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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.
Determination of amino acid profile of mare milk produced in the highlands of the Kyrgyz Republic during the milking season. This study was carried out to determine the influence of milking season on amino acid (AA) profile and chemical composition of milk samples from Kyrgyz native breed mares under traditional pasture conditions. Milk samples were collected monthly from May to August 2014 from mares grazing at 2,200 m above sea level. The AA composition was determined by precolumn derivatization with diethyl ethoxymethylenemalonate in HPLC and 18 AA were determined in mare milk. The analytical parameters of linearity (0.01-4 μg/mL), precision of the method (0.26-4.88% relative standard deviation), derivatization ...
Initial analytic quality assessment and method comparison of an immunoassay for adrenocorticotropic hormone measurement in equine samples. Equine pituitary pars intermedia dysfunction (PPID) may be diagnosed by measuring baseline plasma adrenocorticotrophic hormone (ACTH). The Immulite 1000 analyzer uses an automated chemiluminescence enzyme assay, previously validated for measuring equine ACTH. Recently, an automated bench-top immunoassay analyzer (AIA-360), designed for analytes in people, became available for veterinary use. Objective: Objectives were to evaluate analytic performance of the AIA immunoassay for measuring equine ACTH, and compare the results with those obtained by the Immulite. Methods: Adrenocorticotrophic horm...
Aptamer based peptide enrichment for quantitative analysis of gonadotropin-releasing hormone by LC-MS/MS. Over recent years threats to racing have expanded to include naturally occurring biological molecules, such as peptides and proteins, and their synthetic analogues. Traditionally, antibodies have been used to enable detection of these compounds as they allow purification and concentration of the analyte of interest. The rapid expansion of peptide-based therapeutics necessitates a similarly rapid development of suitable antibodies or other means of enrichment. Potential alternative enrichment strategies include the use of aptamers, which offer the significant advantage of chemical synthesis onc...
On the mineral core of ferritin-like proteins: structural and magnetic characterization. It is generally accepted that the mineral core synthesized by ferritin-like proteins consists of a ferric oxy-hydroxide mineral similar to ferrihydrite in the case of horse spleen ferritin (HoSF) and an oxy-hydroxide-phosphate phase in plant and prokaryotic ferritins. The structure reflects a dynamic process of deposition and dissolution, influenced by different biological, chemical and physical variables. In this work we shed light on this matter by combining a structural (High Resolution Transmission Electron Microscopy (HRTEM) and Fe K-edge X-ray Absorption Spectroscopy (XAS)) and a magneti...
A quantitative approach to analysing cortisol response in the horse. The cortisol response to glucocorticoid intervention has, in spite of several studies in horses, not been fully characterized with regard to the determinants of onset, intensity and duration of response. Therefore, dexamethasone and cortisol response data were collected in a study applying a constant rate infusion regimen of dexamethasone (0.17, 1.7 and 17 μg/kg) to six Standardbreds. Plasma was analysed for dexamethasone and cortisol concentrations using UHPLC-MS/MS. Dexamethasone displayed linear kinetics within the concentration range studied. A turnover model of oscillatory behaviour accu...
Evaluation of methodological aspects of digestibility measurements in ponies fed different grass hays. Methodological aspects of digestibility measurements of feedstuffs for equines were studied in four Welsh pony geldings consuming four grass-hay diets in a 4 × 4 Latin square design. Diets contained either a low (L), medium (M), high (H), or very high (VH) ADF content (264, 314, 375, or 396 g·kg DM, respectively). Diets were supplemented with minerals, vitamins, and TiO (3.9 g Ti·d). Daily feces excreted were collected quantitatively over 10 consecutive days and analyzed for moisture, ash, ADL, AIA, and titanium (Ti). Minimum duration of total fecal collection (TFC) required for an accurate...
