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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.
Detection using chamber digital PCR with a DNA extraction-free method for gene-doping control. Gene doping, which entails the administration of transgenes, poses a serious threat to the integrity of equine sports and also raises both ethical and regulatory concerns. Current methods used for the detection of such doping often necessitate the extraction of DNA from plasma, which can be time-consuming and labour-intensive. To overcome this limitation, we developed a direct chamber digital PCR (cdPCR) method that enables transgene detection in equine plasma without the need for DNA purification. Using the equine erythropoietin (EPO) transgene as a model, we validated the assay by analysing ...
Investigation of peptide cross reactivity in equine plasma using two adrenocorticotropic hormone immunoassays. This study aimed to further define and quantify possible cross-reactive peptides when measuring plasma adrenocorticotropic hormone (ACTH) concentration in equids. Equine plasma samples were spiked with known concentrations of exogenous manufactured peptides comprising human ACTH, ACTH (corticotropin-like intermediate lobe peptide, CLIP) and ACTH (corticotropin inhibiting peptide, CIP). All samples were assayed in duplicate using Siemens Immulite 2000xpi chemiluminescent assay (CLA) and Tosoh AIA-900 immunoflurorescent assay (IFA). As expected, ACTH was measured by both assays although higher v...
Objective Evaluation of Gait Asymmetries in Traditional Racehorses During Pre-Race Inspection: Application of a Markerless AI System in Straight-Line and Lungeing Conditions. Subtle locomotor asymmetries are common in horses and may go unnoticed during routine pre-race clinical inspections, particularly when based solely on subjective evaluation. This study aimed to describe vertical head and pelvic movement asymmetries in racehorses that passed official pre-race inspections at a traditional racing event. Twenty-four horses were analysed using a markerless AI-based gait analysis system while trotting in-hand and during lungeing in both directions. Asymmetry parameters (HDmin, HDmax, PDmin, and PDmax) were extracted from video recordings, with values ≥0.5 consider...
Modelling Energy Demands of Cross-Country Tests in 2-Star to 5-Star Eventing Competitions. Eventing is an Olympic equestrian discipline comprising dressage, cross-country, and show jumping, with the cross-country phase imposing the greatest physical demands on horses. This study presents a composite model to estimate energy expenditure during the cross-country phase, integrating physiological data (heart rate-derived VO2 and lactate-based anaerobic estimates) with external workload indicators (GPS-derived speed, elevation, and course complexity). Model development was based on 691 rides from 256 horses across 232 events at 2-star to 5-star competition levels. The analysis showed tha...
Exposure of horses to biotoxins, phytoestrogens, and pesticides from different feed materials and supplementary feeds. The occurrence of biotoxins and chemical residues in marketed horse feeds has direct influences on horse health but has not been studied yet. Objective: The study investigated the exposure and health implications of contaminants in various horse feedstuffs available on the European market. Methods: A total of 108 feed samples representing diverse product categories such as hay, processed roughage products, grains, and various supplementary feeds were collected from different European countries and analyzed for contaminants, including mycotoxins, phytoestrogens, pesticides, and veterinary drug ...
Analytical Data Review on an Artificial Intelligence Platform for Doping Control in Horse Racing. In the screening of prohibited substances (PS) in horse biological samples with gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) for doping control, an enormous number of chromatograms are generated. Reviewing these chromatograms to identify suspicious findings requires an extensive manual effort. Recent advancements in Artificial Intelligence (AI) enable its use to classify images into different categories. This can potentially be utilized to perform first-line analysis of chromatograms, which are usually displayed as images, by classifying them...
Suppress or Not to Suppress … CRAFT It: A Targeted Metabolomics Case Study Extracting Essential Biomarker Signals Directly from the Full 1H NMR Spectra of Horse Serum Samples. : There are a few very specific inflammation biomarkers in blood, namely lipoprotein NMe signals of protein clusters (GlycA and GlycB) and a composite resonance of phospholipids (SPC). The relative integrals of these resonances provide clear indication of the unique metabolic changes associated with disease, specifically inflammatory conditions, often related to serious diseases such as cancer or COVID-19 infection. Relatively complicated, yet very efficient experimental methods have been introduced recently (DIRE, JEDI) to suppress the rest of the spectrum, thus allowing measurement of these ...
