeding regime, while 453 genes involved in axon guidance, notch signalling and collagen formation were differentially methylated between cows that received rumen protected methionine and control diet irrespective of their fertility status. Overall, this study indicated that postpartum cows that could potentially become pregnant could be distinguishable based on their endometrial DNA methylation patterns at the time of breeding

Morpho-biometric characterization of indigenous chicken ecotypes in north-western Ethiopia.

Morphological characterization of Animal Genetic Resources is the first step to documenting diversity and designing breed specific breeding programs. The current study characterized the morpho-biometric variation of indigenous chicken ecotypes prevailing in northwestern Ethiopia. A multi-stage purposive, stratified, and random sampling method was employed to select the study areas and chickens. A total of 1200 adult chickens were sampled and characterized for 12 qualitative and 11 quantitative traits. Univariate and multivariate data analysis methods were employed to analyze the data using SAS and R statistical software. Red plumage colour (33.2%), white and red earlobe colour (73.8%) and yellow shank colour (57.0%) were the most predominant colour trait categories. Sex, agro-ecology, location, and the interaction of sex and location had a highly significant (p<0.001) effect on all body measurements. Shank traits were found to have the highest discriminating power in both sexes. The overall classification rates for the female and male sample populations were 57.47% and 69.97%, respectively. The squared Mahalanobis distances between sites were significant (p<0.001) for both sexes. The longest distance was obtained between North Achefer and Banja (19.25) and between North Achefer and Dembecha (16.80) in female and male chickens, respectively. In female chickens, canonical variates 1 (CAN 1) and 2 (CAN 2) explained 82% of total variation and distinctly separated the sample populations of North Achefer and Jawi from others. In male chickens, 90% of the total variance is explained by CAN1, CAN2, and CAN3, which distinctly separate the sample populations of the North Achefer, Sinan, and Jawi, among others. Using cluster analysis, the indigenous chickens found in the study area could be classified into four ecotypes: ecotype 1 (Banja, Dembecha, and Aneded), ecotype 2 (North Achefer), ecotype 3 (Sinan), and ecotype 4 (Jawi)

Potential role of microRNAs in mammalian female fertility

Since the first evidence for the involvement of microRNAs (miRNAs) in various reproductive processes through conditional knockout of DICER, several studies have been conducted to investigate the expression pattern and role of miRNAs in ovarian follicular development, oocyte maturation, embryo development, embryo-maternal communication, pregnancy establishment and various reproductive diseases. Although advances in sequencing technology have fuelled miRNA studies in mammalian species, the presence of extracellular miRNAs in various biological fluids, including follicular fluid, blood plasma, urine and milk among others, has opened a new door in miRNA research for their use as diagnostic markers. This review presents data related to the identification and expression analysis of cellular miRNA in mammalian female fertility associated with ovarian folliculogenesis, oocyte maturation, preimplantation embryo development and embryo implantation. In addition, the relevance of miRNAs to female reproductive disorders, including polycystic ovary syndrome (PCOS), endometritis and abnormal pregnancies, is discussed for various mammalian species. Most importantly, the mechanism of release and the role of extracellular miRNAs in cell-cell communication and their potential role as non-invasive markers in female fertility are discussed in detail. Understanding this layer of regulation in female reproduction processes will pave the way to understanding the genetic regulation of female fertility in mammalian species

Endogenous and Exogenous Modulation of Nrf2 Mediated Oxidative Stress Response in Bovine Granulosa Cells: Potential Implication for Ovarian Function

Nrf2 is a redox sensitive transcription factor regulating the expression of antioxidant genes as defense mechanism against various stressors. The aim of this study is to investigate the potential role of noncoding miRNAs as endogenous and quercetin as exogenous regulators of Nrf2 pathway in bovine granulosa cells. For this cultured granulosa cells were used for modulation of miRNAs (miR-28, 153 and miR-708) targeting the bovine Nrf2 and supplementation of quercentin to investigate the regulatory mechanisms of the Nrf2 antioxidant system. Moreover, cultured cells were treated with hydrogen peroxide to induce oxidative stress in those cells. Our results showed that, oxidative stress activated the expression of Nrf2 as a defense mechanism, while suppressing the expression of those miRNAs. Overexpression of those miRNAs resulted in downregulation of Nrf2 expression resulted in higher ROS accumulation, reduced mitochondrial activity and cellular proliferation. Quercetin supplementation showed its protective role against oxidative stress induced by H2O2 by inducing the expression of antioxidant enzymes. In conclusion, this study highlighted the involvement of miR-153, miR-28 and miR-708 in regulatory network of Nrf2 mediated antioxidant system in bovine granulosa cells function. Furthermore, quercetin at a low dose played a protective role in bovine granulosa cells against oxidative stress damage