Transcriptome Analysis of Non‐Coding RNAs in Livestock Species: Elucidating the Ambiguity

The recent remarkable development of transcriptomics technologies, especially next generation sequencing technologies, allows deeper exploration of the hidden landscapes of complex traits and creates great opportunities to improve livestock productivity and welfare. Non-coding RNAs (ncRNAs), RNA molecules that are not translated into proteins, are key transcriptional regulators of health and production traits, thus, transcriptomics analyses of ncRNAs are important for a better understanding of the regulatory architecture of livestock phenotypes. In this chapter, we present an overview of common frameworks for generating and processing RNA sequence data to obtain ncRNA transcripts. Then, we review common approaches for analyzing ncRNA transcriptome data and present current state of the art methods for identification of ncRNAs and functional inference of identified ncRNAs, with emphasis on tools for livestock species. We also discuss future challenges and perspectives for ncRNA transcriptome data analysis in livestock species

Transcriptome adaptation of the bovine mammary gland to diets rich in unsaturated fatty acids shows greater impact of linseed oil over safflower oil on gene expression and metabolic pathways

Differentially expressed genes implicated in apoptosis of cows in LSO treatment as compared to the same cows on the control diet. Expression direction of several genes predicted to decrease apoptosis. (DOCX 35 kb

Étude de l'état immunitaire des vaches laitière atteintes de la paratuberculose bovine

La paratuberculose bovine, ou maladie de Johne, est une maladie inflammatoire intestinale chronique provoquant d’importantes pertes économiques chez les producteurs de ruminants du monde entier. Que ce soit chez la vache laitière ou de boucherie, ces pertes sont causées majoritairement par une diminution de la capacité de reproduction, la baisse de production laitière et l’amaigrissement des vaches qui perdent ainsi beaucoup de valeur à l’abattage, en plus d’être sujettes à une réforme précoce. Outre les pertes économiques, le potentiel de transmission à l’humain est un facteur non négligeable en plus d’un risque de contamination de la chaîne alimentaire. Cette maladie est causée par une bactérie intracellulaire obligatoire nommée Mycobacterium avium subspecies paratuberculosis (MAP). II n’existe actuellement aucune stratégie efficace pour combattre l’infection chez les animaux atteints. L’évolution lente de la maladie fait en sorte que les signes cliniques apparaissent tardivement, soit plusieurs années (4 à 7 ans) après l’infection initiale. Au cours de cette progression, les animaux infectés commencent à excréter le pathogène dans leur environnement. Les animaux atteints deviennent infectieux et peuvent contaminer d’autres congénères, ainsi que leur propre veau. Afin de permettre aux producteurs d’éliminer les vaches atteintes avant qu’elles n’atteignent ce stade, il s’avère important d’établir un diagnostic précoce. Actuellement, ce n’est qu’en phase sous-clinique avancée que les tests diagnostiques sont plus sensibles, soit 2 à 3 ans après le début des excrétions fécales chez les animaux infectés. L'incompréhension du manque de sensibilité des tests de dépistage et de l'évolution de cette maladie justifient les efforts de recherche dans ce domaine en vue de mieux comprendre les réponses immunitaires impliquées dans cette maladie. En effet, une meilleure connaissance des processus d’inflammation chronique pourrait aider à développer des outils diagnostiques complémentaires. Nos résultats suggèrent une dérégulation de la réponse immunitaire. Ainsi, en étudiant les composantes et caractéristiques du sang provenant de vaches infectées, il nous a été possible d’observer que les niveaux de cytokines plasmatiques telles l’interleukine 17 et l'ostéopontine se trouvent sécrétées à différents niveaux chez les vaches atteintes de paratuberculose bovine. De plus, l'analyse de la capacité de leur sérum à soutenir efficacement la prolifération des cellules mononucléées du sang périphérique révèle que le sérum de vaches infectées interfère pour atténuer la prolifération cellulaire. II semble qu’un constituant du sérum provoque une diminution de la réponse immunitaire chez les vaches malades. Les résultats offrnt une appréciation des dérèglements immunitaires provoqués par la paratuberculose bovine

Étude de l'état immunitaire des vaches laitière atteintes de la paratuberculose bovine

