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International audience; AbstractBackgroundBovine paratuberculosis is a contagious disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), with adverse effects on animal welfare and serious economic consequences. Published results on host genetic resistance to MAP are inconsistent, mainly because of difficulties in characterizing the infection status of cows. The objectives of this study were to identify quantitative trait loci (QTL) for resistance to MAP in Holstein and Normande cows with an accurately defined status for MAP.ResultsFrom MAP-infected herds, cows without clinical signs of disease were subjected to at least four repeated serum ELISA and fecal PCR tests over time to determine both infected and non-infected statuses. Clinical cases were confirmed using PCR. Only cows that had concordant results for all tests were included in further analyses. Positive and control cows were matched within herd according to their birth date to ensure a same level of exposure to MAP. Cows with accurate phenotypes, i.e. unaffected (control) or affected (clinical or non-clinical cases), were genotyped with the Illumina BovineSNP50 BeadChip. Genotypes were imputed to whole-genome sequences using the 1000 Bull Genomes reference population (run6). A genome-wide association study (GWAS) of MAP status of 1644 Holstein and 649 Normande cows, using either two (controls versus cases) or three classes of phenotype (controls, non-clinical and clinical cases), revealed three regions, on Bos taurus (BTA) chromosomes 12, 13, and 23, presenting significant effects in Holstein cows, while only one of those was identified in Normande cows (BTA23). The most significant effect was found on BTA13, in a short 8.5-kb region. Conditional analyses revealed that only one causal variant may be responsible for the effects observed on each chromosome with the ABCC4 (BTA12), CBFA2T2 (BTA13), and IER3 (BTA23) genes as good functional candidates.ConclusionsA sequence-based GWAS on cows for which resistance to MAP was accurately defined, was able to identify candidate variants located in genes that were functionally related to resistance to MAP; these explained up to 28% of the genetic variance of the trait. These results are very encouraging for efforts towards implementation of a breeding strategy aimed at improving resistance to paratuberculosis in Holstein cows.

Additional file 1: Fig. S1.. correlation clustering run on the methylation percentages at CpGs10 covered in at least 22 samples per group. Fig. S2: heatmap run on CpGs discriminant between sperm and somatic cells. Fig. S3: annotation of fertility-related DMRs relative to different genome features. Fig. S4: average DNA methylation at individual LINE and LTR repeats after alignment of the RRBS sequences on a Repbase artificial genome. Fig. S5: pyrosequencing validation of the upstream region of LBX1 gene. Fig. S6: fertility, semen functional parameters and semen sample characteristics in correctly classified and misclassified bulls. Fig. S7: PCA run on the methylation percentages at DMCs, with or without the imputation of missing values. Fig. S8: semen functional parameters before and after correction for the batch effect.

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Metabolomics has been increasingly used in animal and food sciences. Animal health is one of the most important factor that can also alter animal integrity and welfare. Some studies have already investigated the link between health and metabolic profile of dairy animals. These studies in metabolomics often consider a single type of sample using a single analytical platform (nuclear magnetic resonance or mass spectrometry). Only few studies with multi-platform approaches are also used with a single or a multi type of sample, but they mainly consider dairy cows’ metabolome although dairy goats present similar diseases, that it could be interesting to detect early to preserve animal health and milk production. This study aims to create a metabolic atlas of goat plasma, milk and feces, based on healthy animals. Our study describes a standard operating procedure for three goat matrices: blood plasma, milk, and feces using multiple platforms (NMR (1H), UHPLC (RP)-MS and UHPLC (HILIC)-MS) that follows a unique sample preparation procedure for each sample type to be analyzed on multi-platforms basis. Our method was evaluated for its robustness and allowed a better characterization of goat metabolic profile in healthy conditions.

International audience; Mycobacterium avium subsp. paratuberculosis is the etiological agent of Johne's disease in ruminants. Here, we report the annotated draft genome sequences of 142 M. avium subsp. paratuberculosis strains that were isolated from dairy cattle in France between 2014 and 2018. The genomes of these strains were sequenced using Illumina technology.

