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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.
Evaluation of Blood Glucose and Lactate Concentrations in Mule and Equine Foals. Information about mule physiology is scarce. Glucose and lactate serve as prognostic tools in neonates; thus, real-time evaluations would be beneficial. Our main objective was to measure glucose and lactate concentrations from healthy mule and equine foals from birth to 720 hours. Glucose and lactate concentrations were evaluated with a benchtop Randox Daytona analyzer (LAB) using plasma and with an Accutrend Plus system (ACP) using whole blood. Data were analyzed using PROC MIXED (P < .05), intraclass coefficient correlation and Bland-Altman analysis. Glucose and lactate concentrations fro...
Ethanol-Soluble Carbohydrates of Cool-Season Grasses: Prediction of Concentration by Near-Infrared Reflectance Spectroscopy (NIRS) and Evaluation of Effects of Cultivar and Management. Ethanol-soluble carbohydrates (ESCs) of cool-season grasses include mono- and disaccharides and sometimes short-chain fructans, which may exacerbate the risk of pasture-associated laminitis. A calibration for prediction of ESC concentrations by near-infrared reflectance spectroscopy (NIRS) was developed from 323 samples of four cool-season grass species (orchardgrass, Kentucky bluegrass, tall fescue, and perennial ryegrass) across 10 cultivars collected in central Kentucky in the morning and afternoon over two growing seasons. The calibration, which had accuracy above 95%, was used to predict ...
Comprehensive characterization of the peroxisome proliferator activated receptor-δ agonist GW501516 for horse doping control analysis. According to international sport institutions, the use of peroxisome proliferator activated receptor (PPAR)-δ agonists is forbidden at any time in athlete career due to their capabilities to increase physical and endurance performances. The (PPAR)-δ agonist GW501516 is prohibited for sale but is easily available on internet and can be used by cheaters. In the context of doping control, urine is the preferred matrix because of the non-invasive nature of sampling and providing broader exposure detection times to forbidden molecules but often not detected under its native form due to the organi...
Determination of vitamin E and its metabolites in equine urine using liquid chromatography-mass spectrometry. Equine neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM) is a hereditary, deteriorating central nervous disease in horses. Currently, the only way to confirm eNAD/EDM is through a postmortem histological evaluation of the central nervous system. Vitamin E, specifically the isoform alpha-tocopherol (α-TP), is known to protect eNAD/EDM susceptible horses from developing the clinical phenotype. While vitamin E is an essential nutrient in the diet of horses, there are no diagnostic tests able to quantitate vitamin E and its metabolites in urine. An ultra-performance liquid chromat...
A validated method for the quantification of IOX-2, a potent prolyl hydroxylase inhibitor in equine urine and plasma using liquid chromatography-high-resolution mass spectrometry. IOX-2 is a potent inhibitor of enzyme prolyl hydroxylases-2 (PHD) that plays a critical role in regulating hypoxia inducible factor (HIF) abundance and oxygen homeostasis. Federation for Equestrian Sports has listed HIF activators as prohibited substances to prevent their usage in doping. Consequently, it became essential to develop adequate knowledge and testing methods to detect it in equine sports drug testing samples. The validated method utilizes ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry in order to detect extremely low concentration of the ...
Investigation of Equine In Vivo and In Vitro Derived Metabolites of the Selective Androgen Receptor Modulator (SARM) ACP-105 for Improved Doping Control. Selective Androgen Receptor Modulators (SARMs) have anabolic properties but less adverse effects than anabolic androgenic steroids. They are prohibited in both equine and human sports and there have been several cases of SARMs findings reported over the last few years. The aim of this study was to investigate the metabolite profile of the SARM ACP-105 (2-chloro-4-[(3-endo)-3-hydroxy-3-methyl-8-azabicyclo[3.2.1]oct-8-yl]-3-methylbenzonitrile) in order to find analytical targets for doping control. Oral administration of ACP-105 was performed in horses, where blood and urine samples were collect...
Doping detection in animals: A review of analytical methodologies published from 1990 to 2019. Despite the impressive innate physical abilities of horses, camels, greyhounds, or pigeons, doping agents might be administered to these animals to improve their performance. To control these illegal practices, anti-doping analytical methodologies have been developed. This review compiles the analytical methods that have been published for the detection of prohibited substances administered to animals involved in sports over 30 years. Relevant papers meeting the search criteria that discussed analytical methods aiming to detect and/or quantify doping substances in animal biological matrices pu...
