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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.
Genetic characteristics and phylogenetic relationship of Parascaris spp. from Equus zebra, E. caballus, and E. asinus. The equine Parascaris spp. is large, parasitic nematodes, and predominantly focuses on the intestine of foals and young weanlings. There are two roundworms, Parascaris equorum and Parascaris univalens, recognized among equine hosts. In this study, all fifty-nine Parascaris worms were harvested from three different equine hosts (twenty specimens from Equus zebra, twenty specimens from E. caballus, and nineteen specimens from E. asinus). The ribosomal gene (ITS) and mitochondrial genes (cox1 and nadh1) were amplified to identify and genetically characterize these worms. Analysis of ITS sequences...
Satellite DNA at the Centromere is Dispensable for Segregation Fidelity. The typical vertebrate centromeres contain long stretches of highly repeated DNA sequences (satellite DNA). We previously demonstrated that the karyotypes of the species belonging to the genus are characterized by the presence of satellite-free and satellite-based centromeres and represent a unique biological model for the study of centromere organization and behavior. Using horse primary fibroblasts cultured in vitro, we compared the segregation fidelity of chromosome 11, whose centromere is satellite-free, with that of chromosome 13, which has similar size and a centromere containing long s...
Effects of extender, cryoprotectants and thawing protocol on motility of frozen-thawed stallion sperm that were refrozen for intracytoplasmic sperm injection doses. We examined the effects of different freezing extenders, cryoprotectant agents (CPA) and initial thawing temperatures for preparing doses of refrozen stallion sperm for intracytoplasmic sperm injection (ICSI). Single ejaculates, from twelve stallions, were frozen in lactose-EDTA-egg yolk extender (LE) with 5% glycerol. In experiment 1, sperm were initially thawed to 5 °C or 37 °C, before being diluted in LE or skim milk-egg yolk extender (SMEY) containing either 5% glycerol (GLY), 5% methylformamide (MF) or 5% of a combination of both (GMF). In experiment 2, frozen sperm were initially t...
Evaluation of automated erythrocyte methodology in new world camelids using the ADVIA 2120 hematology analyzer. Accurate erythrocyte measurements with ADVIA hematology analyzers require isovolumetric cell sphering in one reaction and hemolysis in another. However, camelid erythrocytes are resistant to sphering and osmotic lysis, and no published evaluation of ADVIA methods for camelids exists. Objective: The objectives were to demonstrate whether camelid erythrocytes sphere in the ADVIA red blood cell/platelet (RBC/PLT) reagent and lyse in the ADVIA cyanide HGB reagent, and to determine optimal ADVIA settings for camelids. Methods: Camelid and canine blood were diluted to 1:625 in RBC/PLT reagent and ev...
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...
Isolation and identification of cellulolytic bacteria from gastrointestinal tract of Arabian horse and investigation of their effect on the nutritional value of wheat straw. This study was conducted to isolate and identify the cellulolytic bacteria from gastrointestinal tract of Arabian horse and investigate their effect on the nutritional value of wheat straw (WS). Results: Fresh faeces were collected from four Arabian horses. The cellulose-hydrolytic bacteria were isolated by using a medium amended with carboxymethyl cellulose (CMC). The activity of CMC was determined by measuring the release of reducing sugars from CMC. Among the isolates, four isolates (L11, L12, L2 and Z2) showed maximum hydrolysis capacity. 16S rRNA sequence analysis showed that these isolat...
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...
Pharmacokinetics of chloramphenicol base in horses and comparison to compounded formulations. Chloramphenicol is commonly used in horses; however, there are no studies evaluating the pharmacokinetics of veterinary canine-approved tablets. Studies using different formulations and earlier analytical techniques led to concerns over low bioavailability in horses. Safety concerns about human health have led many veterinarians to prescribe compounded formulations that are already in suspension or paste form. The objective of this study was to evaluate the pharmacokinetics of approved chloramphenicol tablets in horses, along with compounded preparations. The hypothesis was that chloramphenico...
