The Baroque horse breeds, originating from Southern Europe, are known for their unique characteristics and roles in High School dressage and ceremonial contexts. These breeds have been shaped by centuries of selective breeding, geographic isolation, and interactions with other equine populations. This study explores the genetic diversity and population structure of Baroque breeds (Lipizzan, Murgese, Lusitano, and Pura Raza Espa & ntilde;ola) alongside eleven relevant equine populations including Baroque-derived, cold-blood and warm/hot-blood breeds. After quality control and subsampling, the final dataset included 440 genotyped individuals. The overall variability was assessed using genetic diversity indices while population structure and genetic relationships were inferred using PCA, ADMIXTURE, Neighbour-net and TreeMix analysis. The results show that the Baroque and Baroque-derived breeds form a cohesive genetic group exhibiting heterogeneous genetic diversity. Within the Baroque breeds, the Pura Raza Espa & ntilde;ola was more closely related to Lusitano while Lipizzan to Murgese. This genetic relatedness conforms with both historical connections and geographic proximity of these breeds. A widespread ancestral component of the Pura Raza Espa & ntilde;ola can be observed in both Baroque and Baroque-derived breeds reflecting its past role in moulding modern breeds. However, the Pura Raza Espa & ntilde;ola showed moderate genetic diversity and positive values of inbreeding suggesting the need for conservation efforts. Our results also highlight a more recent influence in terms of gene exchange of several warm/hot-blood breeds in shaping current Baroque breeds. These findings underscore the uniqueness of Baroque breeds and lays the foundation for clarifying their origin and impact on equine biodiversity.

Pigs are attracting increasing attention as a large-animal model with translational relevance in biomedical research. They share several immunological similarities with humans, although the pig immune system has not been fully characterized yet, also at the level of macrophage polarization. A better understanding of porcine macrophage polarization could improve translational studies, allowing a better interpretation of in vitro and in vivo results. In this study, we performed a detailed characterization of the impact of two poorly studied cytokines [interleukin-1β (IL-1β) and its receptor antagonist IL-1Ra] on porcine monocyte-derived macrophage (moMФ) phenotype and functions. The effects of these IL-1 family members were investigated through microscopy, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and gene expression studies. We observed that stimulation with IL-1β increased CD14, SLA-I, and SLA-II DR expression, and enhanced the release of pro-inflammatory cytokines, particularly IL-6, IL-12, and IL-1α at higher concentrations, and the expression of several antiviral and pro-inflammatory genes, including JUN, FOS, and MAP3K1. The concomitant release of IL-10 and IL-1Ra suggests that IL-1β stimulation may also activate compensatory regulatory feedback mechanisms aimed at limiting excessive inflammation. In contrast, IL-1Ra had limited effects on surface marker expression but increased IL-10 release. A small but significant release of IL-1α was also observed, as well as a trend toward enhanced IL1B expression at 24 h, suggesting a potential regulatory interaction within the IL-1 axis. MoMФ stimulated with IL-1Ra was also able to release high levels of IL-12 in response to stimulation with a TLR-3 ligand [polyinosinic-polycytidylic acid (poly I:C)], with an intensity comparable to moMФ and moMФ stimulated with IL-1β. Our data suggest that porcine macrophages attempt to maintain a balance between IL-1β and IL-1Ra, as a defense mechanism to control inflammation, by counteracting each other's activity. Overall, in this work, we provided a better understanding of porcine macrophage polarization, which could benefit translational studies using this large-animal model.

Milk is a rich source of bioactive extracellular vesicles (EVs), with potential therapeutic functions for intestinal inflammation. A major limitation for the clinical use is their preservation, as they typically require storage at − 80 °C. To address this, our study aimed to develop a stable, room-temperature dry powder formulation of milk EVs (mEVs) that preserves their bioactivity. The mEVs were isolated via differential centrifugation with an EDTA cleaning step and subsequently spray-dried into microparticles using mannitol and leucine as excipients. The resulting microparticles were morphologically characterized as collapsed structures with a volume-weighted mean diameter of 11–20 µm and low aggregation. We found that the drying process did not cause molecular alterations, as the dried mEVs preserved protein and nucleic acid content comparable to that of unprocessed mEVs. Furthermore, the formulation showed minimal cytotoxicity on THP-1 cells (up to 1011 particles/mL), as measured by MTT assay. In a cellular inflammatory model, spray-dried mEVs stored for up to 18 months significantly reduced the gene expression of key pro-inflammatory cytokines (IL1B, IL6, CXCL8, and TNFA) to a degree comparable to unprocessed mEVs. This confirms the long-term functional stability of the dry mEV formulation. Our findings demonstrate that proper spray-drying approaches are a flexible and effective strategies for preserving mEVs, paving the way to the development of EVs loaded microparticles for a broad range of functional and therapeutic applications.

