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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.
Metabolomics in equine sport and exercise. metabolomics is the high-throughput, multiparametric identification and classification of hundreds of low molecular weight metabolites in a biological sample. Ultimately, metabolites are the downstream readouts of cellular signalling, transcriptomic and proteomic changes that can provide a comprehensive view of tissue and organismal phenotype. The popularity of metabolomics in human sport and exercise has been gaining over the past decade and has provided important insights into the energetic demands and mechanistic underpinnings of exercise and training. To the contrary, metabolomics in the f...
Differential Biodegradation Kinetics of Collagen Membranes for Bone Regeneration. Native collagen-based membranes are used to guide bone regeneration; but due to their rapid biodegradation, this treatment is often unpredictable. The purpose of this study was to investigate the biodegradability of natural collagen membranes. Three non-cross-linked resorbable collagen barrier membranes were tested: Derma Fina (porcine dermis), Evolution Standard (equine pericardium) and Duo-Teck (equine lyophilized collagen felt). 10 × 10 mm pieces of membranes were submitted to three different degradation procedures: (1) hydrolytic degradation in phosphate buffer solution, (2) enzyme resist...
Conjugation and Characterization of Latex Particles with Toxoplasma gondii-specific Immunoglobulin Y Antibodies for Diagnostic Aim and Evaluation Efficiency in In Vitro Culture. Toxoplasma gondii is a parasite that causes severe health problems in the world. Toxoplasmosis, an infection caused by T. gondii, leads to high risk of mortality in patients with immunodeficiency, transplantation, and cancer. Besides that, it causes miscarriages in pregnancy, various abnormalities such as hydrocephalus in infants and congenital diseases. Because the clinical indication of the disease is not specific, it is confused with many diseases, and this leads to the necessity of directly detecting the presence of the toxoplasmosis. Therefore, various diagnostic assays are needed for th...
Determination of bromhexine and its metabolites in equine serum samples by liquid chromatography – Tandem mass spectrometry: Applicability to the elimination study after single oral dose. Bromhexine (BH), expectorant used in the treatment of respiratory disorders associated with viscid or excessive mucus, is not permitted for use in the competing horse by many authorities in horseracing and Olympic disciplines. Metabolic studies are of the great importance in anti-doping field because they allow for updating the selection of the most appropriate markers for prohibited substances, such as metabolites present at higher concentration levels and/or lasted for a longer period of time in biological samples than a parent drug. This study describes LC-MS/MS-based method for simultaneou...
Eliminating sweet spot in MALDI-MS with hydrophobic ordered structure as target for quantifying biomolecules. In matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), the analyte is usually distributed unevenly throughout the sample spot. The area with aggregated analyte molecules contributing abundant signal, is termed as "sweet spot", which results in poor detection reproducibility and makes it impossible to quantify analytes without internal standards. We proposed a strategy to eliminate sweet spot in MALDI-MS by using a hydrophobic ordered structure as target. The target is fabricated by creating a hydrophobic silicon nanopillar array and subsequently decorating it uniformly wi...
Enhanced UHPLC-MS/MS screening of selective androgen receptor modulators following urine hydrolysis. Selective androgen receptor modulators (SARMs) represent non-steroidal agents commonly abused in human and animal (i.e. equine, canine) sports, with potential for further misuse as growth promoting agents in livestock-based farming. As a direct response to the real and possible implications of illicit application in both sport as well as food production systems, this study incorporated enzymatic hydrolysis (β-glucuronidase/arylsulfatase) into a previously established protocol while maintaining the minimal volume (200 µL) of urine sample required to detect SARMs encompassing various pharmacop...
Doping control analysis of antipsychotics and other prohibited substances in equine plasma by liquid chromatography/tandem mass spectrometry. Antipsychotics are banned substances and considered by the Fédération Equestrian Internationale (FEI) to have no legitimate use in equine medicine and/or have a high potential for abuse. These substances are also prohibited in horseracing according to Article 6 of the International Agreement on Breeding, Racing and Wagering (published by the International Federation of Horseracing Authorities). Over the years, antipsychotics have been abused or misused in equestrian sports and horseracing. A recent review of literature shows that there is yet a comprehensive screening method for antipsychoti...