Detection of Transdermal Application of Testosterone to Racehorses by Analysis of Urine and Plasma. The use of testosterone in racehorses is predominantly monitored using international urine and plasma concentration-based thresholds and complementary steroid ratios. To date, there has been no published pharmacokinetic study on transdermally applied testosterone products in horses and whether their use could result in adverse analytical findings. Therefore, quantitative analysis of testosterone and epitestosterone in urine and testosterone in plasma samples was performed following a pilot multi-dose transdermal Testogel administration (1 mg/kg once a day for 7 days on clipped skin) to one...
An application of the MR-Horse method to reduce selection bias in genome-wide association studies of disease progression. Genome-wide association studies (GWAS) of disease progression are vulnerable to collider bias caused by selection of participants with disease at study entry. This bias introduces spurious associations between disease progression and genetic variants that are truly only associated with disease incidence. Methods of statistical adjustment to reduce this bias have been published, but rely on assumptions regarding the genetic correlation of disease incidence and disease progression which are likely to be violated in many human diseases. MR-Horse is a recently published Bayesian method to estimate...
Evaluation of a thermal camera in measuring surface temperature in horses. The use of thermal cameras in horses has proven effective for monitoring surface body temperature non-invasively, enabling the early identification of disease and inflammation. This study evaluated the agreement between a thermal camera compared to an infrared thermometer in the measurement of surface temperatures of 60 healthy horses (27 mares, 33 stallions; 7.88 ± 4.01 years; 466 ± 50.1 kg). Measurements were taken at the neck, thorax, and coronary band between 8:00-9:00 a.m., under controlled environmental conditions. Analysis revealed strong correlations between the two methods at the co...
Pharmacokinetics of Chloramphenicol and Chloramphenicol Glucuronide in Horses Following Administration Per Rectum or via Nasogastric Intubation. Chloramphenicol is a broad-spectrum antibiotic used in equine practice. It is known to produce adverse effects of hyporexia/anorexia after oral administration. Administration per rectum (PR) could mitigate the appetite suppression seen with oral administration and allow its use in horses unable to receive oral medications. The objectives of this study were to evaluate the relative bioavailability of chloramphenicol administered PR or via nasogastric tube (NGT) and determine relevant pharmacokinetic/pharmacodynamic parameters and metabolic ratios. Ten healthy, adult horses were administered chl...
Comparative Analysis of Proteomic Characteristics in Seminal Plasma Between Horses and Donkeys. Horses and donkeys, as integral members of the equine family, exhibit distinct reproductive capabilities and characteristics. Seminal plasma, the fluid component of semen, contains a variety of proteins that play critical roles in sperm function and fertility. This study aimed to systematically compare the protein profiles in the seminal plasma of horses and donkeys, thereby elucidating the molecular differences between these two species. The study utilized 4D-DIA proteomics technology to analyze seminal plasma from horses and donkeys and further validated key proteins through Western blot. Ou...
Development and validation of a quantitative UHPLC-HRMS bioanalytical method for equine anti-doping control. The quantification of banned substances in equine antidoping control, especially in racehorse urine, necessitates robust analytical methods with high detection levels due to the extremely low concentrations of the target substances and the significant impact of minor variations on doping test results. Reliable quantification is important for substances near regulatory thresholds, which, if exceeded, are prohibited. This study presents the development and validation of a bioanalytical UHPLC-HRMS method for quantifying doping substances in equine urine, including diazepam and acepromazine with a...
Wastewater surveillance as a tool for understanding West Nile virus transmission and distribution in Oklahoma. West Nile Virus (WNV) is the most widespread and frequently reported mosquito-borne disease in the US, with a high risk of outbreaks. Accurate surveillance of WNV is complicated by many mild or asymptomatic infections, resulting in human cases being under-reported and disease distribution and transmission not being well understood. In this study, we investigated how wastewater surveillance can be used for monitoring WNV in Oklahoma. We analyzed samples collected from wastewater treatment facilities in 16 counties during July 1 to September 31, 2023, for the presence of WNV genetic material and...
Doping Control of Ranitidine in Horses. Ranitidine is a histamine H-receptor antagonist commonly used to treat gastric ulceration in horses. The author's laboratory conducted a study some years ago in the early 2000s on its metabolism as well as its urinary elimination profile in two geldings. With the technology advancement as well as popularity of blood for doping control testing, the laboratory has recently conducted another administration trials of the substance in six horses to study the in vivo metabolism of ranitidine, aiming to identify and reinvestigate the appropriate target(s) for controlling misuse of ranitidine in hors...
