Utilización de herramientas genómicas en el estudio de la arquitectura molecular de caracteres de importancia económica en el ganado ovino = Using genomic tools for the study of the molecular architecture of economically important traits in sheep

266 p.El objetivo general de esta Tesis Doctoral ha sido utilizar las herramientas genómicas disponibles en el estudio de caracteres de importancia económica en el ganado ovino lechero. En concreto, utilizar el OvineSNP50 BeadChip en el mapeo de regiones portadoras de mutaciones causales de enfermedades de herencia mendeliana y utilizar las herramientas de secuenciación masiva paralela en la caracterización de la mutación causal de enfermedades monogénicas y del transcriptoma de la glándula mamaria de la oveja en lactación. Este trabajo ofrece una visión global del transcriptoma mamario de la oveja en lactación, proporcionando información para el desarrollo de futuros análisis detallados de los genes implicados en las rutas significativas detectadas, relacionadas a priori con caracteres productivos, así como el análisis funcional de transcritos no anotados en el genoma de referencia o la identificación de variantes relacionadas con caracteres productivos de interé

Isolation of RNA from milk somatic cells as an alternative to biopsies of mammary tissue for nutrigenomic studies in dairy ewes

11 páginas, 4 tablas, 1 figura.Nutrigenomic studies of mammary lipogenesis in ruminants often rely on the use of mammary tissue (MT) collected either by biopsy or at slaughter. However, isolating RNA from milk would be a useful and cost-effective technique that may avoid distress to the animal and facilitate the collection of samples in time series experiments. This assay was therefore conducted to test the hypothesis that RNA extracted from milk somatic cells (MSC) in dairy sheep would be a feasible alternative to the performance of MT biopsies for nutrigenomic analyses. To meet this objective, 8 lactating Assaf ewes were divided in 2 groups and offered a total mixed ration without supplementation (control) or supplemented with 2.4% dry matter of fish oil, which was known not only to elicit milk fat depression but also to downregulate the expression of some candidate genes involved in mammary lipogenesis. Total RNA was extracted from MSC and biopsied MT to examine whether the potential changes in the abundance of transcripts was similarly detected with both RNA sources. Milk fatty acid profile was also analyzed by gas chromatography, and variations in mRNA abundance were determined by reverse transcription quantitative PCR. Values of RNA integrity number were always ≥7.7. The expected and designed decrease of milk fat concentration with fish oil (−29%), was associated with a lower transcript abundance of genes coding for enzymes involved in fatty acid activation (ACSS1), de novo synthesis (ACACA and FASN), uptake from plasma lipids (LPL), and esterification of fatty acids to glycerol (LPIN1), as well as of a transcription factor that may regulate their expression (INSIG1). Stable mRNA levels were showed in other candidate genes, such as FABP3, GPAT4, or SCD. Changes due to the dietary treatment were similarly detected with both RNA sources (MSC and MT biopsies), which supports the initial hypothesis and would validate the use of milk as an alternative RNA source for nutrigenomic analyses in dairy sheep.This work was supported by the Ministry of Economy and Competitiveness (MINECO, Spain; AGL2014-54587, co-funded by the European Regional Development Fund, European Union). P. G. Toral gratefully acknowledges receipt of a postdoctoral research contract from the MINECO (Juan de la Cierva program).Peer reviewe

Gaining Bioinformatics skills through the End of Degree Project in Veterinary Medicine

Resúmenes IV Congreso VetDoc de Docencia Veterinaria, León 2017 (6-7 de Julio)[ES] El análisis de los datos genómicos se realiza con programas libres diseñados para entorno Linux (Unix), frente a los sistemas operativos más habituales (Windows), y en la utilización de la línea de comandos. Dado la falta de familiarización de los estudiantes del Grado de Veterinaria con este entorno, y previendo la bioinformática como una potencial salida profesional para algunos futuros graduados en Veterinaria, nuestro Grupo de Innovación Docente (GID) de la Universidad de León (ULE), ha realizado varias ofertas de Trabajos de Fin de Grado (TFG) expresamente diseñados para la adquisición de competencias en el campo de la bioinformática

Elucidating fish oil-induced milk fat depression in dairy sheep: Milk somatic cell transcriptome analysis

9 páginas, 4 figuras.In this study, RNA sequencing was used to obtain a comprehensive profile of the transcriptomic changes occurring in the mammary gland of lactating sheep suffering from fish oil-induced milk fat depression (FO-MFD). The milk somatic cell transcriptome analysis of four control and four FO-MFD ewes generated an average of 42 million paired-end reads per sample. In both conditions, less than 220 genes constitute approximately 89% of the total counts. These genes, which are considered as core genes, were mainly involved in cytoplasmic ribosomal proteins and electron transport chain pathways. In total, 117 genes were upregulated, and 96 genes were downregulated in FO-MFD samples. Functional analysis of the latter indicated a downregulation of genes involved in the SREBP signaling pathway (e.g., ACACA, ACSL, and ACSS) and Gene Ontology terms related to lipid metabolism and lipid biosynthetic processes. Integrated interpretation of upregulated genes indicated enrichment in genes encoding plasma membrane proteins and proteins regulating protein kinase activity. Overall, our results indicate that FO-MFD is associated with the downregulation of key genes involved in the mammary lipogenesis process. In addition, the results also suggest that this syndrome may be related to upregulation of other genes implicated in signal transduction and codification of transcription factors.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO; AGL2014-54587-R and AGL2015-66035-R projects, co-funded by the European Regional Development Fund). P.G.T. and B.G.G. are funded by two postdoctoral research contracts from MINECO (Juan de la Cierva -JCI-2012-11836- and Ramón y Cajal -RYC-2012-10230 programs, respectively).Peer reviewe