International audience; Introduction: Paratuberculosis is a chronic infection of the intestine, mainly the ileum, caused by Mycobacterium avium subsp. paratuberculosis (Map) in cattle and other ruminants. This enzootic disease is present worldwide and has a strong impact on the dairy cattle industry. For this species, the typing tools do not make it possible to investigate the genetic diversity of the strains. These limitations can be overcome by the application of Whole Genome Sequencing (WGS), particularly for clonal populations such as Map. WGS analyses can provide comprehensive genetic information, including information on genome evolution, discrimination of closely related strains and virulence determinants.The purpose of the present study was to undertake a whole-genome analysis of Map strains to identify accurate phylogenetic relationships between isolates and establish correlations between genomic traits and epidemiologicaldata within a population of well documented-strains.Materials and methods: A set of 200 animal field strains were selected based on a previous large-scale longitudinal study representative of the French Map population circulating in the West of France. Strains were isolated from bovine of breed Prim’Holstein or Normande naturally infected by Map. For each strain isolated, all information about the animal is available including: breed, herd prevalence, locations, serological status and excretion level. The genomic sequences of Map strains were obtained by Illumina MiSeq sequencing. Genomic sequences were assessed for potential contamination using Kraken2. Cleaned reads were aligned to the reference genome K-10 and SNP were called using FreeBayes. IQ-Tree was used to infer a SNP-based phylogeny.Results, discussion and conclusion: This study provided 200 new genomes of French strains isolates from naturally infected animals. Pangenome analysis of this panel confirmed the degree of Map clonality. SNP analyzes provided accurate phylogeny able to distinguish each strain divided into 4 clusters independently of the breed of the cattle. Interestingly, the phylogeny clearly distinct the two major MLVA profiles. A phylogeny was inferred with French Map isolates and with other Map isolates found across the world. Relations between genetic traits and epidemiological data will be investigated to better understand the transmission dynamics of the disease

International audience; Bovine paratuberculosis is a contagious and incurable disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), with adverse effects on animal welfare and serious economic consequences. In a previous GWAS conducted on 1644 Holstein cows with an accurately defined status for MAP (controls, non-clinical and clinical cases) and whole genome sequences (WGS) imputed using the 6th Run of the 1000 bull genomes (1kBG) project (multibreed population, UMD3.1 reference genome), we identified three genomic regions on chromosomes 12, 13, and 23. The objective of the present study was to conduct further investigations using the 7th Run of the 1kBG, which includes a larger Holstein population with WGS based on the recent ARS-UCD1.2 reference genome. 50K genotypes of the 1644 Holstein cows were imputed to the high-density (HD) level with FImpute and then up to WGS with Minimac using reference sets of 776 and 700 Holstein bulls, respectively. GWAS was carried out with GCTA, testing the individual effect of ~25M variant and accounting for the population structure through a HD-based genomic relationship matrix. After filtering variants for low imputation accuracy (R² < 0.3) or frequency (MAF < 0.001), 4815 variants had significant effects (6 ≤ -log(P) ≤ 34.5) on resistance to MAP. They were located in eight different genomic regions on chromosomes 6, 9, 12, 13 (2 regions), 14, 18, and 23. Two-thirds of these variants (3194) were located in genes, mainly in introns (2170), upstream or downstream regions (907), and exons (101 including 65 non-synonymous). In each region, the variants with the most significant effects were located in the TMPRSS11F, HS6T3, ABCC4, SNTA1, SNTB1, NOTCH4, NFKBTL1, ATP6V1G2, DDX39B, and ELOVL5 genes. This study confirms the three genomic regions we detected before and reveals five novel regions for resistance to MAP.

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Additional file 1: Figure S1. −log(P-value) plotted against the position of variants detected by GWAS (in grey) and conditional GWAS (GWAS_COJO; in blue).

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International audience; Spermatozoa have a remarkable epigenome in line with their degree of specialization, their unique nature and different requirements for successful fertilization. While bull semen is widely used in artificial insemination, the literature describing DNA methylation in bovine spermatozoa is still scarce. In the context of genomic selection, more information on the epigenetic features transferred to the embryo alongside the paternal genetic heritage is necessary, in order to improve semen quality control procedures and bull fertility. We characterized the bull sperm methylome relative to bovine fibroblasts and monocytes using reduced restriction bisulfite sequencing (RRBS). A wide majority of differentially methylated cytosines (DMCs) were less methylated in sperm than in somatic cells. Consistent with previous studies in other species, these hypomethylated DMCs were enriched for genes relevant to the germline differentiation program and sperm functions. The most remarkable observation was a dramatic enrichment for repeats and particularly for satellites, which may partly account for the lower global methylation we observed in bull sperm relative to sperm from other species (LUminometric Methylation Assay). We next investigated whether fertility biomarkers could be identified in the sperm methylome. Based on field fertility indicators, we compared fertile ejaculates, subfertile ejaculates and ejaculates from bulls with a disappointing career in contradiction with genomic predictions (Montbéliarde breed, n=10/group). The median age at production was consistent among groups and the phenotypic characterization revealed no significant differences except for the proportion of motile sperm, which was reduced in disappointing bulls. We identified 4823 DMCs targeting genes involved in signaling pathways, angiogenesis and the response to growth factors. Since DNA methylation is affected by the CpG content of the genome and its alteration by DNA polymorphism, an integrative approach was conducted to identify CpGs and SNPs in correlation, which revealed both direct and indirect interactions between the genome and the epigenome.