Confirmation of a novel, stable estrogen metabolite, as 5a,6a-epoxy-estrone sulfate in stallion blood by LC-MS/MS. Mass spectrometry (MS) has become pivotal for accurately delineating intricate molecular structures for steroids present in minute quantities within biological samples. This study utilized liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) to identify and characterize a 'new' estrogen metabolite, 5α,6α-epoxy-estrone sulfate, in stallion serum from three animals. The estrogen structure was predicted previously using radiolabeled steroids. HRMS/MS, in combination with a seamless sample preparation involving liquid-liquid extraction and chromatographic separation, enabl...
Validation of the Equine Urticaria Activity Score for the assessment of chronic recurrent urticaria in horses. Recurrent urticaria is common in horses. The pathophysiology is poorly understood and treatment options are limited. Often, only glucocorticoids are effective for controlling clinical signs, albeit with potential adverse effects. Studies investigating new treatments need a validated objective scoring system for the grading of skin lesions to assess response. Objective: The aims were to investigate inter- and intraobserver reliability of the Equine Urticaria Activity Score (EqUAS) for the grading of skin lesions in horses with recurrent urticaria, and to examine agreement between experienced an...
The Suitability of Dried Blood Spot Sampling for Pharmacokinetic Studies in Veterinary Medicine. Dried blood spot (DBS) sampling has emerged as a promising microsampling technique in biomedical and clinical research, offering advantages such as reduced invasiveness, minimal blood volume requirements, and enhanced analyte stability. Although well established in human medicine for neonatal screening and diagnostic applications, its potential in veterinary pharmacology remains underexplored. This study investigated the feasibility of using DBS samples to quantify anesthetic agents-ketamine and medetomidine in cats and lidocaine in horses-during routine surgical procedures at a veterinary tea...
Comparison of RNA- and DNA-based 16S amplicon sequencing to find the optimal approach for the analysis of the uterine microbiome. Studies in humans and large animals indicate a relationship between the uterine microbiome composition and endometrial receptivity. Despite many studies have been performed, the analysis of the uterine microbiome remains challenging due to the very low microbial biomass. Studies in other biological systems showed that RNA-based microbiome analysis complements DNA-based results and provides information about active bacteria in a sample. Thus, the aim of this study was to establish a highly sensitive and specific 16S rRNA gene V3-V4 amplicon PCR from equine uterine cytobrush samples and to compa...
Multiplex PCR-Liquid Chromatography High-Resolution Tandem Mass Spectrometry for Simultaneous Detection of Multiple Transgenes in Equine Plasma. The development of gene therapy techniques introduces a potential risk of gene doping, which threatens the integrity of sport. In response to this challenge, we have developed a novel analytical method that employs a multiplex polymerase chain reaction (PCR) in conjunction with liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) for the simultaneous identification of multiple transgenes in equine plasma within a single reaction. The method targets three potential doping transgenes: equine growth hormone 1 (eGH1), equine growth hormone-releasing hormone (eGHRH), and equi...
The Effect of Filtering on Signal Features of Equine sEMG Collected During Overground Locomotion in Basic Gaits. In equine surface electromyography (sEMG), challenges related to the reliability and interpretability of data arise, among other factors, from methodological differences, including signal processing and analysis. The aim of this study is to demonstrate the filtering-induced changes in basic signal features in relation to the balance between signal loss and noise attenuation. Raw sEMG signals were collected from the quadriceps muscle of six horses during walk, trot, and canter and then filtered using eight filtering methods with varying cut-off frequencies (low-pass at 10 Hz, high-pass at 20 Hz...
Deciphering the Underlying Mechanism for Au/ZnO Nanocomposites-Induced Modulation of Structural Features and Thermodynamic Stability of Horse Myoglobin. Au/ZnO nanocomposites (NCs) were synthesized and characterized by using various analytical techniques. Analysis of Au/ZnO NCs effect on H NMR, CD, fluorescence, and absorbance spectra of horse myoglobin (-Mb) at 0.0 and 5.0 M urea (pH 7.4) revealed that the Au/ZnO NCs weaken the heme-globin interactions and also disrupt the secondary/tertiary structure of -Mb. Furthermore, the Au/ZnO NCs effect of weakening the heme-globin interactions and disrupting the protein structures was detected more in the denaturant media than in the aqueous solution. Analysis of the Au/ZnO NCs effect on thermodynamic...