Variant discovery in genes identified as differentially expressed genes between the abomasal lymph node transcriptome of resistant and susceptible adult sheep to teladorsagia circumcincta infection

Gastrointestinal nematode infections are one of the major health issues facing grazing sheep populations and it incurs on major economic losses for sheep breeders. The resistance/susceptibility trait appears to be a highly complex trait (Behnke et al. 2003; Dominik 2005). In sheep resistance to nematode infection shows a moderate level of heritability (range 0.3-0.6) (Stear et al. 2001). Several QTL mapping studies have tried to identify genomic regions and mutations that influence resistance to nematode infection (Atlija et al. 2016; Coltman et al. 2001; Gutiérrez-Gil et al. 2009; Sayers et al. 2005), although the detection of causal mutations for this trait is still a challenge for the research community. The recently available RNA-seq technology provides the opportunity to extract highthroughput transcriptome data from a specific tissue to perform gene quantification, differential gene expression and detection of variants (SNPs and indels), which could be assessed as potential causal mutations (Hudson, Dalrymple, and Reverter 2012). A previous study of our research group has identified a list of 106 differential expression genes (DEGs) based on RNA-Seq dataset obtained from the abomasal lymph nodes of 12 adult sheep, previously classified as resistant or susceptible to GIN infection based on an artificial infection with T. circumcincta larvae Chitneedi et al. (2018). In the present study we present a detailed study of the variants mapping within the list of DEGs previously reported in that study. Thus, the present study provides a list of functionally relevant variants that could underlie the genetic control of resistance/susceptibility to T. circumcincta in adult sheep. Financiado por la Junta de Castilla y León (Conserjería de Educación) y Fondo Social Europeo (Ref. LE248U14). Ejecución: Universidad de León. Dpto. Producción Animal. Investigador Principal: Dr. Beatriz Gutierrez Gi

Exploring the mechanisms of resistance to Teladorsagia circumcincta infection in sheep through transcriptome analysis of abomasal mucosa and abomasal lymph nodes

8 p.The present study exploited the RNA-seq technology to analyze the transcriptome of target tissues affected by the Teladorsagia circumcincta infection in two groups of adult ewes showing different statuses against gastrointestinal nematode (GIN) infection with the aim of identifying genes linked to GIN infection resistance in sheep. For this, based on the accumulated faecal egg count of 18 adult Churra ewes subjected to a first experimental infection with T. circumcincta, six ewes were classified as resistant and six others as susceptible to the infection. These 12 animals were dewormed and infected again. After humanitarian sacrifice of these 12 animals at day 7 post-infection, RNA samples were obtained from abomasal mucosa and lymph node tissues and RNA-Seq datasets were generated using an Illumina HiSeq 2000 sequencer. The distribution of the genes based on their expression level were very similar among the two different tissues and conditions. The differential expression analysis performed with two software (DESeq and EdgeR) only identified common differentially expressed genes (DEGs), a total of 106, in the lymph node samples which were considered as GIN-activated. The enrichment analysis performed for these GIN-activated genes identified some pathways related to cytokine-mediated immune response and the PPARG signaling pathway as well as disease terms related to inflammation and gastro-intestinal diseases as enriched. A systematic comparison with the results of previous studies confirmed the involvement of genes such as ITLN2, CLAC1 and galectins, in the immune mechanism activated against T. circumcincta in resistant sheep. Financiado por la Junta de Castilla y León (Conserjería de Educación) y Fondo Social Europeo (Ref. LE248U14). Ejecución: Universidad de León. Dpto. Producción Animal. Investigador Principal: Dr. Beatriz Gutierrez GilS

Study on the concordance between different SNP‐genotyping platforms in sheep

.Different SNP genotyping technologies are commonly used in multiple studies to perform QTL detection, genotype imputation, and genomic predictions. Therefore, genotyping errors cannot be ignored, as they can reduce the accuracy of different procedures applied in genomic selection, such as genomic imputation, genomic predictions, and false-positive results in genome-wide association studies. Currently, whole-genome resequencing (WGR) also offers the potential for variant calling analysis and high-throughput genotyping. WGR might overshadow array-based genotyping technologies due to the larger amount and precision of the genomic information provided; however, its comparatively higher price per individual still limits its use in larger populations. Thus, the objective of this work was to evaluate the accuracy of the two most popular SNP-chip technologies, namely, Affymetrix and Illumina, for high-throughput genotyping in sheep considering high-coverage WGR datasets as references. Analyses were performed using two reference sheep genome assemblies, the popular Oar_v3.1 reference genome and the latest available version Oar_rambouillet_v1.0. Our results demonstrate that the genotypes from both platforms are suggested to have high concordance rates with the genotypes determined from reference WGR datasets (96.59% and 99.51% for Affymetrix and Illumina technologies, respectively). The concordance results provided in the current study can pinpoint low reproducible markers across multiple platforms used for sheep genotyping data. Comparing results using two reference genome assemblies also informs how genome assembly quality can influence genotype concordance rates among different genotyping platforms. Moreover, we describe an efficient pipeline to test the reliability of markers included in sheep SNP-chip panels against WGR datasets available on public databases. This pipeline may be helpful for discarding low-reliability markers before exploiting genomic information for gene mapping analyses or genomic predictionS