Effect of Preparation Method on the Protein Profile of Equine Amnion Dressings. The protein content of amnion is thought to be the primary contributor to its efficacy as a biological dressing for wounds. Protein elution into antibiotic processing media has been reported, but the effect of antiseptic-based processing methods is unknown. Amniotic membranes were collected from eight healthy mares. Samples were collected after removal of gross debris. Tissues were subsequently divided and processed with either 0.05% chlorhexidine or 2% iodine/0.25% acetic acid. After protein extraction and trypsin digestion, the proteins were labeled with 8-plex iTRAQ tags, combined, and 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...
What makes a fertile sperm? Unique molecular attributes of stallion fertility. Stallions experience lower per-cycle conception rates compared to other livestock species, largely because they are selected for breeding based on athletic prowess and not reproductive fitness. Mares are seasonal breeders, and pregnancies cannot be detected until 10-14 days post cover via transrectal ultrasonography. This means the detection of stallion fertility fluctuations is delayed by at least 2 weeks, which within the short breeding season employed by the thoroughbred horse breeding industry, can prove quite costly. For these reasons, there is increased demand for robust laboratory assay...
Vitrification of stallion sperm using 0.25 ml straws: Effect of volume, concentration and carbohydrates (sucrose/trehalose/raffinose). Sperm vitrification is a rapid freezing method in which carbohydrates are used as cryoprotectants. The aim of this study was to determine the optimal volume, concentration and type of carbohydrates for stallion sperm vitrification using 0.25 ml straws in comparison to conventional freezing. Ejaculates (n = 54) were collected from six stallions. For vitrification, straws were filled with different volumes (30, 70, 100 μl), sperm concentrations (50, 100, 200 × 10 sperm/ml) and extenders containing sucrose (20, 100, 200 mM), trehalose (50, 100, 200 mM) and raffinose (50, 100, 20...
Modified stall-side crossmatch for transfusions in horses. After-hours or out-of-clinic crossmatches are often limited by the lack of access to specialized material and technical expertise. Objective: The goal was to adapt a stall-side crossmatch test for pretransfusion evaluation in horses. Methods: Twelve healthy mares (plasma and blood donors, teaching mares). Methods: In a prospective study, blood from 12 mares was used to compare the results of 132 crossmatches performed with a rapid gel assay to crossmatches performed with a microgel column assay, and with predicted compatibilities based on blood types and detection of antibodies at a reference ...
Fetal-fluid proteome analyses in late-term healthy pregnant mares and in mares with experimentally induced ascending placentitis. Characterisation of fetal fluids in healthy and disease states of pregnant mares can help to unravel the pathophysiology and to identify putative markers of disease. Thus, this study aimed to compare the protein composition of: (1) amniotic and allantoic fluids of healthy mares obtained immediately after euthanasia and (2) allantoic fluid harvested via centesis before and after experimental induction of placentitis via transcervical inoculation of Streptococcus equi ssp zooepidemicus in healthy mares. Fetal fluids were analysed with a high-throughput proteomic technique after in-gel digestion....
Chondrogenic expression and DNA methylation patterns in prolonged passages of chondrocyte cell lines of the horse. We investigated the activity of chondrogenic markers and variation of methylation patterns in equine cartilaginous cells cultivated in monolayer. The transcriptional and epigenetic effect of the long-term culture of chondrocytes has been evaluated using several passages of chondrocyte cell-lines derived from equine articular cartilage. Using 3 genes as endogenous control we tested the expression of 7 genes important for different stages of chondrocyte differentiation and maturation. CpG islands in RUNX3 locus were inspected for the evaluation of differential methylation state of passaged cell-...
A novel selective medium for the isolation of Burkholderia mallei from equine specimens. Burkholderia mallei is a Gram-negative bacterium that causes glanders, a zoonotic disease, especially in equine populations (e.g. horses, donkeys, and mules). B. mallei usually grows slowly on most culture media, and this property makes it difficult to isolate from clinical specimens. One of the problems is that B. mallei is easily overgrown by other bacteria, especially in animal specimens collected from non-sterile sites. The aim of this study was to develop a new selective agar for the laboratory diagnosis of glanders. We formulated a new agar, named BM agar, to enrich B. mallei growth, but...
