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Topic:Analytical Methods
Analytical methods in equine research encompass a variety of scientific techniques and tools used to study and evaluate different aspects of horse health, performance, and physiology. These methods help advance our understanding of equine biology, diagnosing conditions, and improving management practices. Common analytical methods include molecular techniques like PCR and ELISA for detecting pathogens and measuring biomarkers, imaging technologies such as ultrasound and MRI for assessing musculoskeletal health, and statistical models for analyzing genetic data and performance metrics. This page compiles peer-reviewed research studies and scholarly articles that explore the development, application, and impact of various analytical methods in equine science.
Stability of Gastric Fluid and Fecal Microbial Populations in Healthy Horses under Pasture and Stable Conditions. Equine gastrointestinal microbial communities vary across the gastrointestinal tract and in response to diet or disease. Understanding the composition and stability of gastric fluid microbiota in healthy horses is a prerequisite to understanding changes associated with the development of disease. The objective of this study was to describe microbial communities in the gastric fluid and feces of healthy horses longitudinally. Horses were maintained on pasture (6 weeks), stabled (5 weeks), then returned to pasture. A consistent forage diet was provided throughout. Native gastric fluid and feces ...
Lipid and Corticosteroid Biomarkers Under the Influence of Bisphosphonates. Detecting the use of bisphosphonates (BPs) in equine athletes is of interest to regulators and laboratories due to the threat to welfare issues for the potential to provide analgesic effects and manipulating bone structure. The detection of BPs in biological matrices is challenging due to erratic biological elimination and inconsistent analytical recoveries. Therefore, complementary approaches are needed to provide evidence of their misuse in racehorses. BPs have two sub-classes: nitrogenous and non-nitrogenous. This study investigated plasma elimination following administration of one example...
Evaluation of Two Commercial ELISA Kits for Measuring Equine Serum Gastrin Compared to Radioimmunoassay. Gastrin is an important hormone involved in gastric acid secretion. Despite its importance, validated methods other than radioimmunoassay (RIA) to assess serum gastrin concentrations in horses are lacking. This study aims to determine the agreement between ELISA and RIA in quantifying equine serum gastrin concentrations. Serum gastrin concentrations were quantified using two ELISA kits and RIA. Samples (196) from 14 horses at different time points were analyzed using one ELISA kit and RIA, selected samples (7) were analyzed using a second ELISA kit, and the correlation between methods was calc...
Unconventional Animal Species Participation in Animal-Assisted Interventions and Methods for Measuring Their Experienced Stress. The participation of animals during complementary therapy for humans is a developing area. Dogs and horses are the most frequent partner species in animal-assisted services, but several unconventional species have also been involved, although there is a lack of information about the stress they experience caused by AAS. We conducted a comprehensive literature search, analyzing 135 articles with the purpose of investigating the effects of AAS on unconventional species such as guinea pigs, rabbits, farm animals, alpacas, donkeys, reptiles, aquarium fishes, and dolphins. We found that the relevan...
Comparison of Citrated Whole Blood to Native Whole Blood for Coagulation Testing Using the Viscoelastic Coagulation Monitor (VCM Vet™) in Horses. Viscoelastic monitoring of horse coagulation is increasing due to its advantages over traditional coagulation testing. The use of a point-of-care viscoelastic coagulation monitor (VCM Vet™) has been validated for use in horses using native whole blood (NWB) but has not been assessed using citrated whole blood (CWB), a technique that might have advantages in practicality and precision. Blood was collected from 70 horses, tested in duplicate immediately using NWB (T0), and stored at room temperature as CWB for testing in duplicate at 1 (T1) and 4 (T4) hours after venipuncture for comparison to...