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International audience; DNA methylation of cytosines is a critical epigenetic modification in mammals that plays crucial roles in transcriptional regulation, chromatin remodelling and genomic imprinting. Dynamic erasure and reestablishment of DNA methylation marks are required for spermatogenesis and the normal function of mature sperm. DNA methylation is catalysed by DNA methyltransferase enzymes (DNMT) providing either maintenance (DNMT1) or de novo (DNMT3A/B/L) DNA methylation processes. The DNA methylation dynamics during spermatogenesis has been previously described in mice and humans but nothing is known in productive livestock. However, these investigations in ruminants could be helpful to determine epigenetic events likely to be crucial for male fertility. Interestingly, a recent study from our laboratory pointed DNA undermethylation of bull spermatozoa compared to other mammals such as humans, mice, sheep or goats [1]. This result raises the question of the dynamic of DNA methylation in bovine male germline. We thus propose to define and compare the methylation dynamics of bovine and caprine germ cells during spermatogenesis. Analyses of 5mC immunohistochemistry were performed on adult testis sections to illustrate the presence of DNA methylation marks in various spermatogenic cells. As purification of the different spermatogenic populations is required to characterize DNA methylation more precisely, we firstly developed a flow cytometry-based method (Hoechtst-FACS) for cell sorting from goat testis. Fraction enrichments were evaluated using stage-specific markers by real time qPCR analyses. In addition, de novo DNA methyltransferase expression was studied on the different fractions of purified spermatogenic cells, giving some clues on DNA methylation dynamics. In the future, spermatogenic cells will be purified from both caprine and bovine testes, and together with gene expression analyses (RNA-sequencing), DNA methylation will be determined for each cell types by Reduced-Representation Bisulfite Sequencing (RRBS)

Background Spermatozoa have a remarkable epigenome in line with their degree of specialization, their unique nature and different requirements for successful fertilization. Accordingly, perturbations in the establishment of DNA methylation patterns during male germ cell differentiation have been associated with infertility in several species. While bull semen is widely used in artificial insemination, the literature describing DNA methylation in bull spermatozoa is still scarce. The purpose of this study was therefore to characterize the bull sperm methylome relative to both bovine somatic cells and the sperm of other mammals through a multiscale analysis. Results The quantification of DNA methylation at CCGG sites using luminometric methylation assay (LUMA) highlighted the undermethylation of bull sperm compared to the sperm of rams, stallions, mice, goats and men. Total blood cells displayed a similarly high level of methylation in bulls and rams, suggesting that undermethylation of the bovine genome was specific to sperm. Annotation of CCGG sites in different species revealed no striking bias in the distribution of genome features targeted by LUMA that could explain undermethylation of bull sperm. To map DNA methylation at a genome-wide scale, bull sperm was compared with bovine liver, fibroblasts and monocytes using reduced representation bisulfite sequencing (RRBS) and immunoprecipitation of methylated DNA followed by microarray hybridization (MeDIP-chip). These two methods exhibited differences in terms of genome coverage, and consistently, two independent sets of sequences differentially methylated in sperm and somatic cells were identified for RRBS and MeDIP-chip. Remarkably, in the two sets most of the differentially methylated sequences were hypomethylated in sperm. In agreement with previous studies in other species, the sequences that were specifically hypomethylated in bull sperm targeted processes relevant to the germline differentiation program (piRNA metabolism, meiosis, spermatogenesis) and sperm functions (cell adhesion, fertilization), as well as satellites and rDNA repeats. Conclusions These results highlight the undermethylation of bull spermatozoa when compared with both bovine somatic cells and the sperm of other mammals, and raise questions regarding the dynamics of DNA methylation in bovine male germline. Whether sperm undermethylation has potential interactions with structural variation in the cattle genome may deserve further attention. Electronic supplementary material The online version of this article (10.1186/s12864-018-4764-0) contains supplementary material, which is available to authorized users.