La paratuberculose bovine, ou maladie de Johne, est une maladie inflammatoire intestinale chronique provoquant d’importantes pertes économiques chez les producteurs de ruminants du monde entier. Que ce soit chez la vache laitière ou de boucherie, ces pertes sont causées majoritairement par une diminution de la capacité de reproduction, la baisse de production laitière et l’amaigrissement des vaches qui perdent ainsi beaucoup de valeur à l’abattage, en plus d’être sujettes à une réforme précoce. Outre les pertes économiques, le potentiel de transmission à l’humain est un facteur non négligeable en plus d’un risque de contamination de la chaîne alimentaire. Cette maladie est causée par une bactérie intracellulaire obligatoire nommée Mycobacterium avium subspecies paratuberculosis (MAP). II n’existe actuellement aucune stratégie efficace pour combattre l’infection chez les animaux atteints. L’évolution lente de la maladie fait en sorte que les signes cliniques apparaissent tardivement, soit plusieurs années (4 à 7 ans) après l’infection initiale. Au cours de cette progression, les animaux infectés commencent à excréter le pathogène dans leur environnement. Les animaux atteints deviennent infectieux et peuvent contaminer d’autres congénères, ainsi que leur propre veau. Afin de permettre aux producteurs d’éliminer les vaches atteintes avant qu’elles n’atteignent ce stade, il s’avère important d’établir un diagnostic précoce. Actuellement, ce n’est qu’en phase sous-clinique avancée que les tests diagnostiques sont plus sensibles, soit 2 à 3 ans après le début des excrétions fécales chez les animaux infectés. L'incompréhension du manque de sensibilité des tests de dépistage et de l'évolution de cette maladie justifient les efforts de recherche dans ce domaine en vue de mieux comprendre les réponses immunitaires impliquées dans cette maladie. En effet, une meilleure connaissance des processus d’inflammation chronique pourrait aider à développer des outils diagnostiques complémentaires. Nos résultats suggèrent une dérégulation de la réponse immunitaire. Ainsi, en étudiant les composantes et caractéristiques du sang provenant de vaches infectées, il nous a été possible d’observer que les niveaux de cytokines plasmatiques telles l’interleukine 17 et l'ostéopontine se trouvent sécrétées à différents niveaux chez les vaches atteintes de paratuberculose bovine. De plus, l'analyse de la capacité de leur sérum à soutenir efficacement la prolifération des cellules mononucléées du sang périphérique révèle que le sérum de vaches infectées interfère pour atténuer la prolifération cellulaire. II semble qu’un constituant du sérum provoque une diminution de la réponse immunitaire chez les vaches malades. Les résultats offrnt une appréciation des dérèglements immunitaires provoqués par la paratuberculose bovine

Co-Expression Network and Pathway Analyses Reveal Important Modules of miRNAs Regulating Milk Yield and Component Traits

Co-expression network analyses provide insights into the molecular interactions underlying complex traits and diseases. In this study, co-expression network analysis was performed to detect expression patterns (modules or clusters) of microRNAs (miRNAs) during lactation, and to identify miRNA regulatory mechanisms for milk yield and component traits (fat, protein, somatic cell count (SCC), lactose, and milk urea nitrogen (MUN)) via miRNA target gene enrichment analysis. miRNA expression (713 miRNAs), and milk yield and components (Fat%, Protein%, lactose, SCC, MUN) data of nine cows at each of six different time points (day 30 (D30), D70, D130, D170, D230 and D290) of an entire lactation curve were used. Four modules or clusters (GREEN, BLUE, RED and TURQUOISE) of miRNAs were identified as important for milk yield and component traits. The GREEN and BLUE modules were significantly correlated (|r| > 0.5) with milk yield and lactose, respectively. The RED and TURQUOISE modules were significantly correlated (|r| > 0.5) with both SCC and lactose. In the GREEN module, three abundantly expressed miRNAs (miR-148a, miR-186 and miR-200a) were most significantly correlated to milk yield, and are probably the most important miRNAs for this trait. DDR1 and DDHX1 are hub genes for miRNA regulatory networks controlling milk yield, while HHEX is an important transcription regulator for these networks. miR-18a, miR-221/222 cluster, and transcription factors HOXA7, and NOTCH 3 and 4, are important for the regulation of lactose. miR-142, miR-146a, and miR-EIA17-14144 (a novel miRNA), and transcription factors in the SMAD family and MYB, are important for the regulation of SCC. Important signaling pathways enriched for target genes of miRNAs of significant modules, included protein kinase A and PTEN signaling for milk yield, eNOS and Noth signaling for lactose, and TGF β, HIPPO, Wnt/β-catenin and cell cycle signaling for SCC. Relevant enriched gene ontology (GO)-terms related to milk and mammary gland traits included cell differentiation, G-protein coupled receptor activity, and intracellular signaling transduction. Overall, this study uncovered regulatory networks in which miRNAs interacted with each other to regulate lactation traits

Integration of miRNA and mRNA Co-Expression Reveals Potential Regulatory Roles of miRNAs in Developmental and Immunological Processes in Calf Ileum during Early Growth

