Widespread horse-based mobility arose around 2,200 BCE in Eurasia.

Horses revolutionized human history with fast mobility1. However, the timeline between their domestication and widespread integration as a means of transportation remains contentious2-4. Here we assemble a large collection of 475 ancient horse genomes to assess the period when these animals were first reshaped by human agency in Eurasia. We find that reproductive control of the modern domestic lineage emerged ~2,200 BCE (Before Common Era), through close kin mating and shortened generation times. Reproductive control emerged following a severe domestication bottleneck starting no earlier than ~2,700 BCE, and coincided with a sudden expansion across Eurasia that ultimately resulted in the replacement of nearly every local horse lineage. This expansion marked the rise of widespread horse-based mobility in human history, which refutes the commonly-held narrative of large horse herds accompanying the massive migration of steppe peoples across Europe ~3,000 BCE and earlier3,5. Finally, we detect significantly shortened generation times at Botai ~3,500 BCE, a settlement from Central Asia associated with corrals and a subsistence economy centered on horses6,7. This supports local horse husbandry before the rise of modern domestic bloodlines.

Genomic characterization of equine influenza A subtype H3N8 viruses by long read sequencing and functional analyses of the PB1-F2 virulence factor of A/equine/Paris/1/2018.

Equine influenza virus (EIV) remains a threat to horses, despite the availability of vaccines. Strategies to monitor the virus and prevent potential vaccine failure revolve around serological assays, RT-qPCR amplification, and sequencing the viral hemagglutinin (HA) and neuraminidase (NA) genes. These approaches overlook the contribution of other viral proteins in driving virulence. This study assesses the potential of long-read nanopore sequencing for fast and precise sequencing of circulating equine influenza viruses. Therefore, two French Florida Clade 1 strains, including the one circulating in winter 2018-2019 exhibiting more pronounced pathogenicity than usual, as well as the two currently OIE-recommended vaccine strains, were sequenced. Our results demonstrated the reliability of this sequencing method in generating accurate sequences. Sequence analysis of HA revealed a subtle antigenic drift in the French EIV strains, with specific substitutions, such as T163I in A/equine/Paris/1/2018 and the N188T mutation in post-2015 strains; both substitutions were in antigenic site B. Antigenic site E exhibited modifications in post-2018 strains, with the N63D substitution. Segment 2 sequencing also revealed that the A/equine/Paris/1/2018 strain encodes a longer variant of the PB1-F2 protein when compared to other Florida clade 1 strains (90 amino acids long versus 81 amino acids long). Further biological and biochemistry assays demonstrated that this PB1-F2 variant has enhanced abilities to abolish the mitochondrial membrane potential ΔΨm and permeabilize synthetic membranes. Altogether, our results highlight the interest in rapidly characterizing the complete genome of circulating strains with next-generation sequencing technologies to adapt vaccines and identify specific virulence markers of EIV.

A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitochondrial DNA variants.

BACKGROUND: Mitochondrial DNA is remarkably polymorphic. This is why animal geneticists survey mitochondrial genomes variations for fundamental and applied purposes. We present here an approach to sequence whole mitochondrial genomes using nanopore long-read sequencing. Our method relies on the selective elimination of nuclear DNA using an exonuclease treatment and on the amplification of circular mitochondrial DNA using a multiple displacement amplification step. RESULTS: We optimized each preparative step to obtain a 100 million-fold enrichment of horse mitochondrial DNA relative to nuclear DNA. We sequenced these amplified mitochondrial DNA using nanopore sequencing technology and obtained mitochondrial DNA reads that represented up to half of the sequencing output. The sequence reads were 2.3 kb of mean length and provided an even coverage of the mitochondrial genome. Long-reads spanning half or more of the whole mtDNA provided a coverage that varied between 118X and 488X. We evaluated SNPs identified using these long-reads by Sanger sequencing as ground truth and found a precision of 100.0%; a recall of 93.1% and a F1-score of 0.964 using the Twilight horse mtDNA reference. The choice of the mtDNA reference impacted variant calling efficiency with F1-scores varying between 0.947 and 0.964. CONCLUSIONS: Our method to amplify mtDNA and to sequence it using the nanopore technology is usable for mitochondrial DNA variant analysis. With minor modifications, this approach could easily be applied to other large circular DNA molecules.

Genotype imputation accuracy in multiple equine breeds from medium- to high-density genotypes.