DNA methylation of cytosines is a critical epigenetic modification in mammals that plays crucial roles in transcriptional regulation, chromatin remodelling and genomic imprinting. Dynamic erasure and reestablishment of DNA methylation marks are required for spermatogenesis and the normal function of mature sperm. DNA methylation is catalysed by DNA methyltransferase enzymes (DNMT) providing either maintenance (DNMT1) or de novo (DNMT3A/B/L) DNA methylation processes. The DNA methylation dynamics during spermatogenesis has been previously described in mice and humans but nothing is known in productive livestock. However, these investigations in ruminants could be helpful to determine epigenetic events likely to be crucial for male fertility. Interestingly, a recent study from our laboratory pointed DNA undermethylation of bull spermatozoa compared to other mammals such as humans, mice, sheep or goats [1]. This result raises the question of the dynamic of DNA methylation in bovine male germline. We thus propose to define and compare the methylation dynamics of bovine and caprine germ cells during spermatogenesis. Analyses of 5mC immunohistochemistry were performed on adult testis sections to illustrate the presence of DNA methylation marks in various spermatogenic cells. As purification of the different spermatogenic populations is required to characterize DNA methylation more precisely, we firstly developed a flow cytometry-based method (Hoechtst-FACS) for cell sorting from goat testis. Fraction enrichments were evaluated using stage-specific markers by real time qPCR analyses. In addition, de novo DNA methyltransferase expression was studied on the different fractions of purified spermatogenic cells, giving some clues on DNA methylation dynamics. In the future, spermatogenic cells will be purified from both caprine and bovine testes, and together with gene expression analyses (RNA-sequencing), DNA methylation will be determined for each cell types by Reduced-Representation Bisulfite Sequencing (RRBS)

is a pdf file containing supplementary methods, supplementary references, eight supplementary tables and four supplementary figures. Table S1. reference genomes used for in silico analyses and origin of the files used for annotation. Table S2. in silico characterization of bovine reduced restriction (RR) genomes generated using different size selection criteria. Table S3. comparison of RR genomes obtained with a 40-290 bp selection size window in different species. Table S4. primers and PCR conditions used to generate the pyrosequencing templates. Table S5. pyrosequencing primers. Table S6. library characterization, mapping efficiency on the bovine genome (UMD3.1), coverage and average methylation in RRBS libraries. Table S7. results of comparisons between tissues by RRBS. Table S8. mapping efficiency on a Repbase artificial bovine genome, coverage and average methylation in RRBS libraries. Figure S1. Bootstrap analysis of global CCGG methylation in bull sperm from four different breeds. Figure S2. Genotyping of bull sperm and blood samples. Figure S3. Average methylation percentages for CpGs 5-500 and CpGs > 500 in each cell type, in reads uniquely aligned on a Repbase bovine artificial genome. Figure S4. Pyrosequencing of CpGs hypermethylated in sperm. (PDF 1840 kb)

Le spermatozoïde a longtemps été considéré comme un taxi, protégeant et transportant son patrimoine génétique jusqu’à la délivrance dans l’ovocyte. Mais depuis quelques années de nouveaux arguments, autour des mécanismes épigénétiques, tels que les petits ARN non codants (sncRNA), proposent une nouvelle vision de la fonction du spermatozoïde par son implication dans le développement précoce de l’embryon (Gapp et al., 2014 ; Kotaja 2014 ; Boissonnas et al., 2013 ; Stopka et al., 2003 ; Liu et al., 2012). Or, chez les animaux de rente et en particulier chez les bovins, le contrôle de la fertilité est un élément central pour une diffusion optimale du progrès génétique. L’analyse des paramètres fonctionnels, traduisant la qualité du spermatozoïde, a longtemps été considérée comme une source importante de biomarqueurs du pouvoir fécondant de la semence. Mais malgré des résultats intéressants, la prédiction de la fertilité reste aujourd’hui inefficace (Sellem et al., 2015). C’est pourquoi la recherche de nouveaux biomarqueurs, tels que les sncRNA du spermatozoïde, a été entrepris. Notre étude a été conduite afin d’une part d’établir un catalogue exhaustif du contenu en sncRNA du spermatozoïde bovin et d’autre part de mettre en évidence de potentiels miRNA liés à la fertilité des taureaux. De l'ARN total a été extrait de 40 éjaculats provenant de taureaux Holstein et Montbeliard avec des fertilités contrastées, en utilisant un nouveau protocole amélioré. Deux contrôles qualité ont été effectués sur l'ARN extrait, afin de mesurer les quantités d'ARN (technologie Qubit) et de vérifier si un miARN de référence (miR125) a pu être détecté par RTqPCR. Les 40 banques NGS ont été préparées avec une sélection de taille (