Sarcoids are the most common cutaneous tumors in horses, representing up to 90% (35%–90%) of skin neoplasms. Mostly caused by Bovine Papillomavirus (BPVs) infections, sarcoids are highly resistant to therapy and prone to recurring, posing a significant threat to equine health. The aim of this study is to explore molecular pathogenetic mechanisms underlying the development of equine sarcoids, by applying transcriptomic approach. After testing samples for viral DNA, both mRNA and small RNA expression was analyzed via high-throughput Illumina sequencing comparing 12 sarcoids and 12 healthy skin samples as controls. Differentially expressed genes (DEGs), DE miRNAs (sarcoids vs. controls) and miRNA-DEG couples with opposite expression trends, were retrieved and subjected to a functional analysis. Over 6K DEGs emerged, 3620 down-regulated and 2415 up-regulated along with 145 DE miRNAs, 56 downregulated and 89 upregulated. Among the enriched biological processes for DEGs, some were related to growth factors production and collagen binding, cell migration and proliferation, tissue morphogenesis and inflammatory response. Interestingly, “Pathways in cancer” and “Hippo signaling pathway” were enriched KEGG pathways for the miRNA-DEG couples. Our data identified a great transcription discrepancy between sarcoid lesions and healthy skin with an overall enrichment for processes related to cellular transformation. RNA-seq sequencing depth allowed the search for candidate chimeric transcripts associated with viral integration events. Chimeric RNAs can influence gene regulation and may contribute to tumor growth and immune modulation. Via computational analysis we identified six fusion loci in tumor samples and in two sarcoid margins, with the most frequent event involving WNT10B and FKBP11. This fusion, detected in 6/10 sarcoids, is of particular interest since WNT10B activates the WNT/β-catenin cascade, while FKBP11 has been implicated in osteosarcoma progression. Although functional validation is ongoing, this represents the first report of chimeric transcripts in equine sarcoids, opening new perspectives on BPV-driven oncogenesis.

Phenolic compounds from olive mill wastewater (OMWW) have a strong antioxidant capacity, so there is increasing interest in using them in feed for livestock, including pigs. This study tested the effects of dietary supplementation with a polyphenol extract from OMWW for female Landrace × Duroc heavy finishing pigs. There were three groups: the control diet (C group), the control diet supplemented with 74 ppm of OMWW polyphenols (P-LOW group), and the control diet supplemented with 225 ppm of OMWW polyphenols (P-HIGH group). Each experimental group comprised 45 pigs (n = 15 × 3 replicates), for a total of 135 pigs. The effects of the phenolic extract were assessed in vivo (growth performance) and postmortem (backfat thickness; pubertal status; histopathology of the liver, ovary, uterus, fat, and muscle; morphometry of the liver, ovary, and uterus; antioxidant status in the blood, muscle, and liver; effects on the quality and physicochemical characteristics of the raw meat). There were no significant differences between the treatments regarding the growth performance traits, histopathological and morphometric findings, and backfat thickness. However, there was an increase in 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity in the liver of the P-HIGH group, alongside higher serum paraoxonase activity and ferric reducing antioxidant power. Meat quality analysis showed that cooking loss and redness (a*) decreased, while yellowness (b*) increased in the P-LOW and P-HIGH groups, indicating that OMWW polyphenols influenced the structure and water retention capacity of the meat. Additional research is required to better understand the role of dietary OMWW polyphenols in relation to the technological quality and antioxidant state of pork meat.

