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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 intra-articular gene therapy in horses using quantitative real time PCR in synovial fluid and plasma. Gene therapy promotes the expression of missing or defective genes and can be curative following administration of a single dose. Gene therapy is prohibited in equine athletes by regulatory bodies due to the high potential for abuse and novel analytical methods are needed for detection. The goal of this study was to detect the administration of an experimental gene therapy: a recombinant adeno-associated viral vector (rAAV) carrying a transgene for the anti-inflammatory cytokine IL-10 (rAAV-IL10). Twelve horses were randomly assigned to receive an intra-articular injection of rAAV-IL10 or phos...
Point-of-Care Diagnostics in Equine Practice. Point-of-care testing (POCT) refers to benchtop diagnostic modalities that have been translated into portable and easy-to-use formats suitable for patient-side use. Recent advances in diagnostic technology have allowed the development of a growing collection of POCT assays available to equine practitioners. Advantages include rapid results that reduce initial guesswork and promote diagnosis-targeted patient care, which may ultimately provide better clinical outcomes. Small handheld devices comprise most POCT technologies, providing qualitative or quantitative determination of an increasing ran...
Label-free Proteomics for Discovering Biomarker Candidates for Controlling Krypton Misuse in Castrated Horses (Geldings). Recent advances in label-free quantitative proteomics may support its application in identifying and monitoring biomarkers for the purpose of doping control in equine sports. In this study, we developed a workflow of label-free quantitative proteomics to propose plasma protein biomarkers in horses after administration with krypton (Kr), a potential erythropoiesis-stimulating agent. Plasma proteomes were profiled by using nanoliquid chromatography-high-resolution mass spectrometry. An in-house mass spectral library consisting of 1121 proteins was compiled using samples collected from geldings (...
Optimised method for determination hypoglycine A in maple plant material by multidimensional gas chromatography/mass spectrometry. In 2018, more than 50 cases of horse death by equine atypical myopathy (AM) were reported in the Czech Republic. This disease is often associated with the toxin hypoglycine A (HGA), which is found in several maple plant materials. To monitor this toxin in products of these trees that grow in or around horse pastures, a rapid and inexpensive analytical method that can provide the required accuracy is needed. Until now, maple samples have been prepared for gas chromatography using time-consuming methods, with preparation processes taking longer than 1 h. In this work, a shorter method (25 mi...
Salivary cortisol measurement in horses: immunoassay or LC-MS/MS? The aim of the present study was to measure salivary cortisol concentrations of horses before and after hypothalamic-pituitary-adrenal (HPA) axis stimulation by means of liquid chromatography-tandem-mass spectrometry (LC-MS/MS) and an immunoassay (cELISA) for method comparison. Nine clinically healthy horses participated in the study. An ACTH stimulation test was performed. Saliva samples were collected before (T0) and 60 (T60) min after intravenous injection of 1 μg/kg BW synthetic ACTH1-24. LC-MS/MS was assessed for the determination of equine salivary cortisol. The results of these measur...
Using Inductively Coupled Plasma Mass Spectrometry to Assess Essential and Performance-Enhancing Metals in the Urine of Racehorses. Recently, an increased tendency to use various metals has been observed in the sports competition fields. Many of these metals and their organic complexes reportedly have good pharmacologic, therapeutic and performance-enhancement uses; they are banned or recommended as controlled medications in competitive sports. The objective of this research was to determine the concentration of pharmacologically relevant metals in urine samples collected from racehorses at various sport events, develop a method and assess the concentrations of above metals using inductively coupled plasma mass spectrometr...
Practical Tips on Sample Handling for Hematology, Chemistry, and Cytology Testing for Equine Patients:: Getting More Bang for your Buck. Clinical pathology results are only as good as the quality of samples and accompanying information submitted to the diagnostic laboratory. The frustration of nondiagnostic or equivocal test results can often be avoided by taking the time to follow sample handling and submission guidelines. This article discusses preanalytical errors that commonly affect the accuracy of hematology, chemistry, and cytology testing, and offers practical tips for preventing these errors and maximizing diagnostic yield.