A sensitive HPLC-MS/MS method for the detection, resolution and quantitation of cathinone enantiomers in horse blood plasma and urine. Resolution of cathinone enantiomers in equine anti-doping analysis is becoming more important to distinguish the inadvertent ingestion of plant-based products from those of deliberate administration of designer synthetic analogs. With this in mind, a rapid and sensitive method was developed and validated for the detection, resolution and quantitative determination of cathinone enantiomers in horse blood plasma and urine. The analytes were recovered from the blood plasma and urine matrices by using a liquid-liquid extraction after adjusting the pH to 9. The recovered analytes were derivatized w...
Systemic detectability of dexamethasone and prednisolone after eye drop application in horses. Equine sport agencies list steroids as prohibited substances for competing horses. Objective: The objective of this study was to investigate if the controlled substances dexamethasone and prednisolone are detectable in equine serum and urine samples during and after treatment with eye drops and if this can generate a positive doping test. Methods: Prospective cohort study. Methods: The study cohort included 11 horses. One eye of the horses was treated with either dexamethasone (Maxitrol 0.1%, n = 5 eyes) or prednisolone (Pred forte 1%, n = 6 eyes) eye drops 3 times daily for 14 days. Dexa...
Detection and longitudinal distribution of GW1516 and its metabolites in equine hair for doping control using liquid chromatography/high-resolution mass spectrometry. GW1516 is a peroxisome proliferator-activated receptor-δ (PPAR-δ) agonist that is banned in horseracing and equestrian sports. Long-term detection and longitudinal distribution of GW1516 in the mane of a horse are reported for the first time and this hair analysis could prolong the detection window of GW1516 for doping control. Methods: Mane hairs were divided into three segments (0-7, 7-15, and >15 cm from the cut end) and completely pulverized and homogenized for analysis. The pulverized hair samples were extracted with methanol followed by further purification and the extracts were a...
Applicability of commercial clinical chemistry test kits for horse serum. Validation of a test method is critical for confirming that the test can generate accurate and precise data. Although commercial biochemical test kits exist there are no specific and validated commercial clinical chemistry test kits designed for horses. The aim of this study was to validate commercial clinical chemistry test kits designed for a human serum for use in horses. Results: Blood samples were collected from 29 apparently healthy adult male horses and pooled serum was prepared. Validation comprises replication and recovery experiments. Total observable error (TE), sigma (σ) metrics, ...
Metabolic study of GW1516 in equine urine using liquid chromatography/electrospray ionization Q-Exactive high-resolution mass spectrometry for doping control. The use of GW1516, a peroxisome proliferator-activated receptor δ (PPAR δ) agonist, is strictly prohibited in both horseracing and equestrian competitions. However, little is known about its metabolic fate in horses. To the best of our knowledge, this is the first reported metabolic study of GW1516 in equine urine. Methods: Urine samples obtained from a thoroughbred after nasoesophageal administration with GW1516 were protein-precipitated and the supernatants were subsequently analyzed by liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) with a Q-E...
Towards compound identification of synthetic opioids in nontargeted screening using machine learning techniques. The constant evolution of the illicit drug market makes the identification of unknown compounds problematic. Obtaining certified reference materials for a broad array of new analogues can be difficult and cost prohibitive. Machine learning provides a promising avenue to putatively identify a compound before confirmation against a standard. In this study, machine learning approaches were used to develop class prediction and retention time prediction models. The developed class prediction model used a naïve Bayes architecture to classify opioids as belonging to either the fentanyl analogues, AH...
Quantitative and qualitative analysis of operator inhaled aerosols during routine motorised equine dental treatment. Routine equine odontoplasty is performed by both Veterinary Surgeons and Equine Dental Technicians. The production of aerosolised particulates from motorised equipment has been well documented in human orthodontics but has yet to be investigated in the veterinary industry. Objective: To assess the size, quantity and composition of particulates produced during routine motorised odontoplasty and to model their deposition in the human respiratory tree. Methods: Analytic observational study. Methods: Fifteen-minute routine motorised odontoplasties were performed on cadaver heads with monitoring eq...
Preanalytical variables affecting the measurement of serum paraoxonase-1 activity in horses. Paraoxonase-1 (PON-1) activity is a new inflammatory and oxidative marker. Technical effects and biological factors could affect the accuracy of PON-1 activity measurement. We investigated the effects of storage at different temperatures, repeated freeze-thaw cycles, interferences from hemolytic, lipemic, and icteric samples, and seasonal effects on PON-1 activity in horses. We evaluated 2 substrates with an automated spectrophotometer. Ten equine serum samples were stored under different conditions. Although storage at room (21°C) or refrigeration (4°C) temperature induced a statistically s...