Genotype imputation is now a key component of genomic analyses as it increases the density of available genotypes within a population. However, many factors can influence imputation accuracy. The aim of this study was to assess and compare the accuracy of imputation of high-density genotypes (Affymetrix Axiom Equine genotyping array, 670,806 SNPs) from two moderate-density genotypes (Illumina Equine SNP50 BeadChip, 54,602 SNPs and Illumina Equine SNP70 BeadChip, 65,157 SNPs), using single-breed or multiple-breed reference sets. Genotypes were available from five groups of horse breeds: Arab (AR, 1,207 horses), Trotteur Français (TF, 979 horses), Selle Français (SF, 1,979 horses), Anglo-Arab (AA, 229 horses) and various foreign sport horses (FH, 209 horses). The proportions of horses genotyped with the high-density (HD) chip in each breed group were 10% in AA, 15% in AR and FH, 30% in TF and 57% in SF. A validation set consisting of one-third of the horses genotyped with the HD chip was formed and their genotypes deleted. Two imputation strategies were compared, one in which the reference population consisted only of horses from the same breed group as in the validation set, and another with horses from all breed groups. For the first strategy, concordance rates (CRs) ranged from 97.8% (AR) to 99.0% (TF) and correlations (r²) from 0.94 (AR) to 0.99 (TF). For the second strategy, CR ranged from 97.4% (AR) to 98.9% (TF) and r² from 0.93 (AR) to 0.99 (TF). Overall, the results show a small advantage of within-breed imputation compared with multi-breed imputation. Adding horses from different breed groups to the reference population does not improve the accuracy of imputation. Imputation provides an accurate means of combining data sets from different genotyping platforms, now necessary with the increasing use of the recently developed Affymetrix Axiom Equine genotyping array.

Understanding the response to endurance exercise using a systems biology approach: combining blood metabolomics, transcriptomics and miRNomics in horses.

BACKGROUND: Endurance exercise in horses requires adaptive processes involving physiological, biochemical, and cognitive-behavioral responses in an attempt to regain homeostasis. We hypothesized that the identification of the relationships between blood metabolome, transcriptome, and miRNome during endurance exercise in horses could provide significant insights into the molecular response to endurance exercise. For this reason, the serum metabolome and whole-blood transcriptome and miRNome data were obtained from ten horses before and after a 160 km endurance competition. RESULTS: We obtained a global regulatory network based on 11 unique metabolites, 263 metabolic genes and 5 miRNAs whose expression was significantly altered at T1 (post- endurance competition) relative to T0 (baseline, pre-endurance competition). This network provided new insights into the cross talk between the distinct molecular pathways (e.g. energy and oxygen sensing, oxidative stress, and inflammation) that were not detectable when analyzing single metabolites or transcripts alone. Single metabolites and transcripts were carrying out multiple roles and thus sharing several biochemical pathways. Using a regulatory impact factor metric analysis, this regulatory network was further confirmed at the transcription factor and miRNA levels. In an extended cohort of 31 independent animals, multiple factor analysis confirmed the strong associations between lactate, methylene derivatives, miR-21-5p, miR-16-5p, let-7 family and genes that coded proteins involved in metabolic reactions primarily related to energy, ubiquitin proteasome and lipopolysaccharide immune responses after the endurance competition. Multiple factor analysis also identified potential biomarkers at T0 for an increased likelihood for failure to finish an endurance competition. CONCLUSIONS: To the best of our knowledge, the present study is the first to provide a comprehensive and integrated overview of the metabolome, transcriptome, and miRNome co-regulatory networks that may have a key role in regulating the metabolic and immune response to endurance exercise in horses.

Developing a 670k genotyping array to tag ~2M SNPs across 24 horse breeds.