International audience; New sperm function in embryo development have emerged recently, relying on their small noncoding RNAs (sncRNAs) content. Indeed, involvement of sperm-borne miRNAs in mouse epigenetic inheritance has been evidenced and paternal sncRNAs have been shown to be dispensable for fertilization but crucial for the development of zygotes and 2 cells-embryos. Our study was conducted to unravel the sncRNA content from bull frozen sperm cells and identify miRNA associated with fertility. Total RNA was extracted from 40 ejaculates originating from Holstein and Montbeliard bulls with contrasting fertility, using a novel enhanced protocol. The two quality controls were done on RNA to measure RNAs quantities (Qubit technology) and check whether a reference miRNA (miR125) could be detected by RTqPCR. NGS sequencing libraries were prepared using small RNA (

International audience; The sperm cell has been generally considered as a cab driver, carrying and protecting his genetic heritage, but a new vision of its functions has emerged recently. Indeed, sperm cells also carry small noncoding RNAs (sncRNAs) into oocytes during fertilization. Although their roles remain largely unclear, involvement of sperm-borne miRNAs in mouse epigenetic inheritance has been evidenced and paternal miRNAs and/or endo-siRNAs have been shown to be dispensable for fertilization but crucial for the transcriptomic homeostasis and the developmental of zygotes and 2 cells-embryos. This study aimed at unraveling the sncRNA content from bull frozen sperm cells and identifying miRNA or piRNAs associated with fertility. Total RNA has been produced from 40 ejaculates originating from Holstein and Montbeliard bulls with contrasting fertility. To achieve this task, a novel protocol has been developed to ensure good quality and reproducibility. Quality control was ensured by total RNA quantification using the RNA HS Qubit Assay kit, as well as miR125 quantitation by RTqPCR (miRCURY LNA Universal RT microRNA PCR kit; Exiqon). Starting from 77 million of frozen sperm cells on average, RNA yield was 51 ± 15ng, leading to miR125 Cycle Threshold values in a 19 - 20 range. Only small RNA (

Pheromones: a response to future sustainable farming. The example of the male effect. 26. Annual Meeting of the European-Chemoreception-Research-Organization (ECRO)

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International audience; Spermatogenesis involves structural modifications including epigenetic remodeling of the genome. It allows the sperm genome to be compacted and protected, a prerequisite to the transmission of paternal genetic heritage to the next generation. Therefore, any environmental changes that impact epigenetic remodeling during spermatogenesis may alter sperm function and male fertility. DNA methylation is instrumental in this epigenetic remodeling, and alterations of methylation profiles have been associated with male infertility. While bull semen is widely used in artificial insemination, the literature describing DNA methylation in bovine sperm is scarce. The aim was to characterize the sperm methylome in cattle. The methods used were contrasted in terms of resolution (global rate vs base-level resolution) and genome coverage (genome-wide vs candidate regions). Global quantification by LUminometric Methylation Assay (LUMA) in six species revealed a species-specific behavior of DNA methylation in sperm. Methylation level was lower in bovine semen (~45%) than in other species (~70 to 80%), and this level was not affected by cryopreservation. Somatic cells showed similar level in all species. Breed-dependent variations were detected, suggesting genetic factors in the determination of methylation level. To map DNA methylation at a genome-wide scale, we used reduced representation bisulfite sequencing (RRBS) and Methylated-DNA Immunoprecipitation (MeDIP-chip). Differentially methylated regions between tissues (tDMRs) were identified when sperm, liver, fibroblasts and monocytes were compared. Some of these tDMRs were in the vicinity of genes enriched in important pathways for sperm physiology, and most of them were hypomethylated in sperm. Pyrosequencing validation of these tDMR is currently ongoing.

miRNA gene body coverage. The read coverage of known miRNAs genes per library. On the Y axis coverage measured by Pearson’s skewness coefficients. All transcripts were scaled into 100 nt and the length is denoted on the X axis (RSeQC v.2.6.1). (PDF 33 kb)

