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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.
Focal adhesion kinases and calcium/calmodulin-dependent protein kinases regulate protein tyrosine phosphorylation in stallion sperm. Protein tyrosine phosphorylation (PY) is a hallmark of sperm capacitation. In stallion sperm, calcium inhibits PY at pH 7.8). These results show for the first time a role for calcium/calmodulin-dependent kinases in PRKA-dependent sperm PY; a non-PRKA-dependent pathway regulating sperm PY; and the apparent involvement of the FAK family of protein tyrosine kinases downstream in both pathways.
Analysis of soluble CD14 and its use as a biomarker in neonatal foals with septicemia and horses with recurrent airway obstruction. Soluble CD14 (sCD14) binds bacterial lipopolysaccharide (LPS) and acts as an anti-inflammatory LPS-inhibitor in vivo. In humans, sCD14 is one of the soluble biomarkers used for various inflammatory diseases and conditions, however, sCD14 assays have not yet been evaluated in horses. Here, we developed and optimized a bead-based assay for the quantification of sCD14 in horses. The assay was then used to determine native sCD14 concentrations in serum from healthy and septic foals, in the colostrum of healthy mares and in plasma from adult horses with recurrent airway obstruction (RAO) and contro...
Proteomic characterization and cross species comparison of mammalian seminal plasma. Seminal plasma contains a large protein component which has been implicated in the function, transit and survival of spermatozoa within the female reproductive tract. However, the identity of the majority of these proteins remains unknown and a direct comparison between the major domestic mammalian species has yet to be made. As such, the present study characterized and compared the seminal plasma proteomes of cattle, horse, sheep, pig, goat, camel and alpaca. GeLC-MS/MS and shotgun proteomic analysis by 2D-LC-MS/MS identified a total of 302 proteins in the seminal plasma of the chosen mammali...
Simple tube centrifugation for processing platelet-rich plasma in the horse. This study evaluated the quality and bacteriologic safety of platelet-rich plasma (PRP) produced by 3 simple, inexpensive tube centrifugation methods and a commercial system. Citrated equine blood collected from 26 normal horses was processed by 4 methods: blood collection tubes centrifuged at 1200 and 2000 × g, 50-mL conical tube, and a commercial system. White blood cell (WBC), red blood cell (RBC), and platelet counts and mean platelet volume (MPV) were determined for whole blood and PRP, and aerobic and anaerobic cultures were performed. Mean platelet concentrations ranged from 1.55- to 2...
A new genomic tool, ultra-frequently cleaving TaqII/sinefungin endonuclease with a combined 2.9-bp recognition site, applied to the construction of horse DNA libraries. Genomics and metagenomics are currently leading research areas, with DNA sequences accumulating at an exponential rate. Although enormous advances in DNA sequencing technologies are taking place, progress is frequently limited by factors such as genomic contig assembly and generation of representative libraries. A number of DNA fragmentation methods, such as hydrodynamic sharing, sonication or DNase I fragmentation, have various drawbacks, including DNA damage, poor fragmentation control, irreproducibility and non-overlapping DNA segment representation. Improvements in these limited DNA scissi...
Decorin-PEI nanoconstruct attenuates equine corneal fibroblast differentiation. To explore (i) the potential of polyethylenimine (PEI) nanoparticles as a vector for delivering genes into equine corneal fibroblasts (ECFs) using green fluorescent protein (GFP) marker gene, (ii) whether PEI nanoparticle-mediated decorin (DCN) gene therapy could be used to inhibit fibrosis in the equine cornea using an in vitro model. Methods: Polyethylenimine-DNA nanoparticles were prepared at nitrogen-to-phosphate (N-P) ratio of 15 by mixing 22 kDa linear PEI and a plasmid encoding either GFP or DCN. ECFs were generated from donor corneas as previously described. Initially, GFP was introduc...
Fast and sensitive analysis of dermorphin and HYP6-dermorphin in equine plasma using liquid chromatography tandem mass spectrometry. Dermorphin and HYP(6) -dermorphin are hepta-peptides and natural opioids originally isolated from the skin of South American frogs. They are more potent than morphine but less likely to produce drug tolerance and addiction. These properties make them ideal candidates for the doping of racehorses to enhance performance during competition. Dermorphin was recently classified as a Class I drug by Racing Commissioners International (RCI), indicating that it is a banned substance in equine athletes. To enforce this ban, a fast and sensitive method was developed for dermorphin and HYP(6)-dermorphin a...
