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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 of the selective androgen receptor modulator S-23 and its metabolites in equine urine and plasma following oral administration. S-23 is an arylpropionamide selective androgen receptor modulator that has been investigated in animal models for use as a male hormonal contraceptive but is not yet available therapeutically. S-23 is available alongside other selective androgen receptor modulators (SARMs) to purchase online via uncontrolled sites, sold as supplement products. It has been detected in several human doping cases, highlighting the importance of identifying the best analytical targets for equine doping control. The purpose of this study was to investigate the detection of S-23 and its phase I metabolites in equine...
Systematic evaluation supports the use of ELISA for quantification of calprotectin in equine feces, a first step toward noninvasive quantification of intestinal inflammation in horses. To optimize and evaluate methods for the detection of the inflammatory biomarkers myeloperoxidase (MPO) and calprotectin (CP) in equine feces by ELISA. Methods: Healthy horses (n = 28) and horses with intestinal inflammation (n = 10). Methods: Feces were suspended in buffer to create fecal supernatant. Serum and fecal supernatant were analyzed using ELISA kits validated for the detection of MPO and CP in equine serum. Assay validation steps included intra- and interassay variability (coefficient of variation [CV]), dilution linearity, spike recovery, and sample type correlation. Variations in ...
The detection of ACTH and insulin in equine plasma by solid-phase extraction and micro-flow LC/MSMS. Previous liquid chromatography/mass spectrometry (LC/MS) methods for the detection of insulin and other similar peptide hormones in equine plasma relied on the use of antibody affinity extraction. As a result, these methods were not suitable for routine high-throughput analysis. A solid-phase extraction (SPE) method incorporating size exclusion as well as reversed-phase interactions allows the selective extraction of peptide hormones such as adrenocorticotropic hormone (ACTH), insulin and their synthetic analogues from equine plasma with approximately 80% extraction efficiencies. This extracti...
Evaluation of Non-Invasive Sampling Techniques for the Molecular Surveillance of Equid Herpesviruses in Yearling Horses. Equid alphaherpesvirus 1 (EHV-1) is a highly contagious respiratory tract pathogen of horses, and infection may be followed by myeloencephalopathy or abortion. Surveillance and early detection have focused on PCR assays using less tolerated nasal swabs. Here, we assess non-invasive non-contact sampling techniques as surveillance tools in naturally equid gammaherpesvirus 2-shedding horses as surrogates for EHV-1. Horses were individually housed for 10 h periods on 2 consecutive days. Sampling included nasal swabs, nostril wipes, environmental swabs, droplet-catching devices, and air sampling. T...
Detection of sildenafil and its 9 metabolites in a post-race horse urine sample: A case report. The use of prohibited substances in horse racing is a major concern that jeopardizes both the fairness of competitions and the health of horses. This problem can stem from the use of licensed drugs for animal health, as well as unlicensed substances. Horse doping laboratories monitor the potential use of these substances in racehorses within the framework of regulations set by the International Federation of Horse Racing Authority. In this context, sildenafil and its major metabolite n-desmethyl sildenafil were detected in a post-race horse urine sample sent to the Pendik Veterinary Control In...
Radiographic texture of the trabecular bone of the proximal phalanx in horses with metacarpophalangeal osteoarthritis. Osteoarthritis (OA) is a prevalent condition in horses, leading to changes in trabecular bone structure and radiographic texture. Although fractal dimension (FD) and lacunarity have been applied to quantify these changes in humans, their application in horses remains nascent. This study evaluated the use of FD, bone area fraction (BA/TA), and lacunarity in quantifying trabecular bone differences in the proximal phalanx (P1) in 50 radiographic examinations of equine metacarpophalangeal joints with varying OA degrees. In the dorsopalmar view, regions of interest were defined in the trabecular bo...
Broad-spectrum and sensitive screening of more than 1000 compounds in equine urine using liquid chromatography/high-resolution mass spectrometry. To uphold the integrity of horseracing and equestrian sports, it is critical for an equine doping control laboratory to develop a comprehensive screening method to cover a wide range of target substances at the required detection levels in equine urine. Methods: The procedure involved the enzymatic hydrolysis of 3 mL urine samples followed by solid-phase extraction using HF Bond Elut C18 cartridge. The resulting extracts were then separated on a C18 reversed-phase column and analyzed using liquid chromatography/high-resolution mass spectrometry (LC/HRMS) in both electrospray ionization positiv...
