Identification of sperm proteins as biomarkers of field fertility in Holstein-Friesian bulls used for artificial insemination.

Despite passing stringent quality control, bulls used in artificial insemination can vary significantly in their fertility, emphasizing the need for reliable markers of sperm quality. This study aimed to identify sperm proteins acting as biomarkers of fertility in 2 different populations of dairy bulls classified based on their field fertility. Semen was collected and cryopreserved from: 54 Holstein bulls located in Ireland, classified according to fertility indexes as low fertility (LF, n = 23), medium fertility (n = 14), or high fertility (HF, n = 17); and 18 Holstein bulls located in Denmark, classified as LF (n = 8) or HF (n = 10). The proteome was measured through liquid chromatography-mass spectrometry and data were analyzed with the R software. Differentially abundant proteins between HF and LF bulls and biomarker proteins were determined through a modified t-test and random forest, respectively, selecting 301 differentially abundant proteins and 34 biomarker proteins. The predictive ability of the 34 biomarkers was evaluated employing support vector machine as the classifier, using their abundance levels in the Irish bulls to train the model and in the Danish bulls for validation. The prediction accuracy was 94.4%, with only one HF bull misclassified, corresponding to the lowest fertility index bull in the HF group. The biomarkers more abundant in sperm of HF bulls enriched axoneme assembly and sperm motility (false discovery rate <0.05), according to functional analysis. In conclusion, a robust model coupled with the application of appropriate bioinformatic tools allowed the identification of functionally relevant sperm proteins predictive of the fertility of Holstein bulls used in artificial insemination.

Genetic tools to improve reproduction traits in dairy cattle.

Fertility is a major concern in the dairy cattle industry and has been the subject of numerous studies over the past 20 years. Surprisingly, most of these studies focused on rough female phenotypes and, despite their important role in reproductive success, male- and embryo-related traits have been poorly investigated. In recent years, the rapid and important evolution of technologies in genetic research has led to the development of genomic selection. The generalisation of this method in combination with the achievements of the AI industry have led to the constitution of large databases of genotyping and sequencing data, as well as refined phenotypes and pedigree records. These resources offer unprecedented opportunities in terms of fundamental and applied research. Here we present five such examples with a focus on reproduction-related traits: (1) detection of quantitative trait loci (QTL) for male fertility and semen quality traits; (2) detection of QTL for refined phenotypes associated with female fertility; (3) identification of recessive embryonic lethal mutations by depletion of homozygous haplotypes; (4) identification of recessive embryonic lethal mutations by mining whole-genome sequencing data; and (5) the contribution of high-density single nucleotide polymorphism chips, whole-genome sequencing and imputation to increasing the power of QTL detection methods and to the identification of causal variants.

Reproductive technologies and genomic selection in dairy cattle.

Genomic tools are now available for most livestock species and are used routinely for genomic selection (GS) in cattle. One of the most important developments resulting from the introduction of genomic testing for dairy cattle is the application of reasonably priced low-density single nucleotide polymorphism technology in the selection of females. In this context, combining genome testing and reproductive biotechnologies in young heifers enables new strategies to generate replacement and elite females in a given period of time. Moreover, multiple markers have been detected in biopsies of preimplantation stage embryos, thus paving the way to develop new strategies based on preimplantation diagnosis and the genetic screening of embryos. Based on recent advances in GS, the present review focuses on new possibilities inherent in reproductive technologies used for commercial purposes and in genetic schemes, possible side effects and beneficial impacts on reproductive efficiency. A particular focus is on the different steps allowing embryo genotyping, including embryo micromanipulation, DNA production and quality assessment.

Detection of quantitative trait loci for resistance to gastrointestinal nematode infections in Creole goats.

This study aimed to identify regions of the genome affecting resistance to gastrointestinal nematodes in a Creole goat population naturally exposed to a mixed nematode infection (Haemonchus contortus, Trichostrongylus colubriformis and Oesophagostomum columbianum) by grazing on irrigated pasture. A genome-wide quantitative trait loci (QTL) scan was performed on 383 offspring from 12 half-sib families. A total of 101 microsatellite markers were genotyped. Traits analysed were faecal egg count (FEC), packed cell volume (PCV), eosinophil count and bodyweight (BW) at 7 and 11 months of age. Levels of activity of immunoglobulin A (IgA) and activity of immunoglobulin E (IgE) anti-Haemonchus contortus L3 crude extracts and adult excretion/secretion products (ESPs) were also analysed. Using interval mapping, this study identified 13 QTL for parasite resistance. Two QTL linked with FEC were found on chromosomes 22 and 26. Three QTL were detected on chromosomes 7, 8 and 14 for eosinophil counts. Three QTL linked with PCV were identified on chromosomes 5, 9 and 21. A QTL for BW at 7 months of age was found on chromosome 6. Lastly, two QTL detected on chromosomes 3 and 10 were associated with IgE anti-L3, and IgE anti-ESP was linked with two QTL on chromosomes 1 and 26. This study is the first to have identified regions of the genome linked with nematode resistance in a goat population using a genome scan. These results provide useful tools for the understanding of parasite resistance in small ruminants.