Enhanced sensitivity of an antibody competitive blocking enzyme-linked immunosorbent assay using Equine arteritis virus purified by anion-exchange membrane chromatography. In an effort to improve a competitive blocking enzyme-linked immunosorbent assay (cELISA) for antibody detection to Equine arteritis virus (EAV), antigen purified by anion-exchange membrane chromatography capsule (AEC) was evaluated. Virus purification by the AEC method was rapid and easily scalable. A comparison was made between virus purified by the AEC method with that obtained by differential centrifugation based on the following: 1) the relative purity and quality of EAV glycoprotein 5 (GP5) containing the epitope defined by monoclonal antibody 17B7, and 2) the relative sensitivity of a c...
An analytical ergonomic risk evaluation of body postures during daily cleaning tasks in horse stables. Musculoskeletal disorders (MSDs) are a common reason for health problems and sick leave in many professions. A work arena where MSDs have not been sufficiently studied is stable work. Here many heavy and repetitive work tasks are performed manually with old-fashioned working tools. Objective: The aim of this study was to use an analytical approach to identify, evaluate and grade ergonomic problems in physical work performed during daily activities in horse stables and relate these to MSDs among the workers and to the design of the tools. Methods: Three common work tasks were studied to identif...
Analysis of metronidazole in equine plasma using liquid chromatography/tandem mass spectrometry and high-resolution accurate mass spectrometry. Treatment of racehorses with bicarbonate solutions to manage acidosis and muscle cramps prior to competition is banned in Pennsylvania (PA). Use of excess bicarbonate in horses causes diarrhea, requiring treatment with an antibiotic such as metronidazole (MTNZ). At present no method exists for detecting MTNZ in equine plasma. Thus, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the detection, quantification and confirmation of MTNZ was developed. Methods: The analyte was recovered from plasma by liquid-liquid extraction using methyl tert-butyl ether and separated on an ...
The detection of a synthetic Interleukin-1 receptor antagonist peptide in a seized product from a racing stable. A synthetic Interleukin-1 receptor antagonist peptide with the sequence Acetyl-Phe-Glu-Trp-Thr-Pro-Gly-Tyr-Trp-Gln-Pro-Tyr-Ala-Leu-Pro-Leu-OH has been identified in a vial seized during a stable inspection. The use of peptide-based Interleukin-1 receptor antagonists as anti-inflammatory agents has not been previously reported, making this peptide the first in a new class of sports doping peptides. The peptide has been characterized by high-resolution mass spectrometry and a detection method developed based on solid-phase extraction and liquid chromatography - triple quadrupole mass spectrometr...
Total plasma magnesium in healthy and critically ill foals. Abnormalities in total Mg (tMg) concentration in plasma and/or serum are common in critically ill humans, and the association with increased mortality has been documented in several clinical studies in adults and newborns with hypoxic-ischemic encephalopathy. Abnormalities in tMg were studied in hospitalized dogs, cats, and adult horses. Newborn foals were scarcely studied with regard to Mg concentration. The aims of the present study were: (1) to compare two analytical methods for the determination of tMg in plasma: the automated colorimetric method and the atomic absorption spectrometry; (2)...
Conventional, Bayesian, and Modified Prony’s methods for characterizing fast and slow waves in equine cancellous bone. Conventional, Bayesian, and the modified least-squares Prony's plus curve-fitting (MLSP + CF) methods were applied to data acquired using 1 MHz center frequency, broadband transducers on a single equine cancellous bone specimen that was systematically shortened from 11.8 mm down to 0.5 mm for a total of 24 sample thicknesses. Due to overlapping fast and slow waves, conventional analysis methods were restricted to data from sample thicknesses ranging from 11.8 mm to 6.0 mm. In contrast, Bayesian and MLSP + CF methods successfully separated fast and slow waves and provided reli...
Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments. In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluore...
Optimization and validation method to evaluate the residues of β-lactams and tetracyclines in kidney tissue by UPLC-MS/MS. Methods are validated by a process that defines the analytical requirements and confirms that the investigated method is capable of performing consistently. A quantitative and confirmatory method for determining the presence of β-lactam and tetracycline multiresidues in avian, bovine, equine, and swine kidney tissues using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed, optimized, and validated. Analytes were extracted from the kidneys by a mixture of water and acetonitrile, and the extract was then purified with hexane and C18 (dispersive phase). ...