Development of Rose Bengal test against mallein test for rapid diagnosis of equine glanders. Burkholderia mallei, the etiologic agent of the disease known as glanders. Clinical and bacteriological diagnosis of glanders is difficult in the early stages of the disease. Currently, mallein (allergic hypersensitivity test) is used for the diagnosis of glanders. The mallein test requires an experienced laboratory person and lasts 48 h. Therefore, in order to quickly diagnose the disease, especially in areas (such as the borders of the country) that cannot be kept animals, new methods should be used to identify the disease. The Rose Bengal is a serological diagnostic test and has been recom...
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...
Development and evaluation of a one-step multiplex real-time TaqMan® RT-qPCR assay for the detection and genotyping of equine G3 and G14 rotaviruses in fecal samples. Equine rotavirus A (ERVA) is the leading cause of diarrhea in neonatal foals and has a negative impact on equine breeding enterprises worldwide. Among ERVA strains infecting foals, the genotypes G3P[12] and G14P[12] are the most prevalent, while infections by strains with other genomic arrangements are infrequent. The identification of circulating strains of ERVA is critical for diagnostic and surveillance purposes, as well as to understand their molecular epidemiology. Current genotyping methods available for ERVA and rotaviruses affecting other animal species rely on Sanger sequencing and ar...
Altered morphokinetics in equine embryos from oocytes exposed to DEHP during IVM. Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer with endocrine-disrupting properties. In this study, we used an equine model to investigate DEHP concentrations in ovarian follicular fluid (FF), and to determine the effects of exposure of oocytes to potentially toxic concentrations of DEHP during in vitro maturation (IVM) on embryo development and quality. Embryo development was evaluated using time-lapse monitoring (TLM), a photomicroscopic tool that reveals abnormalities in cleavage kinetics unobservable by conventional morphology assessment. Blastocyst bioenergetic/oxidativ...
Glycan Profiling Analysis of Equine Amniotic Progenitor Mesenchymal Cells and Their Derived Extracellular Microvesicles. Equine amniotic mesenchymal cells (eAMCs) are involved in many mechanisms in tissue regenerative processes. Their secreted vesicles are important effectors in a wide array of biological processes, and contribute to in vivo healing of equine tendon lesions and endometrial inflammation. Glycoconjugates are involved in cellular recognition and in the efficient uptake of extracellular vesicles (EVs) by recipient cells. In this study, we evaluated the surface glycosylation pattern of eAMCs and their EVs from the eAMCs released in conditioned medium. We used a microarray procedure in which eAMCs and...
Genetic evaluation of bovine papillomavirus types detected in equine sarcoids in Poland. Equine sarcoids are the most common neoplasms in horses. Bovine papilloma- virus type 1 (BPV-1) is the main viral type identified in equine sarcoids in Europe. Objective: The aim of the present study was to genetically evaluate BPV types based on DNA analyses of the CDS of the L1 gene. The presence of BPV DNA was confirmed by Degenerate Oligonucleotide-Primed Polymerase Chain Reaction (DOP PCR) with FAP59/FAP64 consensus primers. Results: The DNA was detected in 21/40 (52.5%) of clinically diagnosed sarcoids. More than half of 14 isolates (66.7%) shared 100% homology with BPV-1 Deltapapillomav...
Plasticity of endometrial epithelial and stromal cells-A new approach towards the pathogenesis of equine endometrosis. Equine endometrosis, a frequent cause of subfertility, is characterized by periglandular fibrosis, and no treatment exists. Endometrial biopsies not only contain diseased glands, but also contain healthy glands and stroma. Myoepithelial (ME) and myofibroblastic (MF) markers are calponin, smooth muscle actin (SMA), desmin and glial fibrillary acidic protein (GFAP). Epithelial vimentin expression indicates epithelial to mesenchymal transition (EMT). The aim of this immunohistochemical study was to investigate whether biopsies with endometrosis express MF and ME markers and vimentin. Compared to ...