Milk is a primary source of vital nutrients and bioactive components fundamental to the growth and development of both newborn animals and humans. Produced by economically significant livestock species (including cattle, buffaloes, goats, sheep and camels), milk is a complex matrix rich in caseins, vitamins, fats, and proteins. Beyond its classical nutritional profile, milk serves as a pivotal vehicle for milk-derived extracellular vesicles (mEVs). These specialized food-derived EVs (fEVs) exert pleiotropic effects that resonate with the One Health paradigm, linking animal well-being and human nutrition to broader ecosystem stability. mEVs offer unique advantages, such as high biocompatibility and gastrointestinal stability, also rendering them potential therapeutic tools as drug delivery systems. However, challenges remain regarding the standardization of mEVs and the variability of their molecular cargo. This review provides a comprehensive comparative analysis of mEVs across a diverse taxonomic range, including bovines, water buffaloes, yaks, camels, goats, pigs, horses, donkeys, and humans, highlighting their distinct functional signatures. Indeed, a critical issue in mEV research is the isolation process: recommendations to minimize contamination from milk fat globules and casein micelles (which can cover EV signals) are given. Finally, current detection methods and instrumentation, with a specific focus on advancing flow cytometry (FC) approaches are discussed. Key insights include the use of conventional FC (with fluorescence triggering, the necessity of rigorous controls and calibration, and the utility of bead-based assays to overcome resolution limits) and imaging flow cytometry (IFC). In both technical approaches, the application of different EV generic fluorescent markers and the strategic selection of tetraspanins (i.e., CD9, CD63, CD81), is mandatory: we emphasize that selecting the correct antibody clones and accounting for inter-species cross-reactivity are essential steps for ensuring the accuracy and reproducibility of mEV research across mammalian species.

IntroductionIn horses, heat exposure modulates the hypothalamic-pituitary-adrenal axis, autonomic nervous system, and hypothalamic-pituitary-thyroid axis to maintain body temperature and prevent excessive heat accumulation. However, during strenuous exercise under hot and humid conditions, heat production may exceed dissipation, leading to heat stress, anhidrosis, heat stroke, or brain damage.MethodsIncremental field standardized exercise tests (fSETs) provide a reliable approach to assess training and fitness levels. Six Arabian horses from Italia Endurance Stable and Academy were monitored during fSETs performed under heat stress (HS) and thermoneutral (TN) conditions, with blood samples collected before and after each test. Hematocrit, lactate, and biochemical parameters were measured, and total serum RNA was sequenced. A protein-protein interaction (PPI) network of miRNA targets was constructed and analyzed for Gene Ontology (GO) enrichment.ResultsLactatemia and hematocrit were significantly higher in HS vs. TN, while alanine aminotransferase, creatinine, and creatine kinase increased in HS POST vs. PRE fSET. Differentially expressed small RNAs included eca-myomir-206, eca-mir-301, eca-mir-3613-3p, eca-mir-142, and eca-mir-144, which were modulated by temperature and exercise. In POST vs. PRE fSET, enriched terms involved transcriptional regulation, glucose and LDL response, intracellular trafficking, cytoskeleton organization, cardiac conduction, ion channels, and immune regulation. In HS POST vs. PRE fSET, enrichment was observed for positive regulation of dendritic cell cytokine production, negative regulation of inflammation, and attenuation of oxidative stress-induced apoptotic signaling.DiscussionThis study aimed to investigate the molecular features underlying resilience and adaptation to combined heat- and exercise-induced stress in horses. Overall, our findings indicate that heat amplifies the physiological burden of endurance exercise and alters the molecular mechanisms supporting performance and recovery. Circulating small RNAs may act as early signals for homeostatic restoration and could help elucidate adaptive responses to stress, guiding personalized training strategies.

Equine sarcoid is the most common cutaneous neoplasm of equids and represents a distinctive model of virus-associated tumor persistence. Although bovine papillomaviruses, particularly BPV-1 and BPV-2, are recognized as the main etiological agents, viral infection alone does not fully explain the clinical heterogeneity, frequent recurrence, and limited spontaneous regression of these lesions. This review summarizes current evidence on the molecular and cellular mechanisms underlying equine sarcoid pathogenesis, with emphasis on the interaction between BPV infection, host signaling pathways, tumor microenvironment dynamics, and multi-omic evidence of host regulatory networks. BPV oncoproteins, especially E5, promote fibroblast transformation through PDGFβR activation, downstream PI3K/AKT, MAPK and p38 signaling, altered cell survival, and immune evasion mediated by impaired antigen presentation. However, sarcoid persistence appears to depend on broader host-driven processes, including extracellular matrix remodeling, activated fibroblastic and myofibroblastic phenotypes, chronic inflammatory signaling, and ineffective immune clearance. Recent transcriptomic and epigenomic studies further indicate that long non-coding RNAs, DNA methylation changes, circulating microRNAs, and recently identified virus-host chimeric transcripts may contribute to stabilization of the neoplastic phenotype and may represent future biomarkers. Overall, this review proposes a virus-initiated, host-sustained conceptual framework for equine sarcoid pathogenesis, in which viral oncogene activity and host tissue reprogramming cooperate to promote lesion persistence, recurrence, and therapeutic resistance.