Relationships between growth-related traits with carcass and meat quality traits in Spanish Assaf suckling lambs

[EN] Suckling lamb meat is a relevant product in Mediterranean European dairy sheep farms, and the dairy breed Spanish Assaf is widely extended through the Iberian Peninsula. Knowledge of the influence of birth body weight (bBW) and growth rate on suckling lamb carcass and meat quality is scarce, but useful for breeding optimisation and product homogeneity. In turn, these growth-related traits of lambs might be affected by dietary restrictions of their dams. In this study, 34 male Assaf suckling lambs born from two groups of ewes that had been fed diets with different protein levels when they were prepubertal female lambs (17 lambs per group) were analysed. After birth, the suckling lambs were fed ad libitum on milk replacer until their sacrifice (10–12.5 kg live body weight). The quality traits evaluated in carcasses and meat were carcass compactness, fatness and jointing, meat composition, colour, texture and oxidative stability, and fatty acid profile. The dam group did not show significant effects on lamb growth characteristics or carcass and meat quality traits. The bBW factor showed a negative effect on leg subcutaneous fatness and a positive effect on the forequarter and shoulder percentages of the carcass. The bBW also resulted in increased moisture, lipid oxidation stability, and n-3 FA content (lowering the n-6/n-3 ratio) in the meat. Suckling lambs showing very low average daily gain (ADG) tended to present low carcass quality, i.e., higher bone percentage in the loin and low percentages of muscle or fat compared to those showing high ADG. Further studies are needed to confirm and explain the mechanisms of the significant effects reported here for bBW and ADG on the affected quality traits.S

Estudio preliminar del transcriptoma de la mucosa abomasal de ovejas clasificadas como resistentes y susceptibles según la respuesta a una infección experimental con teladorsagia circumcincta

Este proyecto trata de estudiar el transcriptoma de los tejidos de mayor importancia durante la respuesta inmune de ovejas adultas ante la infección por nematodos gastrointestinales, la mucosa abomasal y los ganglios abomasales. El transcriptoma es el conjunto completo de todos los transcritos (regiones del DNA que pasan a RNA mensajero, o mRNA) de una célula, y su cuantificación en un momento fisiológico específico. La identificación de genes diferencialmente expresados en los tejidos afectados por la infección entre ovejas susceptibles y resistentes es un primer paso para la identificación de genes y rutas génicas involucradas en la resistencia/susceptibilidad a este tipo de infecciones. Esta comunicación es el resultado de la investigación dentro del Proyecto: “Análisis del transcriptoma de la mucosa y los ganglios abomasales para la identificación de genes involucrados en la resistencia a los nematodos gastrointestinales en ovejas adultas”. Financiado por la Junta de Castilla y León (Conserjería de Educación) y Fondo Social Europeo (Ref. LE248U14). Ejecución: Universidad de León. Dpto. Producción Animal. Investigador Principal: Dr. Beatriz Gutierrez Gi

Influence of a temporary restriction of dietary protein in prepubertal ewe lambs on first lactation milk traits and response to a mammary gland inflammatory challenge

[EN] This study evaluated the influence of a temporary nutritional protein restriction (NPR) performed, under commercial conditions, in prepubertal female lambs on first lactation milk production traits and the inflammatory response triggered by an inflammatory challenge of the. From 40 Assaf female lambs, we defined a control group (C n = 20), which received a standard diet for replacement lambs and the NPR group (n = 20), which received the same diet but without soybean meal between 3 and 5 months of age. About 150 days after lambing, 24 of these ewes (13 NPR, 11C) were subjected to an intramammary infusion of E. coli lipopolysaccharide (LPS). Our dynamic study identified indicator traits of local (SCC) and systemic (rectal Ta, IL-6, CXCL8, IL-10, IL-36RA, VEGF-A) response to the LPS challenge. The NPR did not show significant effects on milk production traits and did not affect the SCC and rectal Ta after the LPS challenge. However, the NPR had a significant influence on 8 of the 14 plasma biomarkers analysed, in all the cases with higher relative values in the C group. The effects observed on VEGF-A (involved in vasculogenesis during mammary gland development and vascular permeability) and IL-10 (a regulatory cytokine classically known by its anti-inflammatory action) are the most remarkable to explain the differences found between groups. Whereas further studies should be undertaken to confirm these results, our findings are of interest considering the current concern about the future world's demand for protein and the need for animal production systems to evolve toward sustainability.S