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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.
Screening for gene doping transgenes in horses via the use of massively parallel sequencing. Throughout the history of horse racing, doping techniques to suppress or enhance performance have expanded to match the technology available. The next frontier in doping, both in the equine and human sports areas, is predicted to be genetic manipulation; either by prohibited use of genome editing, or gene therapy via transgenes. By using massively-parallel sequencing via a two-step PCR method we can screen for multiple doping targets at once in pooled primer sets. This method has the advantages of high scalability through combinational indexing, and the use of reference standards with altered ...
Evaluating the use of piezo manipulator, laser or their combination for blastocoel cavity puncture to improve cryopreservation outcomes of large equine embryos. The main difficulty of large equine embryo cryopreservation is the replacement of blastocoel fluid with cryoprotectant solution. The objective of this study was to improve the cryopreservation of large equine embryos with PMAP and/or LAP. Embryos were collected via the non-surgical transcervical procedure and divided into three groups based on their size (A ≤ 300 µm, 300 µm<B 300 µm). However, more research is required to find the best method for embryos ≥700 µm.
Pharmacokinetic properties of metamizole active metabolites in Northeastern Brazilian donkeys (Equus asinus). Metamizole (MT), also known as dipyrone, is an analgesic and antipyretic drug labeled for use in humans and domestic animals in some countries. As with other drugs, the administration of MT in donkeys is based on studies carried out with horses. In the present report, we aimed to determine the pharmacokinetics of the two main metamizole active metabolites (N-methyl-4-aminoantipyrine [MAA] and 4-aminoantipyrine [AA]) following 10 (M ) and 25 mg/kg (M ) IV metamizole doses in Northeast Brazilian donkeys (n = 10). Blood was collected at predetermined times within over 48 h; MAA and AA plasma ...
Sperm-bound antisperm antibodies are associated with poor cryosurvival of stallion spermatozoa. Different stallions exhibit a high level of variation in the ability of their sperm to survive cryopreservation. A large fraction of stallions show poor post-thaw sperm motility, and their semen is not suitable for commercial freezing. In this study, we hypothesized that the presence of sperm-bound antisperm antibodies (ASAs) was associated with poor cryosurvival of stallion sperm. Our objective was to assess the level of ASA binding to stallion sperm, and determine if it was associated with good or poor sperm cryosurvival. In Experiment 1, cooled shipped semen from 27 stallions was frozen usi...
Development of a 19-plex short tandem repeat typing system for individual identification and parentage testing of horses (Equus caballus). Individual identification of horses for pedigree verification and registration is important for the sustainable development of the horse industry. Horse individual identification and parentage tests commonly use the 17 short tandem repeats (STRs) recommended by the International Society for Animal Genetics (ISAG) and the locus LEX33. While many multiplex STR typing systems have been established for the horse, a sex determining marker is usually absent, and none of them can simultaneously detect all 17 ISAG recommended loci and the locus LEX33. Here, we present a 19-plex STR typing system that ...
In vitro metabolism of the REV-ERB agonist SR-9009 and subsequent detection of metabolites in associated routine equine plasma and urine doping control samples. SR-9009 is a synthetic compound widely available to purchase online as 'supplement' products due to its potential performance-enhancing effects, presenting a significant threat with regard to doping control in sport. In vitro metabolism with equine liver microsomes was performed to identify potential targets for detection of SR-9009. Six metabolites were identified, with the most abundant consisting of N-dealkylated metabolites (M1-M3). The addition of the identified metabolites to high-resolution accurate mass databases resulted in a positive finding for the N-dealkylated metabolite M1 of SR-...
Differential Expression Pattern of Retroviral Envelope Gene in the Equine Placenta. Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses, which have coevolved with vertebrate genomes for millions of years. The conservation of ERV genes throughout evolution suggests their beneficial effects on their hosts' survival. An example of such positive selection is demonstrated by the syncytin gene, which encodes a protein with affinity for various mammalian placentas that is involved in the formation of syncytiotrophoblasts. Although the horse has an epitheliochorial placenta, in which the fetal trophoblasts are simply apposed to the intact uterine epithelium, ...