BACKGROUND: To date, genome-scale analyses in the domestic horse have been limited by suboptimal single nucleotide polymorphism (SNP) density and uneven genomic coverage of the current SNP genotyping arrays. The recent availability of whole genome sequences has created the opportunity to develop a next generation, high-density equine SNP array. RESULTS: Using whole genome sequence from 153 individuals representing 24 distinct breeds collated by the equine genomics community, we cataloged over 23 million de novo discovered genetic variants. Leveraging genotype data from individuals with both whole genome sequence, and genotypes from lower-density, legacy SNP arrays, a subset of ~5 million high-quality, high-density array candidate SNPs were selected based on breed representation and uniform spacing across the genome. Considering probe design recommendations from a commercial vendor (Affymetrix, now Thermo Fisher Scientific) a set of ~2 million SNPs were selected for a next-generation high-density SNP chip (MNEc2M). Genotype data were generated using the MNEc2M array from a cohort of 332 horses from 20 breeds and a lower-density array, consisting of ~670 thousand SNPs (MNEc670k), was designed for genotype imputation. CONCLUSIONS: Here, we document the steps taken to design both the MNEc2M and MNEc670k arrays, report genomic and technical properties of these genotyping platforms, and demonstrate the imputation capabilities of these tools for the domestic horse.

Novel equine tissue miRNAs and breed-related miRNA expressed in serum.

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 inter-cellular 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. 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. 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.

Next-generation sequencing identifies equine cartilage and subchondral bone miRNAs and suggests their involvement in osteochondrosis physiopathology.

BACKGROUND: MicroRNAs (miRNAs) are an abundant class of small single-stranded non-coding RNA molecules ranging from 18 to 24 nucleotides. They negatively regulate gene expression at the post-transcriptional level and play key roles in many biological processes, including skeletal development and cartilage maturation. In addition, miRNAs involvement in osteoarticular diseases has been proved and some of them were identified as suitable biomarkers for pathological conditions. Equine osteochondrosis (OC) is one of the most prevalent juvenile osteoarticular disorders in horses and represents a major concern for animal welfare and economic reasons. Its etiology and pathology remain controversial and biological pathways as well as molecular mechanisms involved in the physiopathology are still unclear. This study aims to investigate the potential role of miRNAs in equine osteochondrosis (OC) physiopathology.Short-read NGS technology (SOLID™, Life Technologies) was used to establish a comprehensive repertoire of miRNA expressed in either equine cartilage or subchondral bone. Undamaged cartilage and subchondral bone samples from healthy (healthy samples) and OC-affected (predisposed samples) 10-month Anglo-Arabian foals were analysed. Samples were also subjected or not to an experimental mechanical loading to evaluate the role of miRNAs in the regulation of mechano-transduction pathways. Predicted targets of annotated miRNAs were identified using miRmap. RESULTS: Epiphyseal cartilage and subchondral bone miRNome were defined, including about 300 new miRNAs. Differentially expressed miRNAs were identified between bone and cartilage from healthy and OC foals, as well as after an experimental mechanical loading. In cartilage, functional annotation of their predicted targets suggests a role in the maintenance of cartilage integrity through the control of cell cycle and differentiation, energy production and metabolism as well as extracellular matrix structure and dynamics. In bone, miRNA predicited targets were associated with osteoblasts and osteoclasts differentiation, though the regulation of energy production, vesicle transport and some growth factor signaling pathways. CONCLUSION: Taken together, our results suggest a role of miRNAs in equine OC physiopathology and in the cellular response to biomechanical stress in cartilage and bone. In silico target prediction and functional enrichment analysis provides new insight into OC molecular physiopathology.

Early dispersal of domestic horses into the Great Plains and northern Rockies.

The horse is central to many Indigenous cultures across the American Southwest and the Great Plains. However, when and how horses were first integrated into Indigenous lifeways remain contentious, with extant models derived largely from colonial records. We conducted an interdisciplinary study of an assemblage of historic archaeological horse remains, integrating genomic, isotopic, radiocarbon, and paleopathological evidence. Archaeological and modern North American horses show strong Iberian genetic affinities, with later influx from British sources, but no Viking proximity. Horses rapidly spread from the south into the northern Rockies and central plains by the first half of the 17th century CE, likely through Indigenous exchange networks. They were deeply integrated into Indigenous societies before the arrival of 18th-century European observers, as reflected in herd management, ceremonial practices, and culture.

Mining the equine gut metagenome: poorly-characterized taxa associated with cardiovascular fitness in endurance athletes.