Development and validation of a chiral LC-MS method for the enantiomeric resolution of (+) and (-)-medetomidine in equine plasma by using polysaccharide-based chiral stationary phases. The detection and separation of medetomidine enantiomers from the complex biological matrices poses a great analytical challenge, especially in the field of forensic toxicology and pharmacology. Couple of researchers reported resolution of medetomidine using protein-based chiral columns, but the reported method is quiet challenging and tedious to be employed for routine analysis. This research paper reported a method that enables the enantio-separation of medetomidine by using polysaccharide cellulose chiral column. The use of chiralcel OJ-3R column was found to have the highest potential for ...
Effect of food contamination and collection material in the measurement of biomarkers in saliva of horses. This study aims to evaluate the effect of the presence of food and the material used in a panel of biomarkers in saliva of horses. For the food effect study, clean saliva was incubated with a known amount of food consisting of oats, hay or grass. Significant changes were observed when saliva was incubated with oats for total protein (PÂ =Â .050) and phosphorus (PÂ =Â .008), with grass for total protein (PÂ =Â .037), salivary alpha-amylase (sAA, PÂ =Â .018), total esterase (TEA, PÂ =Â .018), butyrilcholinesterase (BChE, PÂ =Â .037), adenosine deaminase (ADA, PÂ =Â .037), and total bilirubin (PÂ...
Investigation of the metabolism of the selective androgen receptor modulator LGD-4033 in equine urine, plasma and hair following oral administration. LGD-4033 is one of a number of selective androgen receptor modulators (SARMs) that are being developed by the pharmaceutical industry to provide the therapeutic benefits of anabolic androgenic steroids, without the less desirable side effects. Though not available therapeutically, SARMs are available for purchase online as supplement products. The potential for performance enhancing effects associated with these products makes them a significant concern with regards to doping control in sports. The purpose of this study was to investigate the metabolism of LGD-4033 in the horse following oral ...
Validation of an equine serum amyloid A assay with an unusually broad working range. Serum amyloid A (SAA) is a major equine acute phase protein and of great value in detection and monitoring of inflammation. A new immunoturbidometric assay based on monoclonal antibodies (VET-SAA, Eiken Chemical Co., Japan) may be useful for SAA measurements in routine diagnostic laboratories. The aim of the study was to validate the VET-SAA immunoturbidometric assay and use it to measure serum SAA concentrations in a variety of clinical cases. Precision was assessed by intra- and interassay coefficients of variation of repeated measurements of serum pools (low, intermediate, high concentratio...
Bioformation of boldenone and related precursors/metabolites in equine feces and urine, with relevance to doping control. Boldenone (1-dehydrotestosterone) is an exogenous anabolic-androgenic steroid (AAS) but is also known to be endogenous in the entire male horse and potentially formed by microbes in voided urine, the gastrointestinal tract, or feed resulting in its detection in urine samples. In this study, equine fecal and urine samples were incubated in the presence of selected stable isotope labeled AAS precursors to investigate whether microbial activity could result in 1-dehydrogenation, in particular the formation of boldenone. Fecal matter was initially selected for investigation because of its high mic...
Changes in Saliva Analytes Correlate with Horses’ Behavioural Reactions to An Acute Stressor: A Pilot Study. Acute stress induces an array of behavioural reactions in horses that vary between individuals. Attempts to relate behavioural patterns and physiological responses have not always given clear-cut results. Here, we measured the changes in a panel of salivary components: salivary alpha-amylase (sAA), lipase, total esterase (TEA), butyrylcholinesterase (BChE), adenosine deaminase (ADA), and cortisol, and their potential link with horses' behaviours after acute stress. Saliva samples were collected in nine riding horses subjected to a test consisting of opening an umbrella. Saliva sampling was obt...
The effect of counting duration on quantitative fecal egg count test performance. Fecal egg counts are the primary diagnostic tools of equine parasitology and use of the McMaster test and its variants in clinical practice is widely recommended. Manual counting is, however, prone to various sources of human error. For example, in real-world situations analysts can be under significant pressure to process high numbers of samples in a limited time. This practice could affect test result quality, but yet no studies have determined whether this is the case. This study's purpose was to assess the effect of shortened counting duration (from either restricting counting time or coun...
Establishment and application of a 10-plex liquid bead array for the simultaneous rapid detection of animal species. Meat fraud and adulteration incidents occur frequently in almost all regions of the globe, especially with the increase in the world's population. To ensure the authenticity of meat products, we developed a 10-plex xMAP assay to simultaneously detect ten animal materials: bovine, caprine, poultry, swine, donkey, deer, horse, dog, fox and mink. Results: This method was investigated by analyzing DNA extracts from raw muscle, muscle mixtures, meat products and animal feeds. Our results indicated that the species of interest can be identified, differentiated and detected down to 1 g kg in bina...