Potomac horse fever in Ontario: Clinical, geographic, and diagnostic aspects. Clinical findings, geographic locations, laboratory diagnoses, and culture isolation of spp. in Potomac horse fever (PHF) cases diagnosed in Ontario between 2015 and 2019 are described. Forty-six confirmed PHF cases occurred from late June to early September. Of 41 horses admitted to the Ontario Veterinary College, 28 (68%) survived and 13 (32%) were euthanized due to poor prognosis or financial constraints. Most cases were in southern Ontario along the Canada-USA border. Blood and fecal samples from 43 suspect PHF cases were submitted to 2 laboratories for polymerase chain reaction (PCR) tes...
Diagnostic testing patterns for Streptococcus equi subsp. equi in Ontario horses during the years 2008 to 2018. This retrospective study describes testing patterns and the incidence of subsp. in Ontario to assess the utility of laboratory data for surveillance purposes. Laboratory records for equine infectious disease test submissions were extracted from the Animal Health Laboratory (AHL) at the University of Guelph for the years 2008 to 2018. Yearly and seasonal trends in testing and the proportion of tests that returned positive results were assessed. The number of samples submitted for testing decreased over the 11-year period (odds ratio = 0.96, 95% confidence interval: 0.92 to 0.999; = 0.04). ...
Antifungal susceptibility of dermatophytes from racehorses in Japan. Luliconazole (LCZ) is an imidazole antifungal medication that exhibits excellent activity against dermatophytes. As a topical cream and lotion (approved for human use), LCZ has demonstrated a broad spectrum of activity against human dermatophytoses. Objective: This is the first study to investigate the in vitro susceptibility of clinical isolates from horse dermatophytoses to LCZ. Methods: No animals were used in this study. Methods: In the present study, the in vitro susceptibilities of clinical isolates of dermatophytes to LCZ, clotrimazole (CTZ), miconazole (MCZ) and terbinafine (TRF) wer...
What makes a good fecal egg count technique? The first parasite fecal egg counting techniques were described over 100 years ago, and fecal egg counting remains essential in parasitology research as well as in clinical practice today. Several novel techniques have been introduced and validated in recent years, but this work has also highlighted several current issues in this research field. There is a lack of consensus on which diagnostic parameters to evaluate and how to properly design studies doing so. Furthermore, there is a confusing and sometimes incorrect use of terminology describing performance of fecal egg counting techniques, a...
Preliminary Study: Proteomic Profiling Uncovers Potential Proteins for Biomonitoring Equine Melanocytic Neoplasm. Equine melanocytic neoplasm (EMN) is a cutaneous neoplasm and is mostly observed in aged grey horses. This preliminary study aimed to identify potential proteins to differentiate normal, mild and severe EMN from serum proteomic profiling. Serum samples were collected from 25 grey horses assigned to three groups: normal (free of EMN; = 10), mild ( = 6) and severe EMN ( = 9). To explore the differences in proteins between groups, proteomic profiling and analysis were employed. Accordingly, 8241 annotated proteins out of 8725 total proteins were compared between normal and EMN groups and inspect...
Investigation of The Usefulness of Serum Amyloid A in Characterizing Selected Disease Forms of Equine Herpesvirus-1 Infection. The objective of this study was to study the SAA response of horses with various forms of EHV-1 infection. Archived serum samples from 153 horses with various disease forms of EHV-1 infection (48 healthy non-infected horses, 48 subclinically infected horses, 40 horses with respiratory EHV-1 infection and 17 horses with neurological EHV-1 infection) were available for SAA testing. SAA values ranged from 0 to 31 µg/mL (median 0 µg/mL) in healthy horses, from 0 to 2,416 µg/mL (median 8.5 µg/mL) in subclinically infected horses, from 0 to 3,000 µg/mL (median 597 µg/mL) in horse with respirat...
