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Topic:Laboratory Methods
Laboratory methods in equine research encompass a variety of techniques and procedures used to analyze biological samples from horses to study health, disease, genetics, and physiology. These methods include hematological analyses, biochemical assays, molecular biology techniques, and microbiological cultures. Commonly utilized laboratory techniques involve blood tests for complete blood count (CBC) and serum chemistry, polymerase chain reaction (PCR) for genetic and infectious disease studies, and enzyme-linked immunosorbent assays (ELISA) for detecting specific proteins or antibodies. These methods provide valuable data that contribute to understanding equine health and disease mechanisms. This page compiles peer-reviewed research studies and scholarly articles that explore the application, development, and outcomes of laboratory methods in the context of equine research.
Trypanosoma equiperdum Low Molecular Weight Proteins As Candidates for Specific Serological Diagnosis of Dourine. The diagnosis of dourine can be difficult because the clinical signs of this disease in horses are similar to those of surra, caused by . Moreover, and are closely related and, so far, they cannot be distinguished using serological tests. In a previous work, the protein pattern recognized by antibodies from dourine-infected horses and the humoral immune response kinetics were investigated by immunoblotting assay; a total of 20 sera from naturally and experimentally infected horses and from healthy animals were tested. Immunoblotting analysis showed that antibodies from infected horses speci...
Effect of Dental Correction on Fecal Fiber Length in Horses. The aim of the study was to determine if dental correction changes fecal fiber length (FFL) in horses that had never been subjected to dental treatments. Thirty adult horses with mild to moderate dental abnormalities were selected. Dental disorders noted included sharp enamel points, focal overgrowths, shear mouth, step mouth, wave mouth, accentuated transverse ridges, and diastema. The horses were confined to individual stalls and fed Tifton hay (Cynodon spp.) 1.5% bwt/d, divided equally between two feedings. Fecal samples were taken for FFL analysis before (Day 0) and after (Day 15) dental c...
Stallion sperm freezing with sucrose extenders: A strategy to avoid permeable cryoprotectants. The aim of this study was to assess different concentrations of sucrose-based extenders combined with bovine serum albumin (BSA) as an alternative to stallion sperm cryopreservation with permeable cryoprotectants. Semen samples (n = 16) were collected from six stallions. Sperm was cooled, filled in 0.5 mL straws and frozen in nitrogen vapor. Post-thaw sperm kinetic parameters, plasma and acrosome membrane integrity were statistically compared among treatments. In Experiment 1, extenders containing 1% of BSA and different concentrations of sucrose (mmol/L, M): 0, 50, 100, 250, 350 and 450...
A network approach for provisional assay recognition of a Hendra virus antibody ELISA: test validation with low sample numbers from infected horses. Obtaining statistically sound numbers of sera from Hendra virus (HeV)-infected horses is problematic because affected individuals usually die or are euthanized before developing a serum antibody response. As a consequence, test validation becomes a challenge. Our approach is an extension of OIE principles for provisional recognition and included 7 validation panels tested across multiple laboratories that provided estimates for test performance characteristics. At a 0.4 S/P cutoff, 16 of 19 sera from HeV-infected horses gave positive results in the HeV soluble G, indirect ELISA (HeVsG iELISA; ...
Cerebrospinal fluid Lyme multiplex assay results are not diagnostic in horses with neuroborreliosis. The accuracy of the Lyme multiplex assay for the diagnosis of neuroborreliosis in horses is unknown. Objective: To describe Lyme multiplex results in horses with a postmortem diagnosis of neuroborreliosis. The hypothesis was that paired serum and cerebrospinal fluid (CSF) results and a CSF : serum ratio would allow differentiation of horses with neuroborreliosis from those with other neurologic diseases. Methods: Ninety horses that had neurologic examinations, serum and CSF Lyme multiplex analyses, and postmortem examination of the nervous system performed. Methods: Retrospective study. Data...
Analytical validation of a new point-of-care assay for serum amyloid A in horses. Serum amyloid A (SAA) is a major acute phase protein in horses. A new point-of-care (POC) test for SAA (Stablelab) is available, but studies evaluating its analytical accuracy are lacking. Objective: To evaluate the analytical performance of the SAA POC test by 1) determining linearity and precision, 2) comparing results in whole blood with those in serum or plasma, and 3) comparing POC results with those obtained using a previously validated turbidimetric immunoassay (TIA). Methods: Assay validation. Methods: Analytical validation of the POC test was done in accordance with American Society o...