Emerging evidence indicates that the gut microbiome contributes to endurance exercise performance. Still, the extent of its functional and metabolic potential remains unknown. Using elite endurance horses as a model system for exercise responsiveness, we built an integrated horse gut gene catalog comprising ~25 million unique genes and 372 metagenome-assembled genomes. This catalog represents 4179 genera spanning 95 phyla and functional capacities primed to exploit energy from dietary, microbial, and host resources. The holo-omics approach shows that gut microbiomes enriched in Lachnospiraceae taxa are negatively associated with cardiovascular capacity. Conversely, more complex and functionally diverse microbiomes are associated with higher glucose concentrations and reduced accumulation of long-chain acylcarnitines and non-esterified fatty acids in plasma, suggesting increased ß-oxidation capacity in the mitochondria. In line with this hypothesis, more fit athletes show upregulation of mitochondrial-related genes involved in energy metabolism, biogenesis, and Ca2+ cytosolic transport, all of which are necessary to improve aerobic work power, spare glycogen usage, and enhance cardiovascular capacity. The results identify an associative link between endurance performance and gut microbiome composition and gene function, laying the basis for nutritional interventions that could benefit horse athletes.

Antisense oligonucleotides targeting ORF1b block replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The ongoing COVID-19 pandemic continues to pose a need for new and efficient therapeutic strategies. We explored antisense therapy using oligonucleotides targeting the severe acute respiratory syndrome coronavirus (SARS-CoV-2) genome. We predicted in silico four antisense oligonucleotides (ASO gapmers with 100% PTO linkages and LNA modifications at their 5' and 3'ends) targeting viral regions ORF1a, ORF1b, N and the 5'UTR of the SARS-CoV-2 genome. Efficiency of ASOs was tested by transfection in human ACE2-expressing HEK-293T cells and monkey VeroE6/TMPRSS2 cells infected with SARS-CoV-2. The ORF1b-targeting ASO was the most efficient, with a 71% reduction in the number of viral genome copies. N- and 5'UTR-targeting ASOs also significantly reduced viral replication by 55 and 63%, respectively, compared to non-related control ASO (ASO-C). Viral titration revealed a significant decrease in SARS-CoV-2 multiplication both in culture media and in cells. These results show that anti-ORF1b ASO can specifically reduce SARS-CoV-2 genome replication in vitro in two different cell infection models. The present study presents proof-of concept of antisense oligonucleotide technology as a promising therapeutic strategy for COVID-19.

Longitudinal ambulatory measurements of gait abnormality in dystrophin-deficient dogs.

BACKGROUND: This study aimed to measure the gait abnormalities in GRMD (Golden retriever muscular dystrophy) dogs during growth and disease progression using an ambulatory gait analyzer (3D-accelerometers) as a possible tool to assess the effects of a therapeutic intervention. METHODS: Six healthy and twelve GRMD dogs were evaluated twice monthly, from the age of two to nine months. The evolution of each gait variable previously shown to be modified in control and dystrophin-deficient adults was assessed using two-ways variance analysis (age, clinical status) with repeated measurements. A principal component analysis (PCA) was applied to perfect multivariate data interpretation. RESULTS: Speed, stride length, total power and force significantly already decreased (p<0.01) at the age of 2 months. The other gait variables (stride frequency, relative power distributions along the three axes) became modified at later stages. Using the PCA analysis, a global gait index taking into account the main gait variables was calculated, and was also consistent to detect the early changes in the GRMD gait patterns, as well as the progressive degradation of gait quality. CONCLUSION: The gait variables measured by the accelerometers were sensitive to early detect and follow the gait disorders and mirrored the heterogeneity of clinical presentations, giving sense to monitor gait in GRMD dogs during progression of the disease and pre-clinical therapeutic trials.

A New Method for Sequencing the Mitochondrial Genome by Using Long Read Technology.

We describe a protocol to prepare a multiplexed mtDNA library from a blood sample for performing a long read sequencing of the mitochondrial genome. All steps are carefully described to get a high enrichment of mtDNA relative to total DNA extracted from the blood sample. The obtained mutiplexed library allows the production of long sequence mtDNA reads up to 16.5 kbp with a quality enabling variant-calling by using a portable sequencer (MinION, Oxford Nanopore Technologies).

Understanding the Holobiont: Crosstalk Between Gut Microbiota and Mitochondria During Long Exercise in Horse.