In vitro Validation Assessment of a Fecal Occult Blood Protein Test for Horses. A commercially available equine fecal blood test (FBT) claims to be able to detect the presence of blood proteins (albumin and hemoglobin) in manure. The purpose of this study was to determine the FBT test sensitivity, specificity, positive and negative predictive values (PPV, NPV), lower threshold of detection, time to obtain a visual positive result, effect of temperature and result stability in lab and field conditions. The FBT was assessed for its sensitivity and specificity for detecting pure albumin and hemoglobin at two temperatures over a range of concentrations. Time to result was mea...
Factors affecting intracellular calcium influx in response to calcium ionophore A23187 in equine sperm. Exposure to the calcium ionophore A23187 may present a "universal" sperm treatment for IVF, as it bypasses capacitation pathways. However, success in utilizing A23187 is variable, especially in equine spermatozoa. Notably, albumin is used during A23187 treatment but paradoxically is thought to suppress A23187 action. Essentially no critical data are available on the effects of A23187 and albumin concentrations, ratios, or addition protocols on changes in intracellular calcium ([Ca] ) in any cell type. To determine factors that affect the action of A23187 on [Ca] in equine and murine spermatozo...
[Methodical investigation of mineral and trace element concentrations in equine feces with special consideration of the sampling location]. Objective measurements of the mineral supply in horses are rarely performed. As a result, incorrect elements or an improper amount of elements are provided. The analysis of feces could represent a novel method to evaluate the nutritive supply. The prerequisite is a knowledge of methodological factors influencing the mineral concentration in the fecal samples. Within the scope of this investigation, the effects of different kinds of mineral supply and the influence of the sampling location on the concentration of minerals in equine feces samples were analyzed. Additionally, the methodical error...
A preliminary study on the effects of E-Z Mixin® and EquiPlus® extenders supplemented with Edible Bird’s Nest on the quality of chilled Arabian stallion semen. The aim of this study was to evaluate the effects of adding different concentrations of edible bird's nest (EBN) which is secreted by swiftlet birds (), into EquiPlus® and E-Z Mixin® extenders on the quality of chilled Arabian stallion semen at various storage times (0, 24 and 48 h). Ten ejaculates were collected from five stallions, and diluted using the two extenders containing 0% (control), 0.12%, 0.24% and 0.24% of EBN + seminal plasma (SP). All the diluted semen samples were then cooled and stored at 5 °C, and examined at 0, 24 and 48 h. Sperm kinetic parameters were assessed using com...
Nasal colonization and antibiotic resistance patterns of Staphylococcus species isolated from healthy horses in Tripoli, Libya. The present study investigated the colonization rates and antimicrobial susceptibility of Staphylococcus species isolated from the nostrils of healthy horses. A nonselective laboratory approach was applied, followed by confirmation using a Phoenix automated microbiological system. Among the 92 horses included in the study, 48.9% (45/92) carried Staphylococcus species of mostly the coagulase-negative staphylococci (CoNS) type yielding 70 Staphylococcus strains. Of these strains, 37.1% (26/70; 24 CoNS and 2 coagulase-positive staphylococci; CoPS) were identified as methicillin-resistant staphylo...
Circulating activin A during equine gestation and immunolocalization of its receptors system in utero-placental tissues and fetal gonads. Although equine gestation is unique from the standpoint of fetal gonadal enlargement and regression, the activator of this process is still unknown. The present study aimed to show a possible role of activin during equine gestation. In the first experiment, weekly plasma samples from six pregnant mares were used to measure activin A. In the second experiment, eight pregnant mares carrying female (gestational days 110, 140, 180, and 270) and male fetuses (gestational days 120, 180, 225, and 314) were used for immunohistochemistry of activin receptors (IA, IB, IIA, IIB), and their intracellular ...