Cultured equine satellite cells as a model system to assess leucine stimulated protein synthesis in horse muscle. Leucine has been shown to stimulate the mammalian/mechanistic target of rapamycin (mTOR) signaling pathway which plays numerous key regulatory roles in cell growth, survival, and metabolism including protein synthesis in a number of species. However, previous work with equine satellite cells has suggested distinct species differences in regards to physiological effects and the magnitude of responses to growth factors and regulators. Because there is limited research available regarding the role of leucine in regulating equine skeletal muscle protein synthesis, the objective of this study was t...
Lactoferrin increases sperm membrane functionality of frozen equine semen. During cryopreservation, sperm was submitted to an increase in reactive oxygen species generation. This work aimed to improve the quality of frozen equine sperm after the addition of antioxidants lactoferrin (Lf) and catalase (Cat) to a freezing extender. Semen from six stallions was frozen with the extenders: F1) control, INRA 82 freezing extender, F2) F1 + 500 μg/ml Lf and F3) F1 + 200 IU/ml Cat. After thawing, sperm motility parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome-reacted sperm were evaluated with a computer-assisted sperm anal...
Detection of prohibited substances in equine hair by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry – application to doping control samples. The detection of drugs in human hair samples has been performed by laboratories around the world for many years and the matrix is popular in disciplines, such as workplace drug testing. To date, however, hair has not become a routinely utilised matrix in sports drug detection. The analysis of hair samples offers several potential advantages to doping control laboratories, not least of which are the greatly extended detection window and the ease of sample collection and storage. This article describes the development, validation, and utilisation of a sensitive ultra-high performance liquid chro...
Development of an antigen-capture ELISA for the quantitation of equine arteritis virus in culture supernatant. Quantitation of virions is one of the important indexes in virological studies. To establish a sensitive and rapid quantitative detection method for equine arteritis virus (EAV), an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed by using two EAV nucleoprotein monoclonal antibodies (mAbs), 2B9 and 2B3, prepared in this study. After condition optimization, mAb 2B9 was used as the capture antibody, and HRP-labeled 2B3 was chosen as the detecting antibody. The AC-ELISA had a good standard curve when viral particles of the Bucyrus EAV strain were used as a reference stan...
Plasma adrenocorticotropic hormone (ACTH) concentrations in ponies measured by two different assays suggests seasonal cross-reactivity or interference. Analysis of plasma adrenocorticotropic hormone concentration [ACTH] aids diagnosis of pituitary pars intermedia dysfunction (PPID). Comparisons of the validated chemiluminescent-immunoassay (CI) and immunofluorescent (IF) assays are limited. Objective: To compare the results of [ACTH] analysis by CI and IF methods of samples collected in autumn and spring and assess cross-reactivity. Methods: Method comparison. Methods: Plasma from nonlaminitic ponies was analysed concurrently using the IF and CI methods in autumn and the following spring. Diagnostic thresholds for the IF method were derived u...
Short-term tolerance of equine spermatozoa to various abiotic factors. The aim of this study was to determine the effects of various abiotic factors, such as light, physical stress (pipetting) and thermal shock, on the quality of fresh and cooled equine sperm. In experiment I, four sperm aliquots were subjected to different light exposures: (i) protected control samples (CTRL), (ii) exposed to UV light at 10 cm (UV10), (iii) exposed to UV light at 20 cm (UV20) and (iv) exposed to laboratory lighting (LAB). In experiment II, four semen aliquots were subjected to repeated pipetting for 0, 10, 20 and 30 times (CTRL, P10, P20 and P30, respectively). In experiment I...
Simultaneous determination of ibuprofen and its metabolites in complex equine urine matrices by GC-EI-MS in excretion study in view of doping control. A novel assay for the simultaneous determination of ibuprofen (IBU) and its four probable metabolites, 1-hydroxyibuprofen (1-OH IBU), 2-hydroxyibuprofen (2-OH IBU), 3-hydroxyibuprofen (3-OH IBU) and carboxyibuprofen (CBX IBU) in equine urine samples with the application of Gas Chromatography-Electron Ionization-Mass Spectrometry (GC-EI-MS) has been developed and elaborated. The new approach for sample preparation including minimizing matrix effects by the application of weak cation exchange solid-phase extraction together with strong cation exchange solid-phase extraction has been applied. The...
Effect of different shipping temperatures (∼22 °C vs. ∼7 °C) and holding media on blastocyst development after overnight holding of immature equine cumulus-oocyte complexes. Intracytoplasmic sperm injection (ICSI) is an important tool for equine embryo production in both clinical and research settings. In clinical ICSI programs, immature equine cumulus-oocyte complexes (COCs) are often collected at the mare's location and shipped to the ICSI laboratory. To simplify shipment and aid scheduling of subsequent procedures, COCs can be held overnight at room temperature (∼22 °C) before placement into maturation culture, with no detrimental effect on meiotic or developmental competence. A recent study indicated that it might be possible to hold COCs overnight at col...