Postmortem Vitreous Humor Analysis in Dogs, Cats and Horses. Postmortem chemistry can be a useful ancillary technique that the forensic pathologist can use during a death investigation. In stark contrast, there is limited information available for use of postmortem vitreous humor analysis in animals. In order to use postmortem vitreous humor in veterinary forensic investigations, validation of a method to analyze vitreous humor is required. The goal of this study was to determine the precision, bias, TEobs and sigma (σ) of the Element DC chemistry analyzer; assess its precision using the vitreous humor collected postmortem from dogs, cats and horses an...
Metabolism and excretion of the benzodiazepine analogue etizolam in the horse. Etizolam is a benzodiazepine analogue that is approved for use in Japan, Italy and India but has recently appeared as a nonapproved product on the illicit drug market in Europe and North America. Etizolam was identified in a crystalline material seized at a Kentucky racetrack, raising concerns that this drug may have been used in racing. The aim of this study was to characterize the metabolism and excretion of etizolam in horses to generate information on its disposition and to incorporate the correct urinary and serum target analytes into anti-doping screening procedures. Etizolam was adminis...
A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitochondrial DNA variants. Mitochondrial DNA is remarkably polymorphic. This is why animal geneticists survey mitochondrial genomes variations for fundamental and applied purposes. We present here an approach to sequence whole mitochondrial genomes using nanopore long-read sequencing. Our method relies on the selective elimination of nuclear DNA using an exonuclease treatment and on the amplification of circular mitochondrial DNA using a multiple displacement amplification step. Results: We optimized each preparative step to obtain a 100 million-fold enrichment of horse mitochondrial DNA relative to nuclear DNA. We sequ...
Application of Raman Spectroscopy for the Evaluation of Metabolomic Dynamic Analysis in Athletic Horses. Raman spectroscopy is a rapid qualitative and quantitative technique that allows the simultaneous determination of several components, both biomolecules both chemical compounds, in the biological fluids to assess the metabolic status. In this study, the serum composition was evaluated in regularly trained athletic horses using Raman spectroscopy to identify biomarkers of sports performance. Five clinically healthy and regularly trained Italian Saddle horses were subjected to a standardized obstacle course (350 m/minute; eleven 1.25 high jumps) preceded by a warm-up. On the collected sera, at ...
Study design synopsis: Battle in the stable: Bayesianism versus Frequentism. Frequentism dominates scientific practice although Bayesianism may provide an alternative, especially when analysing data from complex, high-dimensional models. The key differences between Bayesianism and Frequentism are highlighted in the introduction. Next, I review the different stages of Bayesian statistical reasoning in a research setting, explain the key concepts and illustrate them with toy examples taken in equine veterinary medicine. An extension to more complex models (Bayes network) is introduced and guidelines are offered as a conclusion.
Evaluation of dried blood spots as an alternative sample matrix for equine antidoping analysis. Controlling the abuse of prohibited substances such as anabolic steroids, selective androgen receptor modulators, β-adrenoceptor agonists, and blood doping agents is of great interest to racing authorities. The use of dried blood spots (DBS) as an alternative sampling approach may be a feasible approach for controlling the use of these agents. To assess the feasibility of using DBS in equine blood, an 11-min liquid chromatography-mass spectrometry method was developed on a triple quadrupole mass spectrometer following extraction from Whatman 903 DBS cards. A total of 50 compounds across multi...
Equine metabolism of the selective androgen receptor modulator AC-262536 in vitro and in urine, plasma and hair following oral administration. AC-262536 is one of a number of selective androgen receptor modulators that are being developed by the pharmaceutical industry for treatment of a range of clinical conditions including androgen replacement therapy. Though not available therapeutically, selective androgen receptor modulators are widely available to purchase online as (illegal) supplement products. The growth- and bone-promoting effects, along with fewer associated negative side effects compared with anabolic-androgenic steroids, make these compounds a significant threat with regard to doping control in sport. The aim of this st...
Bioaccumulation of Mineral Elements in Different Biological Substrates of Athletic Horse from Messina, Italy. The objective of this study was to evaluate the levels and the potential bioaccumulation of vanadium (V), chromium (Cr), cobalt (Co), copper (Cu), zinc (Zn), cadmium (Cd), lead (Pb), and bismuth (Bi) in horses from the industrial risk area of Sicily (Italy). Different biological substrates (whole blood, serum; tail and mane) and samples of hay, concentrate and water provided to the horses were processed by means of Thermo Scientific iCAP-Q ICP-MS spectrometer for mineral concentration. One-way analysis of variance (ANOVA) was applied to show the differences in various trace elements in the bio...