Endurance exercise has a dramatic impact on the functionality of mitochondria and on the composition of the intestinal microbiome, but the mechanisms regulating the crosstalk between these two components are still largely unknown. Here, we sampled 20 elite horses before and after an endurance race and used blood transcriptome, blood metabolome and fecal microbiome to describe the gut-mitochondria crosstalk. A subset of mitochondria-related differentially expressed genes involved in pathways such as energy metabolism, oxidative stress and inflammation was discovered and then shown to be associated with butyrate-producing bacteria of the Lachnospiraceae family, especially Eubacterium. The mechanisms involved were not fully understood, but through the action of their metabolites likely acted on PPARγ, the FRX-CREB axis and their downstream targets to delay the onset of hypoglycemia, inflammation and extend running time. Our results also suggested that circulating free fatty acids may act not merely as fuel but drive mitochondrial inflammatory responses triggered by the translocation of gut bacterial polysaccharides following endurance. Targeting the gut-mitochondria axis therefore appears to be a potential strategy to enhance athletic performance.

Accelerometers Provide Early Genetic Selection Criteria for Jumping Horses.

The aim of this study was to evaluate the genetic component of the locomotor jumping ability, via a wearable accelerometer sensor, and to estimate the genetic correlation with performance in competition, to introduce such criteria in selection schema. A sample of 1,056 young 3-year-old horses were equipped with a 3-dimensional accelerometer during a free jumping test, in regular breeding shows from 2015 to 2017. Seven variables were extracted from the dorso-ventral acceleration curve for the last three jumps over a double bar obstacle of 1.15 m for the front pole and 1.20 m for the back pole with a 1.20 m spread. Variables were the peaks of forelimbs, hindlimbs, and landing acceleration, the duration between peaks at take-off, the peak of forelimb acceleration and start of jump, jump duration and duration between the beginning of the impact of forelimbs and the peak at landing. During breeding shows, judges scored balance, strength, style, and reactivity for free jumping and jumping tests under saddle. Jumping competition results were recorded by logarithm of the sum of points earned in each competition. All horses in official competitions were included, i.e., 160,257 horses born in 1997 with a total of 649,491 annual performances. An animal mixed model with complete pedigree over four generations (353,236 horses) were used with fixed effects of jumping test location and date, morning/afternoon, gender, month of birth, rank of jump for accelerometric data, effect of year of competition, combined with age and gender for competition results. As a result, jump duration was the most heritable and repeatable for jump variables: h 2 = 0.16 (0.06), r = 0.52 (0.02), while accelerations were moderately heritable (h 2 = 0.05-0.09, r = 0.39-0.51). Judgement scores were heritable: 0.21 (0.07)-0.33 (0.09) and were highly correlated. Scores during free jumping were genetically correlated to jump duration: 0.71 (0.15)-0.88 (0.16). Both jump duration and judgement scores were genetically correlated to competition performance: 0.59 (0.13) for jump duration, from 0.60 (0.11) to 0.77 (0.12) for scores. Jump duration and judgement scores can be used as early selection criteria. The advantage of the accelerometric measurement is its objectivity and the ease of recording.

Publisher Correction: Unraveling the effects of the gut microbiota composition and function on horse endurance physiology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Gene expression profiling in equine polysaccharide storage myopathy revealed inflammation, glycogenesis inhibition, hypoxia and mitochondrial dysfunctions.

BACKGROUND: Several cases of myopathies have been observed in the horse Norman Cob breed. Muscle histology examinations revealed that some families suffer from a polysaccharide storage myopathy (PSSM). It is assumed that a gene expression signature related to PSSM should be observed at the transcriptional level because the glycogen storage disease could also be linked to other dysfunctions in gene regulation. Thus, the functional genomic approach could be conducted in order to provide new knowledge about the metabolic disorders related to PSSM. We propose exploring the PSSM muscle fiber metabolic disorders by measuring gene expression in relationship with the histological phenotype. RESULTS: Genotypying analysis of GYS1 mutation revealed 2 homozygous (AA) and 5 heterozygous (GA) PSSM horses. In the PSSM muscles, histological data revealed PAS positive amylase resistant abnormal polysaccharides, inflammation, necrosis, and lipomatosis and active regeneration of fibers. Ultrastructural evaluation revealed a decrease of mitochondrial number and structural disorders. Extensive accumulation of an abnormal polysaccharide displaced and partially replaced mitochondria and myofibrils. The severity of the disease was higher in the two homozygous PSSM horses.Gene expression analysis revealed 129 genes significantly modulated (p < 0.05). The following genes were up-regulated over 2 fold: IL18, CTSS, LUM, CD44, FN1, GST01. The most down-regulated genes were the following: mitochondrial tRNA, SLC2A2, PRKCalpha, VEGFalpha. Data mining analysis showed that protein synthesis, apoptosis, cellular movement, growth and proliferation were the main cellular functions significantly associated with the modulated genes (p < 0.05). Several up-regulated genes, especially IL18, revealed a severe muscular inflammation in PSSM muscles. The up-regulation of glycogen synthase kinase-3 (GSK3beta) under its active form could be responsible for glycogen synthase (GYS1) inhibition and hypoxia-inducible factor (HIF1alpha) destabilization. CONCLUSION: The main disorders observed in PSSM muscles could be related to mitochondrial dysfunctions, glycogenesis inhibition and the chronic hypoxia of the PSSM muscles.