Effects of two freezing methods and two cryopreservation media on post-thaw quality of stallion spermatozoa. Glycerol-based extenders are widely utilized for freezing equine semen, but media combining methylformamide may better preserve sperm motility and mitochondrial function. Semen is cryopreserved utilizing either a Styrofoam box filled with liquid nitrogen or an automatic freezer. The objective of this experiment was to compare the post-thaw characteristics of the same ejaculates cryopreserved in a Styrofoam box or in an automatic freezer, utilizing a glycerol-based extender (Gent) and an extender that combines methylformamide and glycerol (BotuCrio ). For that, one ejaculate from 30 stallions c...
Use of split-free nano-liquid chromatography-mass spectrometry/high resolution mass spectrometry interface to improve the detection of α-cobratoxin in equine plasma for doping control. Cobra (Naja naja kaouthia) venom contains a toxin called α-cobratoxin (α-Cbtx) containing 71 amino acids (MW 7821 Da) with a reported analgesic power greater than morphine. In 2013, the first analytical method for the detection of α-Cbtx in equine plasma was developed by Bailly-Chouriberry et al, allowing the confirmation of the presence of α-Cbtx at low concentrations (1-5 ng/mL or 130-640 fmol/mL) in plasma samples. To increase the method sensitivity and therefore to improve the detection of α-Cbtx in post-administration plasma samples, a nano-liquid chromatography-mass spectrometry/hi...
High mortality in foals associated with Salmonella enterica subsp. enterica Abortusequi infection in Italy. Salmonella enterica subsp. enterica serovar Abortusequi is frequently reported as a cause of abortion in mares and neonatal septicemia and polyarthritis in Asian and African countries, but only sporadically in Europe and the United States. We report an outbreak of S. Abortusequi in foals in Italy, characterized by high mortality. In a herd of Murgese horses, 10 of 34 newborns died at birth and a further 7 died, after developing severe clinical signs, during the first 10 d of life. Tissue specimens from different organs of 2 dead foals, synovial fluids from 4 sick foals, and vaginal and rectal ...
Molecular Characteristics of the Equine Periodontal Ligament. The equine periodontal ligament (PDL) is a fibrous connective tissue that covers the intra-alveolar parts of the tooth and anchors it to the alveolar bone-it, therefore, provides a similar function to a tendinous structure. While several studies have considered the formation and structure of tendons, there is insufficient information particularly on the molecular composition of the PDL. Especially for the equine PDL, there is limited knowledge concerning the expression of genes commonly regarded as typical for tendon tissue. In this study, the gene expression of, e.g., (, and fibrocartilage m...
The recovery of added nematode eggs from horse and sheep faeces by three methods. Nematode infections in horses are widespread across the world. Increasing levels of anthelmintic resistance, reported worldwide in equine parasites, have led to the creation of programs for the control of nematodes based on faecal egg counts (FEC). To improve nematode egg counting in equine faecal samples and establish whether the matrix of equine faeces or the eggs affect the counts, the analytical sensitivity, accuracy and precision of Mini-FLOTAC (combined with Fill-FLOTAC), McMaster and Cornell-Wisconsin techniques were compared. Known numbers of eggs extracted from equine or ovine faeces ...
Truncation of the Murine Neonatal Fc Receptor Cytoplasmic Tail Does Not Alter IgG Metabolism or Transport In Vivo. The neonatal Fc receptor (FcRn) is involved in IgG metabolism and transport in placental mammals. However, whether FcRn is responsible for IgG transfer from maternal serum to colostrum/milk is controversial. Interestingly, large domestic animals, such as cows, pigs, sheep, and horses, in which passive IgG transfer is exclusively completed via colostrum/milk, all express an FcRn α-chain that is shorter in the cytoplasmic tail (CYT) than its counterparts in humans and rodents. To address whether the length variation has any functional significance, we performed in vitro experiments using the Tr...
Estimation of dietary threonine requirement using plasma amino acid concentrations in mature thoroughbreds. The experiment was conducted to estimate dietary threonine (Thr) requirement using plasma amino acid concentrations as a criterion in mature thoroughbreds. Four adult thoroughbreds were used, and a 4 × 4 Latin square design was used for four dietary Thr levels. Plasma Thr concentration was constant until 0.41%, and then increased rapidly with increasing dietary Thr levels. The Thr requirement was estimated to be 67% of lysine with plasma Thr concentration at four Thr levels.