Comparative pharmacokinetics of phenylbutazone in healthy young-adult and geriatric horses. To evaluate the effects of aging on phenylbutazone (PBZ) disposition in older horses (≥ 25 years old) compared to young adults (4 to 10 years old) by characterizing the pharmacokinetic profile of PBZ and its active metabolite, oxyphenbutazone (OPBZ), following a 2.2-mg/kg dose, IV. We hypothesized that the disposition of PBZ will be affected by age. Methods: 16 healthy horses (8 young adults aged 4 to 10 years and 8 geriatric horses ≥ 25 years old). Methods: Horses were administered a single 2.2-mg/kg PBZ dose, IV. Plasma samples were collected at designated time points and frozen at -80 ...
Evaluation of vasovagal tonus index and electrocardiographic parameters in horses using a new modified base apex lead method. Vasovagal tonus index (VVTI) serves as a straightforward assessment tool for autonomic function during both physiological and pathological conditions, including pregnancy, in horses. Obtaining VVTI through a modified base apex lead system could be a practical and comfortable solution. In this study, we assessed VVTI in horses with respect to training status and electrocardiographic measurements utilizing a novel modified base apex lead technique. Unassigned: A total of 12 Thai native crossbred horses and 12 Arabian horses, all free of cardiac abnormalities, were enrolled in the study. Animals ...
Doppler renal resistivity index in horses: A systematic review. The resistivity index (RI) evaluates haemodynamic based on arterial resistance. Alterations in renal blood flow are important for the early detection of kidney damage, as perfusion might be one of the first affected aspects. Objective: To retrieve published studies on equine renal RI to develop a standardised method of renal ultrasonographic examination and to evaluate the current reference range for the renal RI in horses. Methods: Systematic review. Methods: An electronic search in Science Direct, PubMed, Scopus and Web of Science databases was performed in February 2023 using the terms 'RI'...
Doping control analysis of myo-inositol trispyrophosphate and 10 bisphosphonates in equine plasma by ion chromatography-mass spectrometry and its application to clodronic acid horse administration. Bisphosphonates and myo-inositol trispyrophosphate (ITPP) are two classes of difficult-to-detect polar drugs that are prohibited under the rules of racing. ITPP is a drug capable of increasing the amount of oxygen in hypoxic tissues, and studies have shown that administration of ITPP increases the maximal exercise capacity in mice. The properties of ITPP make it an ideal candidate as a doping agent to enhance performance in racehorses. In recent years, ITPP had indeed been detected in racehorses and confiscated items. As for bisphosphonates, it is especially critical to control their use as si...
Evaluation of pharmacokinetics of metoclopramide administered via subcutaneous bolus and intravenous constant rate infusion to adult horses. To determine the pharmacokinetics (PK) of metoclopramide administered via intravenous continuous rate infusion (IV CRI) and subcutaneous (SC) bolus and evaluate for gastrointestinal motility and adverse side effects. Methods: Experimental study; randomized, crossover design. Methods: Six healthy adult horses. Methods: Each horse received metoclopramide via IV CRI (0.04 mg/kg/h for 24 h) and SC bolus (0.08 mg/kg once), with ≥1 week washout period between. Plasma was analyzed by UPLC-MS/MS. Compartmental modeling was used to determine PK parameters for each treatment; nonparametric sup...
Metagenomic characterization of the equine endometrial microbiome during anestrus. The equine uterus is highly interrogated during estrus prior to breeding and establishing pregnancy. Many studies in mares have been performed during estrus under the influence of high estrogen concentrations, including the equine estrual microbiome. To date, it is unknown how the uterine microbiome of the mare is influenced by cyclicity; while, the equine vaginal microbiome is stable throughout the estrous cycle. We hypothesized that differences would exist between the equine endometrial microbiome of mares in estrus and anestrus. The aim of this study was two-fold: to characterize the reside...
Viscoelastic properties of the equine hoof wall. The equine hoof wall has outstanding impact resistance, which enables high-velocity gallop over hard terrain with minimum damage. To better understand its viscoelastic behavior, complex moduli were determined using two complementary techniques: conventional (∼5 mm length scale) and nano (∼1 µm length scale) dynamic mechanical analysis (DMA). The evolution of their magnitudes was measured for two hydration conditions: fully hydrated and ambient. The storage modulus of the ambient hoof wall was approximately 400 MPa in macro-scale experiments, decreasing to ∼250 MPa with hydration. In con...
Mitochondrial DNA and Y chromosome reveal the genetic structure of the native Polish Konik horse population. Polish Konik remains one of the most important horse breeds in Poland. The primitive, native horses with a stocky body and mouse-like coat color are protected by a conservation program, while their Polish population consists of about 3,480 individuals, representing 16 dam and six sire lines. To define the population's genetic structure, mitochondrial DNA and Y chromosome sequence variables were identified. The mtDNA whole hypervariable region analysis was carried out using the Sanger sequencing method on 233 Polish Koniks belonging to all dam lines, while the Y chromosome analysis was performe...