Quantitative trait loci for resistance to infection in sheep using a live Salmonella Abortusovis vaccine.

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.

Molecular cytogenetics and comparative mapping in goats (Capra hircus, 2n = 60).

Few goat genome analysis projects have been developed in the last 10 years. The aim of this review was to compile and update all available cytogenetic mapping data, according to the last goat chromosome nomenclature, as well as human and cattle whole genome sequences. In particular, human regions homologous to most of the FISH-mapped microsatellites were identified in silico. This new goat cytogenetic map made it possible to refine delineation of conserved segments relative to the human and cattle genomic sequence. These improvements did not lead to detection of major new rearrangements within ruminants but confirmed the good conservation of synteny and the numerous intrachromosomal rearrangements observed between goats and humans.

Identification of new quantitative trait Loci (other than the PRNP gene) modulating the scrapie incubation period in sheep.

Although susceptibility to scrapie is largely controlled by the PRNP gene, we have searched for additional genomic regions that affect scrapie incubation time in sheep, using two half-sib families with a susceptible PRNP genotype and naturally infected by scrapie. Quantitative trait loci were detected on OAR6 and OAR18.

Rapid Discovery of De Novo Deleterious Mutations in Cattle Enhances the Value of Livestock as Model Species.

In humans, the clinical and molecular characterization of sporadic syndromes is often hindered by the small number of patients and the difficulty in developing animal models for severe dominant conditions. Here we show that the availability of large data sets of whole-genome sequences, high-density SNP chip genotypes and extensive recording of phenotype offers an unprecedented opportunity to quickly dissect the genetic architecture of severe dominant conditions in livestock. We report on the identification of seven dominant de novo mutations in CHD7, COL1A1, COL2A1, COPA, and MITF and exploit the structure of cattle populations to describe their clinical consequences and map modifier loci. Moreover, we demonstrate that the emergence of recessive genetic defects can be monitored by detecting de novo deleterious mutations in the genome of bulls used for artificial insemination. These results demonstrate the attractiveness of cattle as a model species in the post genomic era, particularly to confirm the genetic aetiology of isolated clinical case reports in humans.

A genetic linkage map of the male goat genome.

This paper presents a first genetic linkage map of the goat genome. Primers derived from the flanking sequences of 612 bovine, ovine and goat microsatellite markers were gathered and tested for amplification with goat DNA under standardized PCR conditions. This screen made it possible to choose a set of 55 polymorphic markers that can be used in the three species and to define a panel of 223 microsatellites suitable for the goat. Twelve half-sib paternal goat families were then used to build a linkage map of the goat genome. The linkage analysis made it possible to construct a meiotic map covering 2300 cM, i.e., > 80% of the total estimated length of the goat genome. Moreover, eight cosmids containing microsatellites were mapped by fluorescence in situ hybridization in goat and sheep. Together with 11 microsatellite-containing cosmids previously mapped in cattle (and supposing conservation of the banding pattern between this species and the goat) and data from the sheep map, these results made the orientation of 15 linkage groups possible. Furthermore, 12 coding sequences were mapped either genetically or physically, providing useful data for comparative mapping.

Use of combinations of in vitro quality assessments to predict fertility of bovine semen.