Pig microbiota influences overall animal health, and changes to the diet can modify gut microbiota composition and animal growth rate, bringing about environmental and economic advantages. Dietary polyphenols, due to their antimicrobial and antioxidant properties, may improve gut health. However, their use in monogastric diets is still emerging. The present study investigated the effects of polyphenols derived from an olive mill wastewater (OMWW) extract on gastrointestinal histopathology and morphometry and intestinal microbiota composition. One hundred thirty-five female Landrace x Duroc finishing pigs were randomly allocated to three experimental groups fed: a control (C) diet, or C diet supplemented with 74 ppm (P-LOW) or 225 ppm of OMWW polyphenols (P-HIGH). The experiment lasted 85 days, after which 15 animals per group were slaughtered. Final body weight was 148.1 +/- 4.6 kg. The morphological analysis of the intestine demonstrated a significant increase in the villus height:crypt depth (VH/CD) ratio in the ileum of pigs from P-LOW and P-HIGH groups and the jejunum of pigs from P-HIGH group vs C. 16S amplicon sequencing analysis showed an increase in two indexes of alpha diversity in the caecum of pigs from P-HIGH vs P-LOW. Differential abundance analysis showed that pigs from P-HIGH had a greater abundance of beneficial bacteria, especially Eubacterium and Treponema, and fewer harmful ones, including Fusobacterium, Bacteroides helcogenes, and Corynebacterium urealiticum. The results suggest that OMWW polyphenols may positively influence the pig gut microbiota and overall intestinal health. Further studies are needed to confirm these data.

MicroRNAs (miRNAs) are essential regulators of gene expression, yet few comprehensive databases exist for miRNA expression in non-model species, limiting our ability to characterize their roles in gene regulation, development, and disease. Similarly, isomiRs - length and sequence isoforms of canonical miRNAs with potentially altered regulatory targets and functions - have received even less attention in non-model species, including the horse, leaving a critical gap in our understanding of their biological significance. To address these challenges, we developed an open-source, containerized pipeline for identifying and quantifying miRNAs and isomiRs (FARmiR: Framework for Analysis and Refinement of miRNAs), and an associated interactive browser (AIMEE: Animal IsomiR and MiRNA Expression Explorer). AIMEE was developed to make miRNA expression data more accessible and user-friendly, a feature often lacking from other expression atlases. These tools were developed using equine data but can be readily extended to other species. Using these tools, we aggregated 461 small RNA-seq datasets, spanning 61 distinct tissues, integrating data from public repositories, an American Quarter Horse cohort, and the Functional Annotation of ANimal Genome (FAANG) consortium Thoroughbred samples, predicting 5,781 miRNAs and isomiRs. This work represents the largest systematically curated atlas of equine miRNA expression to date, providing a valuable resource that will enhance our understanding of miRNA and isomiR functions in tissue-specific regulation and ultimately improve biomarker discovery, functional genomics, and precision veterinary medicine.

Horses have undergone extensive natural and artificial selection, shaping the diversity of breeds observed today. Native Italian breeds present unique traits influenced by natural selection, such as adaptation to harsh climates, or hoof strength, but face challenges due to population declines and the reduction of their original breeding purpose. This study focuses on five local Italian breeds: Bardigiano, Haflinger, Maremmano, Murgese, and Italian Heavy Draught Horse, to understand how selection has shaped their populations. A total of 1620 individuals were genotyped with a medium-density SNP chip and remapped to EquCab3. After quality control, where data were filtered based on missing genotypes per SNP (> 0.10) and missing SNPs per sample (> 0.10), 1498 horses and 54,825 SNPs remained for analysis. Population structure and runs of homozygosity (ROH) were identified, and genomic inbreeding coefficients were calculated based on ROH coverage of autosomal SNPs. ROH islands shared by ≥ 70