Identification of metabolomic changes in horse plasma after racing by liquid chromatography-high resolution mass spectrometry as a strategy for doping testing. Recently, the illegal use of novel technologies, such as gene and cell therapies, has become a great concern for the horseracing industry. As a potential way to control this, metabolomics approaches that comprehensively analyze metabolites in biological samples have been gaining attention. However, it may be difficult to identify metabolic biomarkers for doping because physiological conditions generally differ between resting and exercise states in horses. To understand the metabolic differences in horse plasma between the resting state at training centres and the sample collection stage after...
Occupational Exposures in an Equestrian Centre to Respirable Dust and Respirable Crystalline Silica. Sand-based products are regularly used as footing material on indoor equestrian arenas, creating a potential occupational exposure risk for respirable crystalline silica (RCS) for equestrian workers training and exercising horses in these environments. The objective of this study was to evaluate an equestrian worker's personal RCS and respirable dust (RD) exposure. Sixteen personal full-shift RD measurements were collected from an equestrian worker and analysed for RD, quartz and cristobalite. Geometric mean exposures of 0.12 mg m and 0.02 mg m were calculated for RD and RCS concentrations, re...
Investigations of Processing-Induced Structural Changes in Horse Type-I Collagen at Sub and Supramolecular Levels. The aim of this work is to evaluate the effects of different extraction and material processing protocols on the collagen structure and hierarchical organization of equine tendons. Wide and Small Angle X-ray Scattering investigations on raw powders and thin films revealed that not only the extraction and purification treatments, but also the processing conditions may affect the extent of the protein crystalline domain and induce a nanoscale "shield effect." This is due to the supramolecular fiber organization, which protects the atomic scale structure from the modifications that occur during f...
Precision and accuracy of a point-of-care glucometer in horses and the effects of sample type. Point-of-care glucometry is used commonly in clinical and research settings; however, accuracy and precision of this method are concerns. The objectives of this study were to determine the accuracy of glucometry in adult horses and the precision of duplicate measurements. Blood samples were collected from 62 horses into one plain syringe, one EDTA tube and three fluoride oxalate (FO) tubes. Immediately after collection, glucose concentrations in whole blood were determined, in duplicate, by glucometry from the syringe (plain whole blood [WB] group), EDTA tube (EDTA group) and one FO tube (FO g...
Irish Equine Industry Stakeholder Perspectives of Objective Technology for Biomechanical Analyses in the Field. Wearable sensing technologies are increasingly used in human and equine gait research to improve ecological validity of research findings. It is unclear how these tools have penetrated the equine industry or what perspectives industry stakeholders' hold in relation to these relatively new devices. Semi-structured interviews were conducted with Irish equine industry stakeholders to understand their perception of objective tools for biomechanical analysis in the field. The study participants came from professional/elite backgrounds in both the sport horse ( = 6) and thoroughbred ( = 6) sectors. ...
Measurement of peripheral muscle oxygen saturation in conscious healthy horses using a near-infrared spectroscopy device. Maintaining adequate muscle tissue oxygenation is of paramount importance during equine general anesthesia. The objectives of this study were to assess the feasibility, reliability and repeatability of near-infrared spectroscopy (NIRS) muscle oximetry using the Inspectra m650 in conscious healthy adult horses. Methods: Prospective, observational study. Methods: A group of 30 healthy client-owned adult horses admitted to the equine hospital between July 2017 and July 2018. Methods: The probe of an Inspectra m650 NIRS tissue oximeter was placed on the hairless surface of five muscle sites (omotr...
A new method for quantifying causative and diagnostic markers of methylenecyclopropylglycine poisoning. Up to now quantification of hypoglycin A in serum and urine in the range of nmols to μmols per liter plus the measurement of accumulated acyl conjugates have been used for the diagnosis of poisoning by fruits or seeds of in humans and animals. A second poison, methylenecyclopropylglycine, however, is known to occur in this material. The objective of our study was to develop and evaluate a method for the quantification of this compound suitable for test materials obtained from animals and man. Methods: Methylenecyclopropylglycine was extracted from serum and urine of a volunteer by a methanoli...