Comparison of two methods for measurement of equine adrenocorticotropin. Accurate measurement of equine adrenocorticotropin (ACTH) is important for the diagnosis of equine pituitary pars intermedia dysfunction (PPID). Several radioimmunoassays (RIAs) and chemiluminescent immunoassays (CIAs) are used for measurement of ACTH concentration in horses; whether these methods yield similar results across a range of concentrations is not determined. We evaluated agreement between a commercial RIA and CIA. Archived plasma samples ( n = 633) were measured with both assays. Correlation between the 2 methods was moderate ( r = 0.49, p < 0.001). Bland-Altman analysis reveale...
Decreased expression of the immediate early protein, ICP4, by deletion of the tegument protein VP22 of equine herpesvirus type 1. VP22 is a major tegument protein of equine herpesvirus type 1 (EHV-1). In the present study, we examined functions of VP22 in EHV-1 replication by viral protein expression analyses in cells infected with the VP22-deficient virus. The expressions of several viral proteins in the cells infected with the VP22-deficient virus were lower than those in the cells infected with the parent virus. One of the weakly expressed proteins was identified as ICP4, which is a major regulatory protein encoded by an immediate early gene of EHV-1. A real-time PCR analysis showed that the mRNA expression of ICP4 wa...
Culture of somatic cells isolated from frozen-thawed equine semen using fluorescence-assisted cell sorting. Nuclear transfer using somatic cells from frozen semen (FzSC) would allow cloning of animals for which no other genetic material is available. Horses are one of the few species for which cloning is commercially feasible; despite this, there is no information available on the culture of equine FzSC. After preliminary trials on equine FzSC, recovered by density-gradient centrifugation, resulted in no growth, we hypothesized that sperm in the culture system negatively affected cell proliferation. Therefore, we evaluated culture of FzSC isolated using fluorescence-assisted cell sorting. In Exp. 1,...
Comparison of commercial RNA extraction kits and qPCR master mixes for studying gene expression in small biopsy tissue samples from the equine gastric epithelium. Gastric tissue biopsy and gene expression analysis are important tools for disease diagnosis and study of the physiology of the equine stomach. However, RNA extraction from gastric biopsy samples is a complex procedure because the samples contain low quantities of RNA and are contaminated with mucous protein and bacterial flora. The objectives of these studies were to compare the performance of RNA extraction methods and to investigate the sensitivity of commercial qPCR master mixes for gene expression analysis of gastric biopsy samples. Three commercial RNA extraction methods (TRIzol, GENEzol...
A simple flow cytometry protocol to determine simultaneously live, dead and apoptotic stallion spermatozoa in fresh and frozen thawed samples. Spermatozoa undergo apoptotic changes during the cryopreservation process. These changes, recently termed spermptosis, resemble the cryopreservation induced delayed onset of cell death observed after thawing of somatic cells. Due to its importance in cryobiology, methods to easily identify spermptotic cells are warranted. In this study, a well-validated method for identification of spermatozoa with caspase 3 activity was compared with use of the combination of Hoechst 33342 (H-42) and ethidium homodimer (Eth-1). Live, dead and apoptotic spermatozoa assessed with each method were compared using...
Normal reference intervals and the effects of sample handling on dynamic viscoelastic coagulometry (Sonoclot) in healthy adult horses. To determine reference intervals and the effect of sample agitation and rest time on Sonoclot analysis in healthy adult horses. Methods: Original prospective study. Methods: University veterinary medical teaching hospital. Methods: Sixty healthy adult horses. Methods: Blood was collected for assessment of complete blood count, serum biochemical analysis, and Sonoclot analysis. Results: Horses were determined to be healthy based upon physical examination, CBC, and serum biochemistry analysis. Blood was analyzed in a glass bead-containing cuvette using the Sienco Sonoclot analyzer following 2 re...
A study on prevalence and molecular characterization of trypanosomal species infecting equines in Lahore region, Pakistan. Trypanosomiasis is an important protozoal disease with a diverse range of susceptible host including human. In the current study, molecular characterization of prevalent species was done through a pan-trypanosome polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP). A total of three hundred (n = 300) equines including horses, donkeys and mules (100 each) were randomly selected and the equine blood samples were subjected to screening for trypanosomes through microhaematocrit centrifuge technique (MHCT), conventional PCR, semi-nested PCR and RFLP. Overall...
Development and validation of an ultrahigh performance liquid chromatography-high resolution tandem mass spectrometry quantification method for hypoglycin A and methylene cyclopropyl acetic acid carnitine in horse serum in cases of atypical myopathy. Atypical myopathy (AM) is a fatal disease in horses presumably caused by hypoglycine A (HGA) from ingested maple seeds and its active metabolite methylene cyclopropyl acetic acid (MCPA). The aim of this study was the development and validation of a rapid and simple assay for HGA and MCPA-carnitine in horse serum and its application to authentic samples. Identification and quantification were carried out by ultra high performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with full-scan/data-dependent MS/MS. Chromatographic separation was performed by isocrat...