Predicting in vivo fertility of bull ejaculates using in vitro-assessed semen quality criteria remains challenging for the breeding industry. New technologies such as computer-assisted semen analysis (CASA) and flow cytometry may provide accurate and objective methods to improve semen quality control. The aim of this study was to evaluate the relationship between semen quality parameters and field fertility of bull ejaculates. A total of 153 ejaculates from 19 Holstein bulls have been analyzed using CASA (postthawing semen motility and morphology) and several flow cytometric tests, including sperm DNA integrity, viability (estimated by membrane integrity), acrosomal integrity, mitochondria aerobic functionality and oxidation. Samples were analyzed both immediately after thawing and after 4 hours at 37 °C. A fertility value (FV), based on nonreturn rate at 56 days after insemination and adjusted for environment factors, was calculated for each ejaculate. Simple and multiple regressions have been used to correlate FV with CASA and flow cytometric parameters. Significant simple correlations have been observed between some parameters and FV (e.g., straight line velocity [µm/s], r(2) = -0.12; polarized mitochondria sperm (%), r(2) = 0.07), but the relation between simple parameter and FV was too week to predict the fertility. Partial least square procedure identified several mathematical models combining flow cytometer and CASA variables and had better correlations with FV (adjusted r(2) ranging between 0.24 and 0.40 [P < 0.0001], depending on the number of included variables). In conclusion, this study suggests that quality assessment of thawed bull sperm using CASA and flow cytometry may provide a reasonable prediction of bovine semen fertility. Additional work will be required to increase the prediction reliability and promote this technology in routine artificial insemination laboratory practice.

Chromosomal localization of sixty autosomal loci in sheep (OVIS ARIES, 2n = 54) by fluorescence in situ hybridization and R-banding.

Sixty autosomal loci (5 type I and 55 type II) from 24 bovine syntenic groups, and previously FISH-mapped to goat and river buffalo chromosomes, were localized by fluorescence in situ on sheep (OVIS ARIES, 2n = 54) chromosomes, thereby notably extending the cytogenetic map of this economically important species. Caprine BAC clones were hybridized to R-banded chromosome preparations. FITC-signals and RBPI- banding (R-banding by late BrdU-incorporation and propidium iodide staining) were simultaneously visualized and captured by a colour CCD-camera. All mapped loci were localized on homoeologous chromosomes and chromosome regions (bands) of sheep, goat and river buffalo, further supporting chromosome and genetic (loci) homoeologies among bovids.

Mapping of 195 genes in cattle and updated comparative map with man, mouse, rat and pig.

Our on-going goal is to improve and update the comparative genome organization between cattle and man but also among the most detailed mammalian species genomes i.e. cattle, mouse, rat and pig. In this work, we localized 195 genes in cattle and checked all human/bovine non-concordant localizations found in the literature. Next, we compiled all the genes mapped in cattle, goat, sheep and pig (2,166) for which the human ortholog with its chromosomal position is known, added corresponding data in mouse and rat, and ordered the genes relatively to the human genome sequence. We estimate that our compilation provides bovine mapping information for about 89% of the human autosomes. Thus, a near complete, overall and detailed picture of the number, distribution and extent of bovine conserved syntenies (regardless of gene order) on human R-banded autosomes is proposed as well as a comparison with mouse, rat and pig genomes.

The river buffalo (Bubalus bubalis, 2n = 50) cytogenetic map: assignment of 64 loci by fluorescence in situ hybridization and R-banding.

Sixty-four genomic BAC-clones mapping five type I (ADCYAP1, HRH1, IL3, RBP3B and SRY) and 59 type II loci, previously FISH-mapped to goat (63 loci) and cattle (SRY) chromosomes, were fluorescence in situ mapped to river buffalo R-banded chromosomes, noticeably extending the physical map of this species. All mapped loci from 26 bovine syntenic groups were located on homeologous chromosomes and chromosome regions of river buffalo and goat (cattle) chromosomes, confirming the high degree of chromosome homeologies among bovids. Furthermore, an improved cytogenetic map of the river buffalo with 293 loci from all 31 bovine syntenic groups is reported.

Genomic structure, polymorphism and expression of the horse alpha-actinin-3 gene.

Gene characterization is an important feature for genome annotation and more particularly for candidate genes that could be selected in domestic species. Associations between an alpha-actinin-3 gene polymorphism and muscle performance were reported in humans involving a nonsense mutation (R577X) and in mice after inactivation of the gene. Here, we characterized the equine alpha-actinin-3 (ACTN3) gene by sequencing and transcript analysis. The cDNA was determined to be 3.47 kb in length with an open reading frame of 2709 bp expectedly encoding a protein 902 amino acids long. The ACTN3 gene is 13.2 kb long and contains 21 exons. The equine ACTN3 gene has a ubiquitous expression but it is overexpressed in skeletal muscles with fast fibers of type IIb. No alternative transcripts were observed. Sequencing the cDNA revealed 8 SNPs, 6 in the coding and 2 in the 3' non-coding regions with no amino acid change and not affecting potential miRNA targets. The equine in silico promoter sequence reveals a structure with two regions similar to those of other mammalian species.