Recent advancements have led to the development of various cooling devices for humans, specifically designed to lower systemic temperatures during sporting activities. It appears that wearable devices, featuring cooling inserts, may mitigate heat stress and enhance performance. The objective of this study was to investigate the safety of a saddle pad with a paraffin oil cooling insert and its effects on physiological and behavioural parameters of ridden horses during a 20-min flatwork trial. Seven horses in active training for show jumping at amateur level underwent two training sessions, one using a standard saddle pad and one using the cooling system. During each trial, the ambient temperature and humidity were recorded in real-time. Physiological parameters were monitored, including heart rate during exercise, while behaviour assessment was conducted using a ridden ethogram. Thermographic imaging was used to evaluate the surface temperature distribution beneath the saddle before and after exercise. Repeated measure ANOVA was used to investigate the differences in the data with (OIL TRIAL) and without (STANDARD TRIAL) the cooling saddle pad. No significant differences were observed in heart rate between the two trials. Ethogram scores did not exceed the threshold for musculoskeletal pain, and thermographic images revealed similar thermal patterns that indicated no alteration in pressure between trials. The prototype saddle pad with paraffin oil insert appears safe and did not cause discomfort.

Training on deep sand is commonly employed in endurance horses, but its physiological adaptation remains poorly characterized. This study aimed to characterize locomotor adaptations during a 7 km controlled-speed canter on deep sand in eighteen endurance horses, to identify heart rate variability (HRV) components, and to investigate changes in hematological variables before and after exercise. Stride frequency (SF) and stride length (SL), HRV, and hematological profiles were recorded during exercise and recovery with a fitness tracker. Associations between maximum speed and locomotor parameters were assessed by linear regression, while Pearson’s correlation assessed HRV relationships, also with physiological parameters. Hematological parameters were assessed with paired t-test before and after training. SL percentage change was the strongest predictor of speed (β = 0.677). HRV analysis revealed delayed parasympathetic reactivation; the parasympathetic recovery index (PNS REC) was correlated with mean RR interval on the ECG (r = 0.968) and heart rate (r = −0.964) during recovery. Post-exercise rectal temperature showed correlations with HRV recovery indices. Hematological evaluation revealed post-exercise increases in red blood cell count, hematocrit, hemoglobin, and corpuscular indices. SL plays a predominant role in achieving higher speeds on deep sand, while PNS REC emerges as a practical and accessible marker of autonomic recovery and fatigue. Horses with enhanced thermoregulation recover better. Hematological results confirm a physiological stress response that may optimize oxygen delivery. Integrating locomotor, cardiovascular, and hematological monitoring may improve management and welfare in endurance training.

Recently, much interest has been raised for the characterization of signaling molecules carried by extracellular vesicles (EVs), which are particularly enriched in milk (mEVs). Such interest is linked to the capability of EVs to cross biological barriers, resist acidification in the gastric environment, and exert modulation of the immune system, mainly through their microRNA (miRNA) content. We characterized the small-RNA cargo of colostrum EVs (colosEVs) and mEVs from Italian Mediterranean buffalo through next generation sequencing. Colostrum (first milking after birth) and milk (day 50 of lactation) were sampled from seven subjects from five farms. ColosEVs and mEVs were subjected to morphological characterization, followed by high-depth sequencing of small RNA libraries produced from total RNA. The main difference was the amount of EV in the two samples, with colostrum showing 10 to 100-fold higher content than milk. For both matrices, miRNA was the most abundant RNA species (95% for colosEVs and 96% for mEVs) and three lists were identified: colosEV-specific, mEV-specific and shared most expressed. Gene ontology (GO) enrichment analysis on miRNA targets highlighted many terms related to the epigenetic, transcriptional and translational regulations across the three lists, with a higher number of enriched terms for colosEV-specific miRNAs. Terms specific to colosEVs were related to "cell differentiation" and "microvillus assembly", while for mEV "cardiac and blood vessel development" and "mitochondria" emergerd. Immune modulation terms were found for both sample-specific miRNAs. Overall, both matrices carry a similar molecular message in terms of biological processes potentially modulated into receiving cells, but there is significant difference in the abundance, with colostrum containing much more EVs than milk. Moreover, colosEVs carry molecules involved in signal transduction, cell cycle and immune response, as for mEVs and EVs of other previously characterized species, but with a special enrichment for miRNAs with epigenetic regulation capacities. These beneficial characteristics of colosEVs and mEVs are essential for the calf and could also be exploited for the therapeutic purposes in humans, although further studies are necessary to measure the sanitization treatment impact on EV conservation, especially in buffalo where milk is consumed almost exclusively after processing.