This study aimed to investigate the potential regulatory roles of miRNAs in calf ileum developmental transition from the pre- to the post-weaning period. For this purpose, ileum tissues were collected from eight calves at the pre-weaning period and another eight calves at the post-weaning period and miRNA expression characterized by miRNA sequencing, followed by functional analyses. A total of 388 miRNAs, including 81 novel miRNAs, were identified. A total of 220 miRNAs were differentially expressed (DE) between the two periods. The potential functions of DE miRNAs in ileum development were supported by significant enrichment of their target genes in gene ontology terms related to metabolic processes and transcription factor activities or pathways related to metabolism (peroxisomes), vitamin digestion and absorption, lipid and protein metabolism, as well as intracellular signaling. Integration of DE miRNAs and DE mRNAs revealed several DE miRNA-mRNA pairs with crucial roles in ileum development (bta-miR-374a—FBXO18, bta-miR-374a—GTPBP3, bta-miR-374a—GNB2) and immune function (bta-miR-15b—IKBKB). This is the first integrated miRNA-mRNA analysis exploring the potential roles of miRNAs in calf ileum growth and development during early life

Transcriptome Analysis of Long Non-Coding RNA in the Bovine Mammary Gland Following Dietary Supplementation with Linseed Oil and Safflower Oil

This study aimed to characterize the long non-coding RNA (lncRNA) expression in the bovine mammary gland and to infer their functions in dietary response to 5% linseed oil (LSO) or 5% safflower oil (SFO). Twelve cows (six per treatment) in mid lactation were fed a control diet for 28 days followed by a treatment period (control diet supplemented with 5% LSO or 5% SFO) of 28 days. Mammary gland biopsies were collected from each animal on day-14 (D-14, control period), D+7 (early treatment period) and D+28 (late treatment period) and were subjected to RNA-Sequencing and subsequent bioinformatics analyses. Functional enrichment of lncRNA was performed via potential cis regulated target genes located within 50 kb flanking regions of lncRNAs and having expression correlation of >0.7 with mRNAs. A total of 4955 lncRNAs (325 known and 4630 novel) were identified which potentially cis targeted 59 and 494 genes in LSO and SFO treatments, respectively. Enrichments of cis target genes of lncRNAs indicated potential roles of lncRNAs in immune function, nucleic acid metabolism and cell membrane organization processes as well as involvement in Notch, cAMP and TGF-β signaling pathways. Thirty-two and 21 lncRNAs were differentially expressed (DE) in LSO and SFO treatments, respectively. Six genes (KCNF1, STARD13, BCL6, NXPE2, HHIPL2 and MMD) were identified as potential cis target genes of six DE lncRNAs. In conclusion, this study has identified lncRNAs with potential roles in mammary gland functions and potential candidate genes and pathways via which lncRNAs might function in response to LSO and SFA

Integration of lncRNA and mRNA Transcriptome Analyses Reveals Genes and Pathways Potentially Involved in Calf Intestinal Growth and Development during the Early Weeks of Life

A better understanding of the factors that regulate growth and immune response of the gastrointestinal tract (GIT) of calves will promote informed management practices in calf rearing. This study aimed to explore genomics (messenger RNA (mRNA)) and epigenomics (long non-coding RNA (lncRNA)) mechanisms regulating the development of the rumen and ileum in calves. Thirty-two calves (≈5-days-old) were reared for 96 days following standard procedures. Sixteen calves were humanely euthanized on experiment day 33 (D33) (pre-weaning) and another 16 on D96 (post-weaning) for collection of ileum and rumen tissues. RNA from tissues was subjected to next generation sequencing and 3310 and 4217 mRNAs were differentially expressed (DE) between D33 and D96 in ileum and rumen tissues, respectively. Gene ontology and pathways enrichment of DE genes confirmed their roles in developmental processes, immunity and lipid metabolism. A total of 1568 (63 known and 1505 novel) and 4243 (88 known and 4155 novel) lncRNAs were detected in ileum and rumen tissues, respectively. Cis target gene analysis identified BMPR1A, an important gene for a GIT disease (juvenile polyposis syndrome) in humans, as a candidate cis target gene for lncRNAs in both tissues. LncRNA cis target gene enrichment suggested that lncRNAs might regulate growth and development in both tissues as well as posttranscriptional gene silencing by RNA or microRNA processing in rumen, or disease resistance mechanisms in ileum. This study provides a catalog of bovine lncRNAs and set a baseline for exploring their functions in calf GIT development

miRNA Regulatory Functions in Farm Animal Diseases, and Biomarker Potentials for Effective Therapies

MicroRNAs (miRNAs) are small endogenous RNAs that regulate gene expression post-transcriptionally by targeting either the 3′ untranslated or coding regions of genes. They have been reported to play key roles in a wide range of biological processes. The recent remarkable developments of transcriptomics technologies, especially next-generation sequencing technologies and advanced bioinformatics tools, allow more in-depth exploration of messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs), including miRNAs. These technologies have offered great opportunities for a deeper exploration of miRNA involvement in farm animal diseases, as well as livestock productivity and welfare. In this review, we provide an overview of the current knowledge of miRNA roles in major farm animal diseases with a particular focus on diseases of economic importance. In addition, we discuss the steps and future perspectives of using miRNAs as biomarkers and molecular therapy for livestock disease management as well as the challenges and opportunities for understanding the regulatory mechanisms of miRNAs related to disease pathogenesis