Analytical validation of a high-sensitivity cardiac troponin T assay in horses. Although cardiac troponin T (cTnT) assays have been used to detect myocardial damage in horses, a cTnT assay has not been analytically validated, to our knowledge. The aims of this study were to estimate the precision of a high-sensitivity cTnT assay in horses and determine the effect of hemolysis on the measured cTnT concentration. Serum samples from horses were mixed in 3 different pools. Pool 1 consisted of samples from 3 healthy horses, pool 2 from 6 horses with heart failure or atypical myopathy, and pool 3 from 10 horses with atypical myopathy. The within- and between-run coefficients of...
Identification of the hair of a Holocene “Yukagir horse” (Equus spp.) mummy. No abstract available
Application of the ASVCP guidelines for the establishment of haematologic and biochemical reference intervals in Icelandic horses in Austria. Despite the increasing popularity of Icelandic horses, published reference intervals (RIs) in this breed are rare. Due to their isolation and their small gene pool, alterations in some variables are likely and some possible breed-specific peculiarities have been described. The purpose of the present study was the establishment of comprehensive RIs in Icelandic horses according to recently published guidelines. In a prospective observational study, blood samples were collected from the jugular vein of 142 Icelandic horses into EDTA and serum tubes. Reference intervals were established for haema...
Quantification of hypoglycin A in serum using aTRAQ(®) assay. Hypoglycin A has been recently identified has the causal agent of atypical myopathy (AM) in horses. Its identification and quantification in equine's biological fluids is thus a major concern to confirm maple poisoning and to provide insight into the poorly understood mechanism of hypoglycin A intoxication. Methods: Quantification of hypoglycin A has been achieved with the aTRAQ kit for amino acid analysis of physiological fluids (AB Sciex). Acquisition method on mass spectrometer has been updated to record the hypoglycin A specific MRM transition. Results: Outlined accuracy profiles demonstra...
Metabolic study of androsta-1,4,6-triene-3,17-dione in horses using liquid chromatography/high resolution mass spectrometry. Androsta-1,4,6-triene-3,17-dione (ATD) is an irreversible steroidal aromatase inhibitor and is marketed as a supplement. It has been reported to effectively reduce estrogen biosynthesis and significantly increase the levels of endogenous steroids such as dihydrotestosterone and testosterone in human. ATD abuses have been reported in human sports. Its metabolism in human has been studied, and the in vitro metabolic study of ATD in horses has been reported, however, little is known about its biotransformation and elimination in horses. This paper describes the in vitro and in vivo metabolism stu...
Comparison of milk oligosaccharides pattern in colostrum of different horse breeds. Colostrum oligosaccharides are known to exhibit prebiotic and immunomodulatory properties. Oligosaccharide composition is species-specific, and equine colostrum has been reported to contain unique oligosaccharides. Therefore, equine oligosaccharides (EMOS) from colostrum from different horse breeds were analyzed by CE-LIF, CE-MS(n), HILIC-MS(n), and exoglycosidase degradation. Sixteen EMOS were characterized and quantified, of which half were neutral and half were acidic. EMOS showed about 63% structural overlap with human milk oligosaccharides, known for their bioactivity. Seven EMOS were not...
Opiorphin analysis in equine plasma and urine using hydrophilic interaction LC-MS. Due to opiorphin's analgesic and antidepressant functions, its illicit use is rumored in some racing jurisdictions. Opiorphin is very difficult to detect due to its hydrophilic nature and rapid degradation in plasma and urine samples. METHODOLOGY & RESULTS: We have developed a sensitive, reliable method for opiorphin detection and confirmation in equine samples, using EDTA to inhibit analyte degradation between the time of collection and analysis. Opiorphin was extracted by weak cation exchange followed by analysis using HILIC-MS/MS. The method was validated and the LOD was determined to b...