Background: MiRNAs regulate multiple genes at the post-transcriptional level and therefore play an important role in many biological processes. It has been suggested that miRNA exported outside the cells contribute to intercellular communication. Consequently, circulating miRNAs are of particular interest and are promising biomarkers for many diseases. The number of miRNAs annotated in the horse genome is much lower compared to model organisms like human and mouse. We therefore aimed to identify novel equine miRNAs for tissue types and breed in serum. [br/] Results: We analysed 71 small RNA-seq libraries derived from nine tissues (gluteus medius, platysma, masseter muscle, heart, liver, cartilage, bone, total blood and serum) using miRDeep2 and miRdentify tools. Known miRNAs represented between 2.3 and 62.9 % of the reads in 71 libraries. A total of 683 novel miRNAs were identified. Breed and tissue type affected the number of miRNAs detected and interestingly, affected its average intensity. A total of 50 miRNAs in serum proved to be potential biomarkers to differentiate specific breed types, of which miR-122, miR-200, miR-483 were over-expressed and miR-328 was under-expressed in ponies compared to Warmbloods. The different miRNAs profiles, as well as the differences in their expression levels provide a foundation for more hypotheses based on the novel miRNAs discovered. [br/] Conclusions: We identified 683 novel equine miRNAs expressed in seven solid tissues, blood and serum. Additionally, our approach evidenced that such data supported identification of specific miRNAs as markers of functions related to breeds or disease tissues.

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Prediction of male fertility remains a major goal to promote animal insemination (AI). The combination of several markers of sperm quality provides promising results but remain still unsatisfactory. The crucial role of epigenetics in sperm functions and fertilization efficiency is now recognized. A better understanding of epigenetic mechanisms such as DNA methylation may highlight new key criteria that could improve prediction models. Recently, an increased methylation level at the imprinted Gnas locus has been observed in infertile boars (Congras et al., 2014). Our study evaluated the relationship between CpG methylation status at this locus and the field fertility (SF) score of Holstein bulls. Firstly, 56 CpG positions of GNAS locus were analyzed by bisulfite conversion and pyrosequencing using gDNA from 25 Holstein bulls. A negative correlation was observed between SF and methylation status. The two most informative CpG were selected for further investigation using an extended cohort (113 bulls; SF mean=-1.6±7.0 [-30 to +7.5]). Low methylation levels were observed (CpG#1: 26.7±3.4%; CpG#2: 14.4±2.4%). High variability between bulls (min/max %: 18.3/34.4 and 9.7/20.8) and bull families was noted (e.g. mean CpG#1 methylation percentage inner bull families ranging from 21.3±0.2% to 32.3±1.9%), suggesting that the genetic background may affect the methylation status. However, the correlation between SF and the methylation level was lossed. More studies will probably be required to explore the link between the GNAS methylation variability and fertility efficiency or embryo developmen. In conclusion, the methylation status at the Gnas locus does not provide accurate information to improve fertility prediction models. However, our results pave the way to study the relationship between genetics and methylation status at specific or genome-wide levels (ANR SeQuaMol).

Spermatogenesis is a long process requiring several epigenetic modifications to obtain a stably packed chromatin. Recent studies suggest that stochastic and environmental disturbances in this genome-wide epigenetic reprogramming may lead to epigenetic aberrations of semen quality, fertility and embryo development (Boissonnas et al., 2013). Establishing reference values and profiles of spermatozoa DNA methylation provides the opportunity to identify biological disturbances in epigenetic marks, as well as protective measures. Here, we present tools to monitor sperm DNA methylation at different scales and to identify individual and temporal variations in the methylation contents and patterns. The global quantification of DNA methylation amount (MeDNA) estimated by LUminometric Methylation Assay, demonstrated a species specificity (bovine, porcine, ovine, human and murine semen). In bovine, the MeDNA wasn’t higher in semen than in somatic tissues as well as between fresh and cryopreserved semen. However, breed-dependent variations of semen MeDNA were found. To identify methylated regions in bovine semen, we used immunoprecipitation of methylated DNA followed by hybridization on a bovine promoter microarray (MeDIP-chip). The microarray targets the upstream region (-2000/+1360bp) of 21416 genes (UMD3.1 assembly). After normalization of the data, enriched probes were identified using ChIPmix (Martin-Magniette et al., 2008). Genomic DNA from sperm (4 fertile bulls), liver (19 females) and fibroblasts (3 females) were analyzed. Differentially Methylated Regions between tissues (T-DMRs) were identified using Spatstat R package. T-DMRs in sperm were enriched for genes involved in the spliceosome (DAVID analysis). Most of theseT-DMRs were demethylated in sperm. We developed successful and relevant tools to analyze the methylation statue of bovine semen. Now we could analyze the environmental impact on the sperm epigenetic profile according to the spermogram and fertility.