Validation of a Commercial ELISA Kit for Non-Invasive Measurement of Biologically Relevant Changes in Equine Cortisol Concentrations. The measurement of fecal cortisol/corticosterone metabolites (FCMs) is often used to quantify the stress response. The sampling method is relatively non-invasive, reduces concern for elevation of cortisol from the sampling method, and has been shown to measure cortisol more consistently without the daily diurnal rhythm observed in blood. Commercial ELISA (enzyme-linked immunoassay) kits offer benefits over previously validated immunoassay methods but lack validation. The objective of this study was to evaluate a commercial ELISA kit (Arbor Assays DetectX Cortisol ELISA kit, K003-H1, Ann Arbor,...
A Comprehensive Analysis of the Genomic and Expressed Repertoire of the T-Cell Receptor Beta Chain in Equus caballus. In this paper, we report a comprehensive and consistent annotation of the locus encoding the β-chain of the equine T-cell receptor (TRB), as inferred from recent genome assembly using bioinformatics tools. The horse TRB locus spans approximately 1 Mb, making it the largest locus among the mammalian species studied to date, with a significantly higher number of genes related to extensive duplicative events. In the region, 136 TRBV (belonging to 29 subgroups), 2 TRBD, 13 TRBJ, and 2 TRBC genes, were identified. The general genomic organization resembles that of other mammals, with a V cluster o...
A multi-task learning model for clinically interpretable sesamoiditis grading. Sesamoiditis is a common equine disease with varying severity, leading to increased injury risks and performance degradation in horses. Accurate grading of sesamoiditis is crucial for effective treatment. Although deep learning-based approaches for grading sesamoiditis show promise, they remain underexplored and often lack clinical interpretability. To address this issue, we propose a novel, clinically interpretable multi-task learning model that integrates clinical knowledge with machine learning. The proposed model employs a dual-branch decoder to simultaneously perform sesamoiditis grading ...
Equine Doping Controls of Thymosin β $$ beta $$ 4: A Population Study and Strategy for Misuse Detection. Thymosin 4 (TB4) is a ubiquitous, highly conserved and abundant peptide among mammals with a critical role in cytoskeleton organization. In spite of its yet non-authorized use as a medicine and being forbidden by the IFHA, the FEI, and the WADA, intelligence and doping control laboratories reported numerous products available online claiming to contain a synthetic acetylated fragment of TB4 or TB4 itself, promoted as a growth factor with regenerative properties. In this article, the first estimation of the endogenous TB4 concentration in racing horses' blood samples was performed through a po...
Equine metabolic investigation of the phosphodiesterase-4 inhibitor ibudilast as a potential performance enhancer. Phosphodiesterase 4 (PDE4) inhibitors are a newer class of drugs that induce bronchodilation and have anti-inflammatory effects, making them susceptible to misuse as performance enhancers in competitive sports. Methods: This study explores the metabolic conversion of PDE4 inhibitor ibudilast in thoroughbred horses after oral administration and in vitro using equine liver microsomes and Cunninghamella elegans. A liquid chromatography-high resolution mass spectrometry method was used to postulate the plausible structures of the detected metabolites. Results: A total of 20 in vivo metabolites wer...
Discovery of Biomarkers of a Recombinant Human Erythropoietin Administration to Thoroughbred Geldings by Label-Free Proteomics. Erythropoiesis-stimulating agents (ESAs) continue to be a significant threat to the integrity of human and equine sports. Besides conventional direct testing, monitoring the biomarkers associated with the effects of ESAs may provide a complementary approach via indirect detection to enhance doping control. In this study, we applied label-free proteomics to discover plasma protein biomarkers in Thoroughbred geldings after administration with a long-acting form of recombinant human erythropoietin (rhEPO), methoxy polyethylene glycol epoetin beta, Mircera. Increased haematocrit, haemoglobin and r...
Pharmacokinetics of Two Formulations of Altrenogest Administered to Mares. Altrenogest is a synthetic progestin that suppresses reproductive behaviours and assists pregnancy maintenance in female horses. Two formulations are available, a 'weekly' intramuscular injection and a daily oral formulation. Altrenogest administration has returned positive swabs for steroids; consequently, using injectable altrenogest in racing mares is prohibited. Oral administration may be permitted in race mares if there is one clear day between dosing and racing. The only pharmacokinetic data available were generated from geldings. Therefore, to assist veterinarians and analysts in determ...