Import of Non-Coding RNAs into Human Mitochondria: A Critical Review and Emerging Approaches.

Mitochondria harbor their own genetic system, yet critically depend on the import of a number of nuclear-encoded macromolecules to ensure their expression. In all eukaryotes, selected non-coding RNAs produced from the nuclear genome are partially redirected into the mitochondria, where they participate in gene expression. Therefore, the mitochondrial RNome represents an intricate mixture of the intrinsic transcriptome and the extrinsic RNA importome. In this review, we summarize and critically analyze data on the nuclear-encoded transcripts detected in human mitochondria and outline the proposed molecular mechanisms of their mitochondrial import. Special attention is given to the various experimental approaches used to study the mitochondrial RNome, including some recently developed genome-wide and in situ techniques.

A First Step Toward Unraveling the Energy Metabolism in Endurance Horses: Comparison of Plasma Nuclear Magnetic Resonance Metabolomic Profiles Before and After Different Endurance Race Distances.

Endurance racing places high demands on energy metabolism pathways. Metabolomics can be used to investigate biochemical responses to endurance exercise in humans, laboratory animals, and horses. Although endurance horses have previously been assessed in the field (i.e., during races) using broad-window Nuclear Magnetic Resonance metabolomics, these studies included several different race locations, race distances, age classes, and race statuses (finisher or elimination). The present NMR metabolomics study focused on 40 endurance horses racing in three race categories over 90, 120, or 160 km. The three races took place in the same location. Given that energy metabolism is closely related to exercise intensity and duration (and therefore distance covered), the study's objective was to determine whether the metabolic pathways recruited during the race varied as a function of the total ride distance. For each horse, a plasma sample was collected the day before the race, and another was collected at the end of the race. Sixteen, 15, and 9 horses raced over 90, 120, and 160 km, respectively. Proton NMR spectra (500 MHz) were acquired for these 80 plasma samples. After processing, the spectra were divided into bins representing the NMR variables and then classified using orthogonal projection on latent structure models supervised by the sampling time (pre- or post-race) or the distance covered. The models revealed that the post-race metabolomic profiles are associated to the total ride distance groups. By combining biochemical assay results and NMR data in multiblock models, we further showed that enzymatic activities and metabolites are significantly associated to the race category. In the highest race category (160 km), there appears to be a metabolic switch from carbohydrate consumption to lipid consumption in order to maintain glycaemia. Furthermore, signs of protein breakdown were more apparent in the longest race category. The metabolic shift seen in the different racing categories could be related to a mixture of three important factors that are the ride distance, the training status and the inherited endurance capacity of the various horses competing.

Unraveling the effects of the gut microbiota composition and function on horse endurance physiology.

An integrated analysis of gut microbiota, blood biochemical and metabolome in 52 endurance horses was performed. Clustering by gut microbiota revealed the existence of two communities mainly driven by diet as host properties showed little effect. Community 1 presented lower richness and diversity, but higher dominance and rarity of species, including some pathobionts. Moreover, its microbiota composition was tightly linked to host blood metabolites related to lipid metabolism and glycolysis at basal time. Despite the lower fiber intake, community type 1 appeared more specialized to produce acetate as a mean of maintaining the energy supply as glucose concentrations fell during the race. On the other hand, community type 2 showed an enrichment of fibrolytic and cellulolytic bacteria as well as anaerobic fungi, coupled to a higher production of propionate and butyrate. The higher butyrate proportion in community 2 was not associated with protective effects on telomere lengths but could have ameliorated mucosal inflammation and oxidative status. The gut microbiota was neither associated with the blood biochemical markers nor metabolome during the endurance race, and did not provide a biomarker for race ranking or risk of failure to finish the race.