Development of an enzyme-linked immunosorbent assay using a recombinant LigA fragment comprising repeat domains 4 to 7.5 as an antigen for diagnosis of equine leptospirosis. Leptospira immunoglobulin (Ig)-like (Lig) proteins are a novel family of surface-associated proteins in which the N-terminal 630 amino acids are conserved. In this study, we truncated the LigA conserved region into 7 fragments comprising the 1st to 3rd (LigACon1-3), 4th to 7.5th (LigACon4-7.5), 4th (LigACon4), 4.5th to 5.5th (LigACon4.5-5.5), 5.5th to 6.5th (LigACon5.5-6.5), 4th to 5th (LigACon4-5), and 6th to 7.5th (LigACon6-7.5) repeat domains. All 7 recombinant Lig proteins were screened using a slot-shaped dot blot assay for the diagnosis of equine leptospirosis. Our results showed that Li...
Region-specific expression of aquaporin subtypes in equine testis, epididymis, and ductus deferens. The process of water movement in the excurrent duct system of the male reproductive tract is pivotal for establishment of male fertility. The objective was to elucidate expression of aquaporin (AQP) water channels in the stallion reproductive tract. Real-time RT-PCR detected expression of AQP0-5 and AQP7-11 in testis, epididymis, and ductus deferens of mature stallions. There were two main expression patterns: (1) higher expression in testis than in epididymis and ductus deferens (AQP0, -4, -5, -8, -10, and -11); and (2) lower expression in testis than in epididymis and ductus deferens (AQP1, ...
Identification of immuno-dominant antigens of Trypanosoma evansi for detection of chronic trypanosomosis using experimentally infected equines. Trypanosoma evansi is the most extensively distributed trypanosome responsible for disease called surra in livestock in many countries including frequent outbreaks in India. The prevalence of this disease is most commonly reported by standard parasitological detection methods (SPDM); however, antibody ELISA is being in practice by locally produced whole cell lysate (WCL) antigens in many countries. In the present investigation, we attempted to identify and purify immuno dominant, infection specific trypanosome antigens from T. evansi proteome using experimentally infected equine serum by immun...
Development of loop-mediated isothermal amplification (LAMP) for detection of Theileria equi. Several approaches have been developed for diagnosis of Theileria equi infection in horses and donkeys but all of them have limitations in practice. Due to numerous strengths including easy operation, cheapness and high sensitivity and specificity, LAMP has been already extensively used for surveillance of a number of diseases. We here set up a LAMP assay based on 18S rRNA gene for T. equi diagnosis. The approach was specific enough to differentiate T. equi from other evolutionary-related protozoa. Moreover, it was sensitive enough that LAMP was capable of detecting as much low as 10 copy targ...
Colour analysis of the equine endometrium: comparison of spectrophotometry and computer-assisted analysis of photographs within the L*a*b* colour space system. The aims of this study were to compare two different methods of quantifying the colour of the luminal surface of the equine endometrium and to relate the results to histopathological evidence of inflammation and fibrosis. The mucosal surfaces of 17 equine uteri obtained from an abattoir were assessed using a spectrophotometer and by computer-assisted analysis of photographs. Values were converted into L(*)a(*)b(*) colour space. Although there was significant correlation between the two methods of quantification, variations in 'brightness', 'red' and 'yellow' values were noted. Within a given u...
Comparison of allogeneic platelet lysate and fetal bovine serum for in vitro expansion of equine bone marrow-derived mesenchymal stem cells. Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapy and tissue engineering approaches. Fetal bovine serum (FBS) is commonly used for in vitro MSC expansion; however, the use of FBS may be associated with ethical, scientific, and safety issues. This study aimed to compare the ability of allogeneic platelet lysate (PL) and FBS to cause equine bone marrow-derived MSC expansion. MSCs were isolated from bone marrow aspirate in media supplemented with either PL or FBS, and cell proliferation properties and characteristics were examined. There were no significant differences...
Duration of equine influenza virus shedding and infectivity in immunised horses after experimental infection with EIV A/eq2/Richmond/1/07. Equine influenza (EI) is a major respiratory disease of horses. Recent outbreaks of EI have demonstrated the ease with which EI virus (EIV) can be transmitted internationally. This study aimed to improve our understanding of EIV shedding after infection of vaccinated horses, which would inform possible changes to current quarantine requirements. Our objectives were to compare commonly used diagnostic tests and to evaluate the relative merits of nasal and nasopharyngeal swabs for detection of EIV in vaccinated and unvaccinated ponies following EIV infection and to use these data to inform optim...
Species of Candida as a component of the nasal microbiota of healthy horses. Respiratory infections are a common problem among equines and occur with variable rates of morbidity and mortality. Although some fungal species are considered primary agents of respiratory tract infections in several mammals, their relevance in respiratory diseases of equines is frequently neglected. In the present study, we performed an active search for Candida spp. in the nasal cavity of horses. The presence of Candida spp. was investigated through the use of nasal swabs that were streaked on culture media. These yeasts were identified through physiological testing and their in vitro antif...