3000 yr-old patterns of mobile pastoralism revealed by multiple isotopes and radiocarbon dating of ancient horses from the Mongolian Altai. Pastoral nomadism is of great cultural and economic importance in several regions of the world today. However, documenting ancient patterns of mobility in societies where pastoralism was central is challenging and requires tailored approaches and methodologies. Here we use strontium, oxygen and carbon isotopic analyses of dental enamel, together with a local strontium isoscape, to reconstruct the mobility patterns of seven domestic horses deposited in a Late Bronze Age grave from western Mongolia. Radiocarbon indicates that the animals were deposited within a short period of time, 3000 years a...
Long-Term Firocoxib Use in Horses. Many horses receive firocoxib over multiple years, and studies evaluating hematologic and biochemical values in these animals are lacking. Objective: To describe the hematologic and biochemical values of horses receiving long-term firocoxib. Methods: A total of 79 horses receiving long-term firocoxib and 153 horses not receiving long-term firocoxib and not presenting for a specific medical problem. Methods: Retrospective study comparing horses receiving firocoxib (F group) and no firocoxib (NF group). Signalment, hematologic, and biochemical values were compared between the two groups. Simple ...
Surrogate analyte approach for the determination of endogenous cortisol, cortisone, DHEA, DHEAS in horse hair and sheep wool by LC-HRMS/MS. Two new quantitative methods for the determination of four stress-related hormones (cortisol, cortisone, dehydroepiandrosterone and dehydroepiandrosterone sulfate) in horse hair and sheep wool by liquid chromatography coupled with hybrid high resolution mass spectrometry were developed and validated. Hormones were extracted overnight from ground samples with methanol after a washing step with the same solvent to remove external contamination. The extract was evaporated and dry residue was solubilized in an acid mixture of methanol and water. The methods were validated according to ICH guidelin...
Pooled Sampling Technique to Improve the Monitoring of Medication Use in the Racehorse Industry. All anti-doping programmes face financial constraints and monitoring trends in medication use or abuse in a population of racehorses can be difficult and expensive. Obtaining biological samples is the primary method of anti-doping control in individual horses or stables of horses but can be invasive and expensive. Another important practice of anti-doping control has been the confiscation of used and filled syringes by regulators for individual forensic analysis. Pooled samples testing involves the testing of multiple individual samples together as one composite sample. This pooled sample appr...
Measurement of eco-efficiency in the horse industry, spatiotemporal evolution and convergence analysis. The horse industry constitutes a vital economic sector in Xinjiang, China. This study quantitatively assesses the sector's sustainable development through eco-efficiency analysis across northern Xinjiang counties from 2001 to 2021. The research employs four analytical methods: the S-SBM model for efficiency measurement, kernel density estimation for distribution analysis, Moran's index for spatial autocorrelation examination, and convergence tests for long-term trend assessment. Results demonstrate a consistent decline in eco-efficiency, decreasing from 0.821 in 2001 to 0.444 in 2021, with an ...
Agreement between subjective gait assessment and markerless video gait-analysis in endurance horses. Subjective evaluation of gait by official endurance veterinarians (OEVs) is used to determine 'fitness-to-compete' in horses participating in endurance competitions. Objective gait analysis systems could aid in quick and verifiable judgements. Objective: To assess the agreement between objective analysis of head and pelvis vertical movement asymmetry performed with a markerless artificial intelligence motion tracking system (AI-MTS) and subjective lameness assessment performed by an accredited FEI OEV to judge horse gaits. Methods: Cross-sectional. Methods: During three endurance competitions,...
Metagenomic and proteomic analyses reveal similar reproductive microbial profiles and shared functional pathways in uterine immune regulation in mares and jennies. This study aims to unveil potential differences in the vaginal and uterine microbiomes in mares and jennies, and to identify possible mechanisms involved in uterine immune homeostasis. The microbiota was characterized using 16S rRNA sequencing, and the uterine proteome was analyzed using UHPLC/MS/MS in 18 samples from healthy mares and 14 from jennies. While taxonomic analysis revealed high interspecies similarities, β-diversity analysis showed distinct clustering, with only two vaginal taxa and five uterine taxa differing between species. Despite compositional differences, PICRUSt analysis s...
Endogenous Nature of Hydrocortisone Acetate in Horse. Hydrocortisone acetate, a synthetic acetate ester of hydrocortisone, was detected in horse blood samples collected from Thoroughbreds. Hydrocortisone acetate is generally considered an indicator for exogenous administration in horses. As hydrocortisone acetate has been previously reported to be endogenous in selected mammals, a proof-of-concept study was performed to evaluate the possible endogenous nature of hydrocortisone acetate in horses by in vitro incubation experiments using homogenized horse brain tissue.