Characterization of total adenosine deaminase activity (ADA) and its isoenzymes in saliva and serum in health and inflammatory conditions in four different species: an analytical and clinical validation pilot study. Measurement of adenosine deaminase (ADA) can provide information about cell-mediated immunity. This report's objective was to study the enzymatic activity of total ADA (tADA) and its isoenzymes ADA1 and ADA2 in canine, equine, porcine, and bovine serum and saliva and their changes in different inflammatory situations in each species. Besides, an automated method for ADA2 measurement was developed and validated. Results: tADA was present in serum and saliva of healthy animals of the four species. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) concentration of 0.47 mM was needed for ADA1 inhibit...
Microwave assisted fast fabrication of zinc/iron oxides based polymeric nanocomposites and evaluation on equine fibroblasts. We aimed to provide a tissue repair material, which can be synthesized rapidly, using polymers mimicking the natural environment in the extra-cellular matrix and metals/minerals. The components should have the potential to be used in tissue repair and simultaneously, reducing the side-effects of the incorporated molecules. It is challenging to manage the dispersibility of ZnO NPs in common solutions like water. Here, we report a novel method for preparing highly dispersible suspensions of ZnO NPs. In contrast to those synthesized by conventional methods, microwave assisted method allowed synth...
Agreement of 2 electrolyte analyzers for identifying electrolyte and acid-base disorders in sick horses. Use of different analyzers to measure electrolytes in the same horse can lead to different interpretation of acid-base balance when using the simplified strong ion difference (sSID) approach. Objective: Investigate the level of agreement between 2 analyzers in determining electrolytes concentrations, sSID variables, and acid-base disorders in sick horses. Methods: One hundred twenty-four hospitalized horses. Methods: Retrospective study using paired samples. Electrolytes were measured using a Beckman Coulter AU480 Chemistry analyzer (PBMA) and a Nova Biomedical Stat Profile (WBGA), respectivel...
Metabolic studies of selective androgen receptor modulators RAD140 and S-23 in horses. This paper describes the studies of the in vitro biotransformation of two selective androgen receptor modulators (SARMs), namely, RAD140 and S-23, and the in vivo metabolism of RAD140 in horses using ultra-high performance liquid chromatography-high resolution mass spectrometry. in vitro metabolic studies of RAD140 and S-23 were performed using homogenised horse liver. The more prominent in vitro biotransformation pathways for RAD140 included hydrolysis, hydroxylation, glucuronidation and sulfation. Metabolic pathways for S-23 were similar to those for other arylpropionamide-based SARMs. The a...
Clinical magnetic resonance image quality of the equine foot is significantly influenced by acquisition system. Investigation of image quality in clinical equine magnetic resonance (MR) imaging may optimise diagnostic value. Objective: To assess the influence of field strength and anaesthesia on image quality in MR imaging of the equine foot in a clinical context. Methods: Analytical clinical study. Methods: Fifteen equine foot studies (five studies per system) were randomly selected from the clinical databases of three MR imaging systems: low-field standing (LF St), low-field anaesthetised (LF GA) and high-field anaesthetised (HF GA). Ten experienced observers graded image quality for entire studies an...
Synthesis and characterization of barbarin, a possible source of unexplained aminorex identifications in forensic science. Aminorex is a US DEA Schedule 1 controlled substance occasionally detected in racing horses. A number of aminorex identifications in sport horses were thought to have been caused by exposure to plant sources of aminorex. Glucobarbarin, found in plants of the Brassicaceae family, has been suggested as a potential proximate chemical source by being metabolized in the plant or the horse to aminorex. In Brassicaceae, glucobarbarin is hydrolyzed by myrosinase to yield barbarin, which serves as an insect repellant and/or attractant and is structurally related to aminorex. The synthesis, purification...
Changes in Salivary Analytes of Horses Due to Circadian Rhythm and Season: A Pilot Study. This study aims to evaluate the circadian and circannual variations in a panel of analytes in horse saliva that have been previously described as biomarkers related to stress and disease, in order to interpret them correctly when they are measured in this species. This panel of analytes integrated cortisol, salivary alpha-amylase (sAA), lipase (Lip), total esterase (TEA), butyrylcholinesterase (BChE), adenosine deaminase (ADA), γ-glutamyl transferase (gGT), creatine kinase (CK), urea, total bilirubin, total protein (TP), and phosphorus. These analytes were measured in saliva obtained from a p...