Chip-based nanoelectrospray ionization with Fourier transform mass spectrometric detection to screen for local anesthetics intended to mask limb sore in walking horses. We report a high-throughput chip-based nanoelectrospray ionization method coupled with Fourier transform mass spectrometry to screen for local anesthetics in samples collected by swabbing. These drugs have been used to mask pain on the limbs of walking horses after forbidden practices of soring or physical abuse. Optimized for lidocaine, the method afforded sub-ppm mass accuracy for nine local anesthetics included in the study. From doped cotton swabs, two third and all of the analytes were detected after adding 10 ng and 100 ng of each drug, respectively. Benzocaine and/or lidocaine were ...
Determination of the unsaturated disaccharides of hyaluronic acid in equine synovial fluid by high-performance liquid chromatography and fluorescence detection. The purpose of this study was to develop and validate an analytical method to determine the presence of hyaluronic acid derived disaccharides in equine synovial fluid. Results: A high-performance liquid chromatography method for the determination of hyaluronic acid derived unsaturated disaccharides in equine synovial fluid was developed and validated. The method is based on the measurement of unsaturated disaccharides released by digestion of linear hyaluronic acid molecules. The method showed linearity (r(2) = 0.996) over the full working concentration range 0.89-30 mg/l. Relative standard ...
Hematologic and biochemical reference intervals for adult Friesian horses from North America. Established breed-specific reference intervals (RI) are an important tool for monitoring the health of horses. There is a lack of published work on breed-specific RI for Friesian horses. Objective: The goal of this project was to determine hematologic and biochemical RI for Friesian horses residing in North America. Methods: Strict inclusion and exclusion criteria were established for selection of reference subjects and for blood specimen collection and handling. Blood samples from 123 healthy, adult (range 3-18 years, median 8 years) Friesian horses of both sexes (70 mares, 45 geldings, and...
Pre-analytical stability of adrenocorticotrophic hormone from healthy horses in whole blood, plasma and frozen plasma samples. The stability of equine adrenocorticotrophic hormone (ACTH) in blood samples is not fully known. The study objectives were to determine ACTH stability (1) in whole blood and plasma over 72 h at either 4 or 21 °C, and (2) in plasma frozen at either -20 or -80 °C over 30 days. Nine horses were sampled and ACTH concentration were measured after storage as whole blood or plasma, at 4, 21, -20 and -80 °C for up to 30 days. The ACTH concentration was significantly reduced at 24 h but remained stable when plasma was frozen at -20 and -80 °C for 30 days. Beyond 24 h, samples stored a...
A Novel Spectral Method for Inferring General Diploid Selection from Time Series Genetic Data. The increased availability of time series genetic variation data from experimental evolution studies and ancient DNA samples has created new opportunities to identify genomic regions under selective pressure and to estimate their associated fitness parameters. However, it is a challenging problem to compute the likelihood of non-neutral models for the population allele frequency dynamics, given the observed temporal DNA data. Here, we develop a novel spectral algorithm to analytically and efficiently integrate over all possible frequency trajectories between consecutive time points. This advan...
A validated UHPLC-MS/MS method to quantify low levels of anabolic-androgenic steroids naturally present in urine of untreated horses. Doping control is a main priority for regulatory bodies of both the horse racing industry and the equestrian sports. Urine and blood samples are screened for the presence of hundreds of forbidden substances including anabolic-androgenic steroids (AASs). Based on the suspected endogenous origin of some AASs, with β-boldenone as the most illicit candidate, this study aimed to improve the knowledge of the naturally present AAS in horse urine. To this extent, a novel ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated according to...
Plasma cholinesterase activity of rats, western grey kangaroos, alpacas, sheep, cattle, and horses. Plasma cholinesterase activity levels of various species may be of interest to toxicologists or pathologists working with chemicals that interfere with the activity of plasma cholinesterase. Methods: We used a pH titration method to measure the plasma cholinesterase activity of six mammalian species. Results: Plasma cholinesterase activity varied up to 50-fold between species: sheep (88 ± 45 nM acetylcholine degraded per ml of test plasma per minute), cattle (94 ± 35), western grey kangaroos (126 ± 92), alpaca (364 ± 70), rats (390 ± 118) and horses (4539 ± 721). Conclusions: We present ...