EDITORIAL article Front. Vet. Sci., 16 February 2024Sec. Livestock Genomics Volume 11 - 2024 | https://doi.org/10.3389/fvets.2024.1369154

This study investigated the effect of environment, horse and type of different field standardised exercise tests (fSET) on incremental lactate concentration and recovery time in endurance horses. Forty-eight endurance horses underwent one or multiple fSET on a 700-m all-weather racetrack. The fSET comprised phase 1 canter at 19.5-21.5 km/h for 19 km (group N), or 37 km split by 20 minutes of rest (group E1) or 37 km (group E2), followed by phase 2 canter at 26-32.6 km/h and phase 3 at 27.7-42.8 km/h, for 1,400 m each for all groups. Blood lactates were recorded after each phase. Linear mixed models were built with blood lactate value and recovery time as dependent variables and horse and environment data, and type of fSET as fixed effect variables, with horse and horse x fSET group as random effects. Data from a total of 130 fSETs were considered; lactate values were 0.78-7.73 mmol/l (median 1.79 mmol/l) for phase 1, 1.70-10.68 mmol/l (median 3.89 mmol/l) for phase 2, and 3.7-20.86 mmol/l (median 8.74 mmol/l) for phase 3. Blood lactate values after phase 1 and phase 2 were significantly lower in group E1 (median 1.49 mmol/l; median 3.12 mmol/l) compared with group N (median 2.77 mmol/l; median 5.28 mmol/l; P < 0.001) and group E2 (median 2.06 mmol/l; median 4.39 mmol/l; P < 0.001). Blood lactate after phase 3 did not differ between groups. There were two medical and 19 minor musculoskeletal issues within 2 hours after the fSETs. Weather temperature affected blood lactate values after phase 1 and 2 (R = 0.62-0.63). Blood lactate after phase 1 (R = 0.93) and 2 (R = 0.66) affected values after phase 2 and 3, respectively. There was a considerable variation among horses and horse x fSET group random effects. Moreover, blood lactate values are affected by both weather conditions and by horse individual characteristics.

Training has a significant effect on the physiology of blood coagulation in humans and in horses. Several hemostatic changes have been reported after exercise in the horse but data available are inconclusive. The aim of this study was to investigate platelet activation and primary platelet-related hemostasis modifications in young never-trained Thoroughbreds in the first incremental training period in order to improve knowledge on this topic. Twenty-nine clinically healthy, untrained, 2-year-old Thoroughbred racehorses were followed during their incremental 4-month sprint exercise training. Blood collection was performed once a month, five times in total (T-30, T0, T30, T60, and T90). Platelet aggregation was measured by light transmission aggregometry in response to various agonists: adenosine diphosphate (ADP), collagen, and calcium ionophore A23187. Platelet function was evaluated using a platelet function analyzer (PFA-100®) using collagen/ADP and collagen/adrenaline cartridges. Nitrite-nitrate (NOx) plasma concentrations were measured via a colorimetric assay to assess in vivo nitric oxide bioavailability. Platelet activation was also investigated through gene expression analyses (selectin P-SELP, ectonucleotidase CD39-ENTPD1, prostaglandin I2 synthase-PTGIS, endothelial nitric oxide synthase 3-NOS3). Differences among the time points were analyzed and mean ± SEM were calculated. Significant modifications were identified compared with T-30, with an increase in platelet aggregation (collagen:32.6 ± 4.8 vs. 21.6 ± 4.9%; ADP: 35.5 ± 2.0 vs. 24.5 ± 3.1%; A23187: 30 ± 4.7 vs. 23.8 ± 4%) and a shorter closure time of C-ADP cartridges (75.6 ± 4.4 vs. 87.7 ± 3.4 s) that tended to return to the baseline value at T90. NOx concentrations in plasma significantly increased after 30 days of the training program compared with the baseline. The first long-term training period seems to induce platelet hyperactivity after 30 days in never-trained Thoroughbreds. Regular physical training reduces the negative effects of acute efforts on platelet activation.