Coconut Water as an Extender Component for Cooled Equine Sperm. The aim of this study was to evaluate the effect of coconut water as a component of extender in different formulations for cooling equine sperm. One ejaculate of fourteen stallions was collected. Sperm was diluted to 50 × 10 sperm/mL using five different extenders: ACP-105: powdered coconut water extender (ACP-105, ACP Biotecnologia, Brazil); ACP-Milk: ACP-105 + 20 g/L of skimmed milk; ACP-EY 2.5%: ACP-105 + 2.5% of egg yolk; ACP-EY 5%: ACP-105 + 5% of egg yolk; and BotuSêmen (Botupharma, Botucatu, Brazil) and cooled in passive cooling device (BotuFlex, Botupharma, Botucatu, Brazil) at 5...
Age related and seasonal changes of plasma concentrations of insulin-like peptide 3 and testosterone from birth to early-puberty in Thoroughbred male horses. The peripheral blood concentrations of insulin-like peptide 3 (INSL3) have been detected in many mammalian species, but the level of INSL3 in horse remains unknown. The objectives were to develop a time-resolved fluorescence immunoassay (TRFIA) to detect INSL3 concentrations from horse blood as well as to determine the age-related and seasonal changes of plasma concentrations of INSL3 and testosterone from birth to early-puberty in Thoroughbred male horse (n = 11). Monthly blood sample and measurement of body weight, height, chest and cannon bone size were done from birth until 16 mo. Th...
Repeated Impacts Diminish the Impact Performance of Equestrian Helmets. During thoroughbred races, jockeys are placed in potentially injurious situations, often with inadequate safety equipment. Jockeys frequently sustain head injuries; therefore, it is important that they wear appropriately certified helmets. The goals of this study are (1) to perform impact attenuation testing according to ASTM F1163-15 on a sample of equestrian helmets commonly used by jockeys in the United States and (2) to quantify headform acceleration and residual crush after repeat impacts at the same location. Seven helmet models underwent impact attenuation testing according to ASTM...
Concentrations of sulphated estrone, estradiol and dehydroepiandrosterone measured by mass spectrometry in pregnant mares. Few studies have provided a longitudinal analysis of systemic concentrations of conjugated oestrogens (and androgens) throughout pregnancy in mares, and those only using immunoassay. The use of liquid chromatography tandem mass spectrometry (LC-MS/MS) will provide more accurate concentrations of circulating conjugated steroids. Objective: To characterise circulating concentrations of individual conjugated steroids throughout equine gestation by using LC-MS/MS. Methods: Longitudinal study and comparison of pregnant mares treated with vehicle or letrozole in late gestation. Methods: Sulphated oe...
A longitudinal study of the faecal microbiome and metabolome of periparturient mares. Periparturient mares are at increased risk of colic including large colon volvulus, which has a high mortality rate. Alterations in colonic microbiota related to either physiological or management changes, or both, that occur at this time have been suggested as potential causes for increased colic risk in this population of horses. Although the effect of management changes on the horse faecal microbiota has been investigated, limited work has been conducted to investigate changes in faecal microbiota structure and function in the periparturient period. The objectives of the current study were ...
Using next-generation sequencing to determine diversity of horse intestinal worms: identifying the equine ‘nemabiome’. Next-generation sequencing of DNA from nematode eggs has been utilised to give the first account of the equine 'nemabiome'. In all equine faecal samples investigated, multiple species of Strongylidae were detected, ranging from 7.5 (SEM 0.79) with 99+% identity to sequences in the NCBI database to 13.3 (SEM 0.80) with 90+% identity. This range is typical of the number of species described previously in morphological studies using large quantities of digesta per animal. However, the current method is non-invasive; relies on DNA analysis, avoiding the need for specialist microscopy identificatio...
Elucidation of the biosynthetic pathways of boldenone in the equine testis. Boldenone is an anabolic-androgenic steroid that is prohibited in equine sports. Urine from the uncastrated male horse contains boldenone that is thought to be of endogenous origin and thus a threshold ('cut-off') concentration has been adopted internationally for free and conjugated boldenone to help distinguish cases of doping from its natural production. The testis is likely to be a source of boldenone. Qualitative analysis was performed on extracts of equine testicular homogenates (n = 3 horses) incubated non-spiked and in the presence of its potential precursors using liquid chromatog...