Senecio ovatus poisoning in a horse – A case report. This study describes a case of poisoning by pyrrolizidine alkaloids in a horse. To the best of the author's knowledge, this is the first confirmed case of Senecio ovatus poisoning. A six-year-old 450-kg Irish cob mare was presented to the Equine Clinic of the University of Veterinary Sciences Brno (Czechia) with symptoms of hepatic encephalopathy, which progressively worsened with time despite intensive therapy and led to euthanasia. A complex diagnostic and therapeutic approach including the post-mortem patoanatomical and histopathological examination is described here. Regar...
High-Throughput Equine Doping Controls on a Trapped Ion Mobility Quadrupole-Time-of-Flight Mass Spectrometer: Technical Considerations of dia/slice/prmPASEF Applied to the Long-Term Detection of Monoclonal Antibodies. Data-independent acquisition (DIA) methods employing a scanning quadrupole were recently described across multiple platforms. These strategies display remarkable performances in untargeted proteomics studies thanks to rapid duty cycles, leading to ultrashort liquid chromatography gradients while maintaining enough data points per peaks when coupled to fast-scanning mass analyzer. In this article, we perform the evaluation of three data acquisition strategies named diaPASEF,slicePASEF, and prmPASEF on a trapped ion mobility spectrometry quadrupole-time-of-flight (TIMS-Q-TOF) mass spectrometer f...
RNA Sequencing of Sperm from Healthy Cattle and Horses Reveals the Presence of a Large Bacterial Population. RNA molecules within ejaculated sperm can be characterized through whole-transcriptome sequencing, enabling the identification of pivotal transcripts that may influence reproductive success. However, the profiling of sperm transcriptomes through next-generation sequencing has several limitations impairing the identification of functional transcripts. In this study, we explored the nature of the RNA sequences present in the sperm transcriptome of two livestock species, cattle and horses, using RNA sequencing (RNA-seq) technology. Through processing of transcriptomic data derived from bovine and...
Extraction and Physicochemical Characterization of Hydroxyapatites From Horse Humerus Bones of Different Ages (1, 3, 6, and 8 Years old) Calcined at Low Temperature. The aim of this work is to investigate the changes in the physicochemical properties of hydroxyapatite (HAp) extracted from horse humerus bones of different ages (1, 3, 6, and 8 years) subjected to low temperature calcination (600°C). Thermal analysis revealed significant mass loss due to water, collagen, organic compounds, carbonates, and age-related magnesium out-diffusion. Higher fat content in older bones contributed to increased mass loss. Phosphorus content remained constant across age groups, while calcium and sodium showed age-related fluctuations. Magnesium levels decreased with ag...
Emerging Human Medications in Racing Animals: Analytical and Regulatory Challenges. Positive identification and reporting of therapeutic substances intended for human consumption in race-day equine and canine samples is a controversial topic. While inadvertent environmental exposure is a potential cause for the presence of these substances in race-day samples, intentional use cannot be ruled out given their therapeutic benefits. Pregabalin is widely prescribed in Australia to treat epilepsy, anxiety, and neuropathic pain in humans; however, it is also increasingly used as a recreational drug. Metformin is commonly used to treat type 2 diabetes in humans. Both pregabalin and m...
Doping Control Analysis of Perfluorocarbons in Equine Plasma by Headspace Gas Chromatography-Tandem Mass Spectrometry. In order to control the potential misuse of perfluorocarbons as an oxygen carrier in equine sports, a simple and sensitive method for detecting perfluorocarbons in equine plasma by gas chromatography/tandem mass spectrometry using negative chemical ionization with methane as reagent gas has been developed and fully validated. The method covers seven perfluorocarbons, which are the active components in blood substitute products, and shows good sensitivity and robustness. Limits of detection as low as 0.01 ng/mL could be achieved by the method. To the best of our knowledge, this is the first r...