In vitro effects of three formulations of hydroxyethyl starch solutions on coagulation and platelet function in horses. To investigate the in vitro effects of 3 hydroxyethyl starch (HES) solutions on viscoelastic coagulation testing and platelet function in horses. Methods: Blood samples collected from 7 healthy adult horses. Methods: Blood samples were diluted with various crystalloid and HES solutions to approximate the dilution of blood in vivo that occurs with administration of a 10 and 20 mL/kg fluid bolus to a horse (1:8 and 1:4 dilutions, respectively). Diluted samples were analyzed through optical platelet aggregometry, platelet function analysis, thromboelastography, and dynamic viscoelastic coagulomet...
Effects of equine bone marrow aspirate volume on isolation, proliferation, and differentiation potential of mesenchymal stem cells. To compare the mesenchymal stem cell (MSC) yield and chondrogenic and osteogenic differentiation from 5- and 50-mL bone marrow aspirates from horses. Methods: Six 2- to 5-year-old mixed-breed horses. Procedures-2 sequential 5-mL aspirates were drawn from 1 ilium or sternebra. A single 50-mL aspirate was drawn from the contralateral ilium, and 2 sequential 50-mL aspirates were drawn from a second sternebra. The MSC yield was determined through the culture expansion process. Chondrogenesis and osteogenesis were evaluated by means of conventional laboratory methods. Results: The second of the 2 s...
Identification of α-cobratoxin in equine plasma by LC-MS/MS for doping control. Cobra venom (Naja kaouthia) contains a toxin called α-cobratoxin (α-Cbtx). This toxin is a natural protein containing 71 amino acids (MW 7821 Da) with a reported analgesic potency greater than morphine. In 2007, in USA, this substance was found in the barns of a thoroughbred trainer and since then till date, the lack of a detection of this molecule has remained a recurring problem for the horseracing industry worldwide. To solve this problem, the first method for the detection of α-cobratoxin in equine plasma has now been developed. Plasma sample (3 mL) was treated with ammonium sulfate at ...
Evidence for absence of equine arteritis virus in the horse population of New Zealand. To summarise investigation and laboratory data collected between 2001 and 2011 to provide evidence that equine arteritis virus is not present in the horse population of New Zealand. Methods: Analysis was carried out on results from laboratory tests carried out at the Ministry for Primary Industries Animal Health Laboratory (AHL) for equine arteritis virus from horses tested prior to being imported or exported, testing of stallions as part of the New Zealand equine viral arteritis (EVA) control scheme and testing as part of transboundary animal disease (TAD) investigations for exclusion of EVA....
Oxytocin stimulated release of PGF2α and its inhibition by a cyclooxygenase inhibitor and an oxytocin receptor antagonist from equine endometrial cultures. Uterine inflammation results in a poor uterine environment and early embryonic loss in the mare due to an inhibition of maternal recognition of pregnancy caused from increased prostaglandin F2α (PGF2α). Oxytocin binds to endometrial cell receptors to activate prostaglandin synthesis. An oxytocin receptor antagonist (Atosiban) and a cyclooxygenase inhibitor (indomethacin) both decrease PGF2α production. The aim of this study was to evaluate the in vitro effects of Atosiban and indomethacin on equine uterine prostaglandin secretion. Equine endometrial explants were harvested on day two of beh...
Expression of enzymes and receptors of leukotriene pathway genes in equine endometrium during the estrous cycle and early pregnancy. The aims of the present study were to elucidate the expression profiles of leukotriene (LT) pathway mRNA transcription and to determine the possible interaction of LT and prostaglandin (PTG) pathways genes in equine endometrium during the estrous cycle and early pregnancy. Endometrial biopsies were obtained from mares on the day of ovulation (d0), at late diestrous (LD, n = 4), and after luteolysis in the estrus phase (AL, n = 4) of the cycle. Biopsies were also taken on Days 14 (P14; n = 4), 18 (P18, n = 4), and 22 (P22, n = 4) during early pregnancy that were comparable days to cyclic sampli...
Validation of the IDS Octeia ELISA for the determination of insulin-like growth factor 1 in equine serum and tendon tissue extracts. Insulin-like growth factor (IGF-1) is an important mediator of tissue repair in horses. Objective: The aim of the study was to evaluate whether IGF-1 could be measured reliably in equine serum and tendon tissue extracts, using an IGF-1 ELISA kit developed for human serum and plasma. Methods: A glycyl-glycine pretreatment protocol of samples was compared with the pretreatment procedure recommended by the manufacturer. Intra- and inter-assay imprecision were evaluated by repeated measurements of equine serum pools. Assay inaccuracy was determined based on the linearity of serially diluted equine...