Plasma concentrations of buprenorphine administered via matrix-type transdermal patches applied at three different anatomical locations in healthy adult horses. Anatomical location-dependent differences in transdermal opioid penetration are well described in human patients. Although this has been investigated in horses with fentanyl, there is no literature available on location-dependent plasma buprenorphine concentrations when administered as a transdermal matrix-type patch. Unassigned: This study aims to compare the plasma concentrations achieved from the matrix-type transdermal buprenorphine patches placed at different anatomical sites (metacarpus, gaskin, and ventral tail base) in healthy adult horses. Unassigned: This is a randomized experimental...
Flowmetry and spectrophotometry for the assessment of intestinal viability in horses with naturally occurring strangulating small intestinal lesions. Ancillary diagnostic methods to enhance the accuracy of viability assessment have not been established for use in clinical practice. Objective: To assess intestinal microperfusion measured by Laser Doppler Flowmetry and Spectrophotometry (LDFS) in naturally occurring small intestinal strangulations of different origins and to compare this between viable and non-viable segments. Methods: Prospective clinical trial. Methods: Forty horses undergoing colic surgery for naturally occurring small intestinal strangulations were included. Tissue oxygen saturation (tSO), haemoglobin (tHB) and blood flow...
Detection times of clodronic acid in horses with orthopedic disease. Clodronic acid is designated as a controlled medication for competition horses by the International Federation for Equestrian Sports and, according to the International Federation of Horseracing Authorities, clodronic acid is not to be administered to racehorses younger than 3.5 years or within 30 days prior to a race. In this study, 35 horses involved in competition were treated with a single dose of 1.53 mg clodronic acid/kg bodyweight intramuscularly. Plasma samples were obtained before treatment and 10, 20, 30, and 40 days post-administration. Clodronic acid concentrations were mea...
Functional infrared thermography imaging can be used to assess the effectiveness of Maxicam Gel® in pre-emptively treating transient synovitis and lameness in horses. Diagnosing and treating lameness in horses is essential to improving their welfare. In equine orthopedic practice, infrared thermography (IRT) can indirectly detect soreness. Non-steroidal anti-inflammatory drugs can treat painful and inflammatory processes in horses. Using IRT, the efficacy of meloxicam (Maxicam Gel) was evaluated in pre-treating transient synovitis in horses induced by a middle carpal joint injection of lipopolysaccharides (LPS) from 055:B5 at a dose of 10 endotoxin units. Unassigned: In a cross-over design, six healthy horses were randomly assigned to receive either 0.6 ...
Measurement of Trace Elements (Zinc, Copper, Magnesium, and Iron) in the Saliva of Horses: Validation Data and Changes in Equine Gastric Ulcer Syndrome (EGUS). The objective of this study was to evaluate the possible use of spectrophotometric assays for the measurement of trace elements, including Zinc (Zn), Copper (Cu), Magnesium (Mg), and iron (Fe) in the saliva of horses and study their possible changes in equine gastric ulcer syndrome (EGUS). EGUS is a highly prevalent disease, with a current high incidence due to the increase in intensive management conditions. There are two EGUS diseases: equine squamous gastric disease (ESGD) and equine glandular gastric disease (EGGD), which can appear individually or together. For this purpose, automated spe...
A fresh look at the SarcoFluor antibody test for the detection of specific antibodies to Sarcocystis neurona for the diagnosis of equine protozoal myeloencephalitis. Equine protozoal myeloencephalitis (EPM) is a challenging disease to diagnose in horses with neurological signs. To optimize contemporary diagnostic testing, including the use of serum:CSF antibody ratios, the SarcoFluor antibody test for Sarcocystis neurona requires revalidation. The SarcoFluor, a previously validated immunofluorescent antibody test (IFAT) for the detection of antibodies specific to S. neurona in serum and cerebrospinal fluid (CSF) of naturally infected horses was analyzed using recent data and considering a serum:CSF antibody ratio threshold. Utilization of serum and CSF pho...
Non-Targeted Detection of Synthetic Oligonucleotides in Equine Serum Using Liquid Chromatography-High-Resolution Mass Spectrometry. There is great concern in equine sport over the potential use of pharmaceutical agents capable of editing the genome or modifying the expression of gene products. Synthetic oligonucleotides are short, single-stranded polynucleotides that represent a class of agents capable of modifying gene expression products with a high potential for abuse in horseracing. As these substances are not covered by most routine anti-doping analytical approaches, they represent an entire class of compounds that are not readily detectable. The nucleotide sequence for each oligonucleotide is highly specific, which m...