Evaluating the Effects of Solid-Phase Cartridge Chemistry on Extraction of Bisphosphonates and ITPP From Equine Plasma. Bisphosphonate drugs and myo-inositol trispyrophosphate are of concern to the racing industry and have been listed as prohibited substances in equine sports. The current bisphosphonate plasma screening analysis employed at the Australian Racing Forensic Laboratory involves the use of sequential solid-phase extraction procedures, passing the samples through a mixed mode cartridge, followed by a weak anion exchange cartridge. The eluates collected following the second extraction are then methylated and analysed by liquid chromatography-mass spectrometry. Under these extraction conditions, some b...
Pharmacokinetics of Ethyl Glucuronide and Ethyl Sulfate and Pharmacodynamic Effects Following Intravenous and Oral Administration of Ethanol to Exercised Horses. Ethanol, a central nervous system depressant and banned substance in horseracing, has reportedly been administered to horses prior to competition to "calm a horse's nerves." In this study, the pharmacokinetics of two metabolites of ethanol were studied to better understand the behavior of this compound in the horse and provide a scientific basis for regulation of its administration. Six horses received a single intravenous (30 mL; 1200 mg) and oral (90 mL; 3600 mg) administration of ethanol (vodka, 40% ABV) in a balanced cross-over design. Blood and urine samples were collected at vari...
Horseshoe effects on equine gait-A systematic scoping review. To provide an overview of available research about effects of horseshoes on equine kinetics and kinematics. Methods: The terms, "horse/equine," "hoof," "shoes/horseshoes," "kinetics," and "kinematics" were searched in PubMed, Web of Science, Center for Agriculture and Bioscience International, and United States Department of Agriculture National Agricultural Library for manuscripts from first availability to 2024. Independent reviewers used preferred reporting items for systematic reviews and meta-analyses guidelines to map and extract evidence-based data from sources. Metrics included partici...
In vitro evaluation of the automated hematology analyzer XN-31 for rapid diagnosis of equine piroplasmosis. Equine piroplasmosis (EP) is a protozoal disease affecting equids, caused by and . EP is conventionally diagnosed using microscopic, molecular, and/or serological methods, which are time-consuming. Consequently, there is a need for faster testing methods. In this study, we evaluated the application of the Sysmex XN-31 automated hematology analyzer, originally a rapid test for detecting malaria in humans, for the diagnosis of EP. The cultured parasites were measured using the XN-31 that had been customized for horse blood samples (XN-31m). The following parameters were evaluated: limit of dete...
Detection of levamisole and its metabolites in horses after oral levamisole administration over seven days. Levamisole is a regulated substance sometimes administered to racehorses to treat equine protozoal myelitis. Metabolites include compound II, aminorex, and pemoline. Aminorex and pemoline are Horseracing Integrity and Safety Authority-banned substances. Previous studies have examined single doses of the drug. This study examined the disposition of levamisole after 7 days of dosing. Methods: 6 healthy Thoroughbred geldings. Methods: Horses were treated with 500 mg (approx 0.91 to 1 mg/kg) of compounded levamisole hydrochloride paste PO every 12 hours for a total of 13 doses over 7 days. Serum a...
Extracted Plasma Cell-Free DNA Concentrations Are Elevated in Colic Patients with Systemic Inflammation. Colic is a common and potentially life-threatening condition in horses; in many cases, it remains challenging for clinicians to determine the cause, appropriate treatment, and prognosis. One approach that could improve patient care and outcomes is identification of novel diagnostic and prognostic biomarkers. Plasma cell-free DNA (cfDNA) is a biomarker that shows promise for characterizing disease severity and predicting survival in humans with acute abdominal pain or requiring emergency abdominal surgery. In horses, we recently determined that extracted plasma cfDNA concentrations are elevated...