Amino acid utilization by the hindlimb of warmblood horses at rest and following low intensity exercise. In particular branched-chain amino acids might limit muscle protein loss in pathological conditions. Little is known on basic amino acid utilization of muscle in horses. Objective: To assess amino acid utilization by the hindlimb of horses at rest and following low intensity exercise. ANIMALS & METHODS: Amino acid uptake by the hindlimb was investigated using the arteriovenous difference technique. Blood from six warmblood mares (mean age 12 ± 3 (SD) years and weighing 538 ± 39 kg) was collected simultaneously from the (transverse) facial artery and from the caudal vena cava. Food was wi...
Efficient propagation of equine viruses in a newly established equine cell line, FHK-13.1 cells. Equine cells are required for isolation of viruses that infect the horse. However, only a few equine cell lines and cell cultures are available so far. Fetal horse kidney (FHK)-Tcl3.1 cell is a novel cell line established by introducing simian virus 40 (SV40) large T antigen. In the present study, the ability to propagate equine viruses was compared between FHK-Tcl3.1 cells and other equine cells. FHK-Tcl3.1 cells efficiently increased many viruses derived from or having pathogenicity to horses and produced high infective titers in culture fluids. These results indicate that FHK-Tcl3.1 cells w...
Pre-selection by double layer density gradient centrifugation improves the fertilising capacity of frozen-thawed, capacitated stallion sperm. The effect of combining double layer density gradient centrifugation (DL-DGC) with different capacitation treatments on the fertilising capacity of frozen-thawed stallion sperm was examined via a heterologous assay involving in vitro-matured, zona pellucida-free bovine oocytes. In a first experiment, aliquots of frozen-thawed stallion sperm were subjected to one of five capacitation treatments without DL-DGC - ionomycin at 1.0μM, 0.1μM, 0.05μM or 0.01μM, or caffeine at 200μg/mL. The fertilising capacity of the semen was then assessed at 18h by staining the above oocytes with 4,6-diamidino...
Investigation of the solubility and the potentials for purification of serum amyloid A (SAA) from equine acute phase serum–a pilot study. Serum amyloid A (SAA) is useful as a diagnostic marker of systemic inflammation in horses, but only heterologous assays based on non-equine calibration and standardization are available for measurements of equine SAA. More accurate measurements could be obtained using purified species-specific SAA in native conformation for assay calibration and standardization. Further knowledge about the biochemical properties of SAA would facilitate a future production of native species-specific calibration material Therefore, the aim of the study was an investigation of the solubility and potentials for pu...
Development of a focus-reduction neutralizing test for detecting equine herpesvirus type-1-neutralizing antibodies. Virus-neutralizing (VN) testing is essential for evaluating virus-specific immunity in equine herpesvirus type-1 (EHV-1) infection. We developed a focus-reduction neutralization test (FRNT) for EHV-1 using 96-well plates for faster large-scale testing with sufficient sensitivity. We used an overlay medium containing Avicel (FMC Biopolymer), a microcrystalline cellulose with lower viscosity than the methylcellulose. The foci were visualized by immuno-staining with anti-EHV-1 gp14 monoclonal antibody. The FRNT successfully detected seroconversion in horses experimentally infected with EHV-1 (n =...
The expression of mucin genes and the presence of mucin gene products in the equine endometrium. In the equine reproductive tract, little is known about mucin gene expression and the role of mucins in barrier function and host-cell interaction. The aims of the study were to identify equine orthologs of mammalian mucin genes using available equine sequence data, to profile expression of equine orthologous mucin genes in the endometrium using reverse transcriptase polymerase chain reaction (RT-PCR), to determine spatial expression patterns of mucin genes using in situ hybridisation, and to confirm the presence of mucin gene products using Western blotting and equine-specific mucin antibodie...
The absence of ER-β results in altered gene expression in ovarian granulosa cells isolated from in vivo preovulatory follicles. Determining the spatial and temporal expression of genes involved in the ovulatory pathway is critical for the understanding of the role of each estrogen receptor in the modulation of folliculogenesis and ovulation. Estrogen receptor (ER)-β is highly expressed in ovarian granulosa cells, and mice lacking ER-β are subfertile due to inefficient ovulation. Previous work has focused on isolated granulosa cells or cultured follicles and, although informative, provides confounding results due to the heterogeneous cell types present including granulosa and theca cells and oocytes and exposure to in...
Detection, quantification, and identification of dermorphin in equine plasma and urine by LC-MS/MS for doping control. Dermorphin is a unique opioid peptide that is 30-40 times more potent than morphine. It was misused and went undetected in horse racing until 2011 when intelligence obtained from a few North American race tracks suggested its use. To prevent such misuse, a reliable analytical method became necessary for detection and identification of dermorphin in post-race horse samples. This paper describes the first liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for such a purpose. Equine plasma and urine samples were pre-treated with ethylenediamine tetra-acetic acid and urea prior to so...