Metabolism, pharmacokinetics and selected pharmacodynamic effects of codeine following a single oral administration to horses. To describe the pharmacokinetics and selected pharmacodynamic variables of codeine and its metabolites in Thoroughbred horses following a single oral administration. Methods: Prospective experimental study. Methods: A total of 12 Thoroughbred horses, nine geldings and three mares, aged 4-8 years. Methods: Horses were administered codeine (0.6 mg kg) orally and blood was collected before administration and at various times until 120 hours post administration. Plasma and urine samples were collected and analyzed for codeine and its metabolites by liquid chromatography-mass spectrometry, and plas...
Water-Soluble Carbohydrates of Cool-Season Grasses: Prediction of Concentrations by Near-Infrared Reflectance Spectroscopy and Evaluation of Effects of Genetics, Management, and Environment. An excess of water-soluble carbohydrates (WSCs) can present a risk for horses prone to pasture-associated laminitis or some other metabolic conditions. Determining WSC concentrations in commonly grazed cool-season grasses, at different times of day and under different fertilization treatments, can help optimize grazing times and management strategies. The goals of this study were to develop a near-infrared reflectance spectroscopy (NIRS) calibration curve for WSC and to apply it to four cool-season grass species. Tall fescue, Kentucky bluegrass, orchardgrass, and perennial ryegrass (10 cultiva...
Investigation of plasma concentrations of paracetamol, metacetamol, and orthocetamol in Japanese racehorses using liquid chromatography-electrospray ionisation-tandem mass spectrometry. Paracetamol is used widely as an over-the-counter analgesic and antipyretic medication for humans, but not for Japanese racehorses. Paracetamol can be an environmental substance, and is found together with its two isomers, metacetamol and orthocetamol, in equine urine. However, the sources and routes of paracetamol exposure remain unclear. To control the misuse of paracetamol, it is appropriate to establish residue limits for paracetamol to differentiate the administration of paracetamol from its environmental levels. In this study, we developed and validated a quantitative method for paraceta...
Iron Biomineral Growth from the Initial Nucleation Seed in L-Ferritin. X-ray structures of homopolymeric human L-ferritin and horse spleen ferritin were solved by freezing protein crystals at different time intervals after exposure to a ferric salt and revealed the growth of an octa-nuclear iron cluster on the inner surface of the protein cage with a key role played by some glutamate residues. An atomic resolution view of how the cluster formation develops starting from a (μ -oxo)tris[(μ -glutamato-κO:κO')](glutamato-κO)(diaquo)triiron(III) seed is provided. The results support the idea that iron biomineralization in ferritin is a process initiating at the l...
Identification of ex vivo catabolites of peptides with doping potential in equine plasma by HILIC-HRMS. Bioactive peptides pose a great threat to sports integrity. The detection of these peptides is essential for enforcing their prohibition in sports. Identifying the catabolites of these peptides that are formed ex vivo in plasma may improve their detection. In the present study, the stability of 27 bioactive peptides with protection at both termini in equine plasma was examined under different incubation conditions, using HILIC coupled to HRMS. Of the 27 peptides, 13 were stable after incubation at 37°C for 72 hr, but the remaining 14 were less stable. Ex vivo catabolites of these 14 peptides ...
Detection of Age-Related Changes in Tendon Molecular Composition by Raman Spectroscopy-Potential for Rapid, Non-Invasive Assessment of Susceptibility to Injury. The lack of clinical detection tools at the molecular level hinders our progression in preventing age-related tendon pathologies. Raman spectroscopy can rapidly and non-invasively detect tissue molecular compositions and has great potential for in vivo applications. In biological tissues, a highly fluorescent background masks the Raman spectral features and is usually removed during data processing, but including this background could help age differentiation since fluorescence level in tendons increases with age. Therefore, we conducted a stepwise analysis of fluorescence and Raman combined s...
Evaluation of the Element point-of-care blood gas analyzer for use in horses. To compare the Element point-of-care (POC) portable blood gas analyzer with a laboratory-based bench-top reference analyzer using whole blood samples obtained from horses presenting to a referral center with various disorders in order to determine agreement between these analyzers. Methods: Prospective clinical study. Methods: The study was conducted at a university teaching hospital at moderate altitude. Methods: One hundred paired samples from 80 horses >1 year of age were collected after obtaining informed client consent. Fifty paired samples were from patients admitted for elective pro...
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...