Biomolecular evidence reveals mares and long-distance imported horses sacrificed by the last pagans in temperate Europe. Horse sacrifice and deposition are enigmatic features of funerary rituals identified across prehistoric Europe that persisted in the eastern Baltic. Genetic and isotopic analysis of horses in Balt cemeteries [1st to 13th centuries CE (Common Era)] dismantle prevailing narratives that locally procured stallions were exclusively selected. Strontium isotope analysis provides direct evidence for long-distance (~300 to 1500 kilometers) maritime transport of Fennoscandian horses to the eastern Baltic in the Late Viking Age (11th to 13th centuries CE). Genetic analysis proves that horses of both sexe...
A laser-Engraved Wearable Electrochemical Sensing Patch for Heat Stress Precise Individual Management of Horse. In point-of-care diagnostics, the continuous monitoring of sweat constituents provides a window into individual's physiological state. For species like horses, with abundant sweat glands, sweat composition can serve as an early health indicator. Considering the salience of such metrics in the domain of high-value animal breeding, a sophisticated wearable sensor patch tailored is introduced for the dynamic assessment of equine sweat, offering insights into pH, potassium ion (K), and temperature profiles during episodes of heat stress and under normal physiological conditions. The device integra...
Nuclear Magnetic Resonance (NMR) Metabolomics: Current Applications in Equine Health Assessment. Metabolomics can allow for the comprehensive identification of metabolites within biological systems, at given time points, in physiological and pathological conditions. In the last few years, metabolomic analysis has gained popularity both in human and in veterinary medicine, showing great potential for novel applications in clinical activity. The aim of applying metabolomics in clinical practice is understanding the mechanisms underlying pathological conditions and the influence of certain stimuli (i.e., drugs, nutrition, exercise) on body systems, in the attempt of identifying biomarkers th...
Designing a Multiplex PCR-xMAP Assay for the Detection and Differentiation of African Horse Sickness Virus, Serotypes 1-9. African horse sickness is a severe and often fatal disease affecting all species of equids. The aetiological agent, African horse sickness virus (AHSV), can be differentiated into nine serotypes. The identification of AHSV serotypes is vital for disease management, as this can influence vaccine selection and help trace disease incursion routes. In this study, we report the development and optimisation of a novel, molecular-based assay that utilises multiplex PCR and microsphere-based technology to expedite detection and differentiation of multiple AHSV serotypes in one assay. We demonstrated t...
Rapid investigating of phase I metabolites of SR9009 in vitro horse liver microsomes via feature-based molecular networking approach: Potential applications in doping control. SR9009, a peroxisome proliferator-activated receptor δ (PPARδ) agonist, is known for its potential benefits in energy homeostasis. It failed to receive the United States Food and Drug Administration (USFDA) approval and its illegal distribution has raised concerns. As a result, it has been classified as a prohibited substance by the World Anti-Doping Agency and the International Federation of Horseracing Authorities (IFHA). This study emphasizes the application of the in-silico molecular networking technology to analyze phase I drug metabolites in horses, distinguishing it from conventional ...
Why the racing industry and equestrian disciplines need to implement population pharmacokinetics: To learn, explain, summarize, harmonize, and individualize. Population pharmacokinetics (POP PK) is a powerful pharmacokinetic tool, which measures quantitatively, and explains the variability in drug exposure and drug effect between individuals. POP PK uses an observational (nonexperimental) approach; it is conducted in the target population living in its normal environment (e.g., farm and race-track). The strength of the POP PK approach lies in its greater relevance for the population studied in its different natural environments than experimental studies carried out in more or less biased laboratory conditions. In clinical settings, it is commonly n...
Endogenous nature of estra-4,9-diene-3,17-dione in entire male horses. Estra-4,9-diene-3,17-dione (dienedione) is an anabolic androgenic steroid (AAS) sold as a bodybuilding supplement. It is prohibited in both human and equine sports. With no report of 4,9-diene configuration in endogenous steroids, dienedione has long been considered a synthetic AAS. Nevertheless, the reoccurring detection of dienedione in colt (entire male horse) urine samples lead to the investigation of its possible endogenous nature in horses. This paper describes (i) the detection of naturally occurring dienedione in colts, (ii) the conjugation study of dienedione and (iii) the population ...
Metabarcoding study to reveal the structural community of strongylid nematodes in domesticated horses in Thailand. Mixed strongylid infections significantly impact equine health and performance. Traditional microscopy-based methods exhibit limitations in accurately identifying strongylid species. Nemabiome deep amplicon sequencing approach previously succeeded in describing the strongylid communities in livestock including equids. However, there are no available studies that describe the structural communities of strongylid parasites in horses in Thailand. Therefore, this study was undertaken encompassing the ITS-2 rDNA metabarcoding assay to characterize strongylid species within horse fecal samples colle...