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Quantitative trait loci (QTL) mapping for susceptibility to a Salmonella Abortusovis vaccinal strain was performed using an experimental design involving 30 Romane sheep sire families (1216 progenies). Nine QTL corresponding to bacterial load, weight variations and antibody response criteria were mapped on eight chromosomes, including the major histocompatibility complex area on chromosome 20. Surprisingly, none was found to be significant in the SLC11A1 region (formerly NRAMP1) that has been shown to influence Salmonella susceptibility in other species.

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Genomics is a scientific discipline concerned with the study of genome structure, function and evolutionary changes. The field relies on systematic approaches to study the genome architecture and DNA sequence (structural genomics) as well as the regulation of genome expression (functional genomics). Resulting knowledge and technological tools may find numerous applications in the equine industry, such as genetic disease screening, identification of animals susceptible to various diseases, training management as well as genetic improvement and breeding. Equine veterinarians have a major role to play in the design and implementation of such work, which in turn should also provide new opportunities. Indeed, veterinarians may benefit from this genomic information to develop genetic counselling, to suggest relevant changes in breeding practices or to design novel treatment strategies.

A genome-wide association study for osteochondrosis (OC) in French Trotter horses was carried out to detect QTL using genotype data from the Illumina EquineSNP50 BeadChip assay. Analysis data came from 161 sire families of French Trotter horses with 525 progeny and family sizes ranging from 1 to 20. Genotypes were available for progeny (n = 525) and sires with at least 2 progeny (n = 98). Radiographic data were obtained from progeny using at least 10 views to reveal OC. All radiographic findings were described by at least 2 veterinary experts in equine orthopedics, and severity indices (scores) were assigned based on the size and location of the lesion. Traits used were a global score, the sum of all severity scores lesions (GM, quantitative measurement), and the presence or absence of OC on the fetlock (FM), hock (HM), and other sites (other). Data were analyzed using 2 mixed models including fixed effects, polygenic effects, and SNP or haplotype cluster effects. By combining results with both methods at moderate evidence of association threshold P < 5 × 10−5, this genome-wide association study displayed 1 region for GM on the Equus caballus chromosome (ECA) 13, 2 for HM on ECA 3 and 14, and 1 for other on ECA 15. One region on ECA 3 for HM represented the most significant hit (P = 3 × 10−6). By comparing QTL between traits at a decreased threshold (P < 5 × 10−4), the 4 QTL detected for GM were associated to a QTL detected for FM or HM but never both. Another interesting result was that no QTL were found in common between HM and FM.

Gastrointestinal nematodes are one of the main health issues in sheep breeding. To identify loci affecting the resistance to Haemonchus contortus, a genome scan was carried out using 1,275 Romane × Martinik Black Belly backcross lambs. The entire population was challenged with Haemonchus contortus in 2 consecutive experimental infections, and fecal egg counts (FEC) and packed cell volumes were measured. A subgroup of 332 lambs with extreme FEC was necropsied to determine the total worm burden, length of female worms, sex ratio in the worm population, abomasal pH, and serum and mucosal G immunoglobulins (IgG) responses. Pepsinogen concentration was measured in another subset of 229 lambs. For QTL detection, 160 microsatellite markers were used as well as the Illumina OvineSNP50 BeadChip that provided 42,469 SNP markers after quality control. Linkage, association, and joint linkage and association analyses were performed with the QTLMAP software. Linkage disequilibrium (LD) was estimated within each pure breed, and association analyses were carried out either considering or not the breed origin of the haplotypes. Four QTL regions on sheep chromosomes (OAR)5, 12, 13, and 21 were identified as key players among many other QTL with small to moderate effects. A QTL on OAR21 affecting pepsinogen concentration exactly matched the pepsinogen (PGA5) locus. A 10-Mbp region affecting FEC after the 1st and 2nd infections was found on OAR12. The SNP markers outperformed microsatellites in the linkage analysis. Taking advantage of the LD helped to refine the locations of the QTL mapped on OAR5 and 13.