Genome-wide association studies for osteochondrosis in French Trotter horses.

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.

A genome scan for QTL affecting resistance to Haemonchus contortus in sheep.

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.

An extended river buffalo (Bubalus bubalis, 2n = 50) cytogenetic map: assignment of 68 autosomal loci by FISH-mapping and R-banding and comparison with human chromosomes.

We report an extended river buffalo (Bubalus bubalis, 2n = 50; BBU) cytogenetic map including 388 loci, of which 68 have been FISH-mapped on autosomes in the present study. Ovine and caprine BAC clones containing both type I loci (known genes) and type II loci (simple sequence repeats (SRs), microsatellite marker, sequence-tagged sites (STSs)), previously assigned to sheep chromosomes, have been localized on R-banded river buffalo chromosomes (BBU), which expands the cytogenetic map of this important domestic species and increases our knowledge of the physical organization of its genome. The loci mapped in the present study correspond to loci already localized on homoeologous cattle (and sheep) chromosomes and chromosome bands, further confirming the high degree of chromosome homoeologies among bovids. The comparison of the integrated cytogenetic maps of BBU2p/BBU10 and BBU5p/BBU16 with those of human chromosomes (HSA) 6 and 11, respectively, identified, at least, nine conserved chromosome segments in each case and complex rearrangements differentiating river buffalo (and cattle) and human chromosomes.

A 12 000-rad whole-genome radiation hybrid panel in sheep: application to the study of the ovine chromosome 18 region containing a QTL for scrapie susceptibility.

Whole-genome radiation hybrid (RH) panels have been constructed for several species, including cattle. RH panels have proven to be an extremely powerful tool to construct high-density maps, which is an essential step in the identification of genes controlling important traits, and they can be used to establish high-resolution comparative maps. Although bovine RH panels can be used with ovine markers to construct sheep RH maps based on bovine genome organization, only some (c. 50%) of the markers available in sheep can be successfully mapped in the bovine genome. So, with the development of genomics and genome sequencing projects, there is a need for a high-resolution RH panel in sheep to map ovine markers. Consequently, we have constructed a 12 000-rad ovine whole-genome RH panel. Two hundred and eight hybrid clones were produced, of which 90 were selected based on their retention frequency. The final panel had an average marker retention frequency of 31.8%. The resolution of this 12 000-rad panel (SheepRH) was estimated by constructing an RH framework map for a 23-Mb region of sheep chromosome 18 (OAR18) that contains a QTL for scrapie susceptibility.

An advanced sheep (Ovis aries, 2n = 54) cytogenetic map and assignment of 88 new autosomal loci by fluorescence in situ hybridization and R-banding.

Presented herein is an updated sheep cytogenetic map that contains 452 loci (291 type I and 161 type II) assigned to specific chromosome bands or regions on standard R-banded ideograms. This map, which significantly extends our knowledge of the physical organization of the ovine genome, includes new assignments for 88 autosomal loci, including 74 type I loci (known genes) and 14 type II loci (SSRs/microsatellite marker/STSs), by FISH-mapping and R-banding. Comparison of the ovine map to the cattle and goat cytogenetic maps showed that common loci were located within homologous chromosomes and chromosome bands, confirming the high level of conservation of autosomes among ruminant species. Eleven loci that were FISH-mapped in sheep (B3GAT2, ASCC3, RARSL, BRD2, POLR1C, PPP2R5D, TNRC5, BAT2, BAT4, CDC5L and OLA-DRA) are unassigned in cattle and goat. Eleven other loci (D3S32, D1S86, BMS2621, SFXN5, D5S3, D5S68, CSKB1, D7S49, D9S15, D9S55 and D29S35) were assigned to specific ovine chromosome (OAR) bands but have only been assigned to chromosomes in cattle and goat.

Cytogenetic mapping of 25 goat mammary gland Expressed Sequence Tags (ESTs).

Today, there is a shift towards a positional candidate approach in the molecular identification of genes. This study reports on an Expressed Sequence Tags (ESTs) mapping initiative in goats, based on sequence information gathered from a previous mammary gland cDNA systematic sequencing project. A total of 25 novel genes was localised cytogenetically on 16 goat chromosomes. Six of these ESTs were found to map to cattle milk QTL regions. These results made it possible to assess the use of ESTs as a shortcut to the molecular identification of some QTLs and as a valuable tool for comparative mapping.