A comprehensive approach to detecting multitudinous bioactive peptides in equine plasma and urine using hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry. Bioactive peptides possess pharmacological effects and can be illicitly used in sports. To deter such misuse, an untargeted method using high resolution mass spectrometry (HRMS) has been developed for comprehensive detection of multitudinous exogenous peptides in equine plasma and urine. Forty-four peptides were extracted using mixed-mode solid-phase extraction (SPE) from plasma and urine, separated with a hydrophilic interaction liquid chromatography (HILIC) column, and detected on an HRMS instrument. Ammonium formate as a mobile phase additive had effects on HILIC retention and charge state ...
The total content of toxic elements in horsehair given the level of essential elements. Elemental status of 214 mares aged 3-7 years from 11 breeds was studied: Arabian purebred (n = 20), Bashkir (n = 20), Kabarda (n = 20), Vyatka (n = 20), Tuva (n = 19), Yakutsk (n = 30), Mezenskaya (n = 20), Thoroughbred (n = 20), Akhal-Teke (n = 20), Russian trotter (n = 15), Soviet Heavy Draft (n = 10) bred in 13 regions of Russia. The research objective is to study the content of chemical elements in hair from the horse's mane, depending on the sum of toxic elements in animal hair expressed in moles. The elemental composition of the hair was defined by atomic emission and mass spectrometry (...
Determination of reference intervals for equine arterial blood-gas, acid-base and electrolyte analysis. To establish reference intervals for arterial blood-gas (ABG), acid-base and electrolyte values from a healthy equine population. Methods: Retrospective clinical study. Methods: A total of 139 client-owned, systemically healthy horses, 1 year of age and older, presented for elective surgical procedures. Methods: Blood samples were collected anaerobically from the transverse facial or common carotid artery of horses breathing room air, prior to administration of preanaesthetic medication. Samples were analysed immediately, without correction for body temperature, using an automated bench-top an...
Evaluation of Two Handheld Point-of-Care Blood Gas Analyzers in Healthy Donkeys. There has been increasing interest in blood gas analysis in donkeys. "Point-of-care (POC) testing" is a diagnostic testing performed on or immediately next to the patient. This study assesses the agreement between two POC blood gas analyzers in donkeys. Arterial and venous blood samples were collected from 17 donkeys and analyzed using a fully automated blood gas analyzer (ABL 700 Series Radiometer, Denmark) (RAD) and two POC blood gas analyzers (i-STAT System; VetStat, Idexx). The parameters revealed by all three devices were submitted to a canonical discriminant analysis, to evaluate which p...
Changes in saliva analytes in equine acute abdominal disease: a sialochemistry approach. The biochemical components of saliva can change in certain pathologies in horses, for example in acute abdominal disease. The aim of this study was (1) to evaluate if a panel of biochemical analytes usually used in serum can be measured in saliva of horses and (2) to study the possible changes of these biochemical analytes in saliva of horses affected by acute abdominal disease. A panel of 23 analytes was analytically validated in saliva of horses and possible changes in these analytes in a pilot study with six healthy horses and six horses with acute abdominal disease were evaluated. The anal...
Paper spray high-resolution accurate mass spectrometry for quantitation of voriconazole in equine tears. Paper spray high-resolution accurate mass spectrometry is a fast and versatile analysis method. This ambient ionization technique enables the quantitation of xenobiotics in complex biological matrices without chromatography or conventional sample extraction. The simplicity, rapidity, and affordability of the paper spray mass spectrometry (PS-MS) method make the technique especially attractive for clinical investigations where fast and affordable sample analysis is crucial. A new PS-MS method for the quantitation of voriconazole in equine tears was developed and validated. For a concentration r...
A rapid and eco-friendly method for determination of the main components of gamma-oryzanol in equestrian dietary and nutritional supplements by liquid chromatography-Tandem mass spectrometry. Gamma-oryzanol (GO) has gained special attention in the equine sports industry in recent years due to its touted properties, including the fact that it may cause anabolic effects on muscle growth and reduce fatigue. Many manufactures offer supplements containing GO as a naturally occurring anabolic substance; however, some producers do not declare its presence in product compositions. Taking into consideration the touted properties of GO, its ambiguous effectiveness and the open character of the Prohibited Substances List established by the Fédération Equestre Internationale, there is an urg...