A genome-wide association study for osteochondrosis (OC) in French Trotter horses was carried out to detect QTL using genotype data from the Illumina EquineSNP50 BeadChip assay. Analysis data came from 161 sire families of French Trotter horses with 525 progeny and family sizes ranging from 1 to 20. Genotypes were available for progeny (n = 525) and sires with at least 2 progeny (n = 98). Radiographic data were obtained from progeny using at least 10 views to reveal OC. All radiographic findings were described by at least 2 veterinary experts in equine orthopedics, and severity indices (scores) were assigned based on the size and location of the lesion. Traits used were a global score, the sum of all severity scores lesions (GM, quantitative measurement), and the presence or absence of OC on the fetlock (FM), hock (HM), and other sites (other). Data were analyzed using 2 mixed models including fixed effects, polygenic effects, and SNP or haplotype cluster effects. By combining results with both methods at moderate evidence of association threshold P < 5 × 10−5, this genome-wide association study displayed 1 region for GM on the Equus caballus chromosome (ECA) 13, 2 for HM on ECA 3 and 14, and 1 for other on ECA 15. One region on ECA 3 for HM represented the most significant hit (P = 3 × 10−6). By comparing QTL between traits at a decreased threshold (P < 5 × 10−4), the 4 QTL detected for GM were associated to a QTL detected for FM or HM but never both. Another interesting result was that no QTL were found in common between HM and FM.

La génomique est une discipline de la biologie qui étudie la structure, le fonctionnement et l'évolution des génomes par des approches systématiques et à haut débit. Elle permet l'acquisition de connaissances à une échelle et à une vitesse qui révolutionne les approches scientifiques classiques. Ces connaissances et les avancées technologiques offrent de multiples perspectives en amélioration génétique des animaux ou en termes de gestion des populations. De même, les applications en biologie prédictive pourraient contribuer à l'évolution des pratiques zootechniques et vétérinaire dans le sens d'une meilleure prise en compte des particularités individuelles. Mais, loin des idées naïves " du tout génomique " des années 1990, en aucun cas elle ne constitue une solution miracle. La complexité du vivant fait que nous sommes encore très loin de comprendre comment la séquence d'ADN dirige la construction et le fonctionnement d'un être vivant. Après avoir présenté les principales caractéristiques du génome et les principales méthodes de la génomique, cet article tentera d'en évaluer les potentialités à moyen terme en vue de la production d'un cheval sain et performant.

La génomique est la science qui étudie la structure, le fonctionnement et l'évolution des génomes, l'ensemble du patrimoine génétique de tout être vivant. Porteuse de nombreux fantasmes dans les années 90, où le tout génomique apparaissait comme une éventuelle solution miracle à bien des maux, cette science permet de mieux comprendre et d'évaluer certains potentiels, mais, comme toute prédiction statistique, ne permet pas encore de garantir avec certitude un résultat individuel, notamment lorsqu'elle s'applique au sport et à la performance d'un cheval. Dans un contexte économique difficile pour les éleveurs, la génomique pourrait apporter des éléments de réponses susceptibles de contribuer au maintien du niveau d'excellence des populations équines françaises et à la compétitivité économique de la filière. Seulement voilà, faute de prise de conscience, les opportunités stagnent, ce qui menace à court terme la recherche équine en la matière. Les applications que permet aujourd'hui la génomique sont encore embryonnaires chez le cheval, limitées par l'insuffisance des recherches et des études de validation, elles-mêmes limitées par le peu d'intérêt manifesté par la filière. Faute d'un dialogue constructif entre d'une part les chercheurs, et d'autre part les cavaliers, éleveurs, vétérinaires et centres équestres, des opportunités resteront peut-être au stade végétatif. Qu'un éleveur ne souhaite pour l'instant pas détecter les anomalies héréditaires dont tous les poulains d'un même parent sur lequel il a investi peuvent souffrir, s'explique, tant par ledit contexte économique difficile que par l'attachement sentimental et passionnel qui le lie au géniteur. Pour autant, le dépistage des affections génétiques n'est pas le seul domaine d'intervention de la génomique, qui connaîtra indubitablement un essor important au niveau international dans des domaines aussi variés que la médecine vétérinaire personnalisée, la caractérisation des aptitudes sportives et l'optimisation de l'alimentation. Encore faudra-t-il que les acteurs de la filière deviennent acteurs du développement de la recherche. Des choix stratégiques, structurants, sont aujourd'hui à faire.

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Diffusion du document : ID 443; absent

Diffusion du document : ID 443; absent