Quantification of osilodrostat in horse urine using LC/ESI-HRMS to establish an elimination profile for doping control. The use of osilodrostat, developed as a medication for Cushing's disease but categorized as an anabolic agent, is banned in horses by both the International Federation of Horseracing Authorities and the Fédération Equestre Internationale. For doping control purposes, elimination profiles of hydrolyzed osilodrostat in horse urine were established and the detectability of free forms of osilodrostat and its major metabolite, mono-hydroxylated osilodrostat (M1c), was investigated. Post-administration urine samples obtained from a gelding and three mares were analyzed to establish the eliminatio...
Metabolic Pathway of Osilodrostat in Equine Urine Established through High-resolution Mass Spectrometric Characterization for Doping Control. Osilodrostat, used to treat Cushing's disease, exhibits an anabolic effect, leading to its classification as a prohibited substance in horseracing and equestrian sports. This study reports the characterization of osilodrostat metabolites in horse urine and elucidates its metabolic pathways for the first time for doping control purposes. Methods: Osilodrostat was administered nasoesophageally to four thoroughbreds (one gelding and three mares) at a dose of 50 mg each. Potential metabolites were extensively screened via our developed generic approach employing differential analysis to identify m...
Effects of storage-handling methods on nutrient analysis of fresh-forage samples. Forage low in nonstructural carbohydrates (NSC) is recommended for insulin dysregulated (ID) horses, indicating the importance of an accurate forage analysis. However, it remains to be fully understood how handling forage samples pre-analysis impacts nutrient values. The objective of this study was to compare the effect of fresh-forage storage-handling methods: microwave-oven (MO; 9kw; 70s then stored at -20℃), room temperature (20℃), 3℃, -20℃, and -80℃. Subsamples collected from a mixed-grass pasture and a ryegrass plot were analyzed for crude protein (CP), water soluble carbohydrat...
Comparison of a hand-held high-end resolution infrared thermography (FLIR P640) and a smartphone infrared thermographic device (FLIR One) for the assessment of skin surface temperature after anaesthetising the median nerve in Healthy horses. Accuracy of a median nerve block is normally assessed by testing skin sensitivity on the medial and dorsal aspects of fetlock and pastern. The present study evaluated subjective and objective analysis of skin surface temperature obtained with two different infrared (IR) thermography cameras (a high-end [FLIR P640] and a smartphone IR thermography device [FLIR One®]) before and after anaesthesia of the median nerve. Thermographic images were obtained at 0, 30, 60 and 90 minutes after performing a median nerve block with 2% mepivacaine hydrochloride. The subjective analysis of thermographic ima...
Comparison of two methods of measuring the urinary protein concentration for the determination of the urinary protein to creatinine ratio in various animal species. Determination of the urinary protein-to-creatinine ratio (UPC) is an important tool in the quantification of proteinuria in animals. However, the result may be affected by the different methods of determining the urinary protein concentration. The aim of this study was to compare the turbidimetric method using benzethonium chloride and the colorimetric method using pyrogallol red in the measurement of the urinary protein concentration in dogs, cats, guinea pigs and horses. A total of 464, 192, 216 and 119 urine samples from dogs, cats, guinea pigs and horses were examined in the study, respect...
Einthoven’s triangle adapted for horses: Proposal for the Delta configuration. Reliable ECGs are crucial for diagnosing arrhythmias, yet a lack of standardization impedes arrhythmia diagnosis and treatment in horses. Objective: To objectively determine an optimal position of Einthoven's triangle for ECG recordings in horses at rest, which can form the basis for standardized ECG recording and improve diagnosis and treatment of arrhythmias. Methods: The study involved 72 healthy, warmblood horses aged between 3 and 20 years. Methods: In view of future 12-lead studies and vectorcardiography, requiring an orthogonal system, Einthoven's triangle was positioned around the he...