A leptin sandwich ELISA kit unusable for domestic animals. An instance of hormone assay method flaw is reported. In this journal Chronobiology International, two papers appeared in which an ELISA method for human serum or plasma was utilized for blood serum of horse and sheep, respectively. From our testing, it is resulted that such method does not work at all for equine, sheep and other animal species. The use of commercial hormone assay kits for heterologous species always needs a careful validation procedure. First, the same hormone molecule by different species could not share enough homology to be regognized by and react with antibodies utilized ...
Presence of surfactant proteins in the uteri and placentae of pregnant mares. Immunohistochemical investigations of the expression of surfactant protein A (SP-A) and surfactant protein D (SP-D) in the uterine and placental tissues of 13 pregnant mares were performed using anti-horse monoclonal primary antibodies. Strong positive reactions for both SP-A and SP-D were observed in the trophoblasts in the microcotyledons of the placentae at 182 to 314 days of gestation; in uterine glandular epithelial cells, faint-to-weak reactions were observed during gestation. This study describes, for the first time, the changes in the SP-A and SP-D expression levels in the endometrium ...
Detection of synovial sepsis in horses using enzymes as biomarkers. Synovial sepsis is a commonly occurring, potentially career-ending or even life-threatening orthopaedic emergency. Diagnosis of synovial sepsis is currently primarily based on synovial fluid analysis, which often leaves diagnostic ambiguity due to overlap of clinicopathological parameters between septic and aseptic inflammatory synovitis. Objective: To evaluate the reliability of lysozyme (LYS), myeloperoxidase (MPO) and elastase (ELT) as biomarkers for synovial sepsis in horses using a photometric assay to measure increased enzyme activity. Methods: Prospective, single-blinded, analytical, cl...
Bullous amyloidosis in a horse: first description in veterinary medicine. Bullous amyloidosis is a rare disease in humans that has not been described in a veterinary species in the peer-reviewed literature. The human disease is characterised by haemorrhagic vesicles and bullae on the skin and mucosae, which form due to amyloid deposition. Objective: To describe the clinical features, laboratory analysis and histopathological features of an unique presentation of bullous disease in a horse. Methods: A 17-year-old thoroughbred mare presented for weight loss and severe oral cavity ulcers. Methods: Investigations involved haematological evaluation, chemistry profiles, g...
Immunological Cross-Reactivity of Proteins Extracted from the Oomycete Pythium insidiosum and the Fungus Basidiobolus ranarum Compromises the Detection Specificity of Immunodiagnostic Assays for Pythiosis. Pythiosis, a life-threatening disease caused by , has been increasingly diagnosed worldwide. A recently developed immunochromatographic test (ICT) enables the rapid diagnosis of pythiosis. During the 3-year clinical implementation of ICT in Thailand, we collected the laboratory reports of 38 animals with suspected pythiosis and detected ICT false-positive results in three horses and a dog with basidiobolomycosis. and cause infections with indistinguishable clinical and microscopic features. This study investigated cross-reactive antibodies by probing and crude extracts and cell-free synthe...
New mAbs facilitate quantification of secreted equine TNF-α and flow cytometric analysis in monocytes and T cells. Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine, that is involved in acute inflammation and is employed as a biomarker of inflammatory diseases in several species for which reliable quantification is available. We aimed to develop suitable tools to quantify TNF-α in equine samples. We generated two new mAbs against equine TNF-α (clones 48 and 292), evaluated their specificity for this cytokine, and confirmed detection of native TNF-α in stimulated equine PBMC. The TNF-α mAbs were paired in a fluorescent bead-based assay for quantification of equine TNF-α. The TNF-α assay had ...
Detection of Anaplasma Phagocytophilum in Horses With Suspected Tick-Borne Disease in Northeastern United States by Metagenomic Sequencing. Metagenomic sequencing of clinical diagnostic specimens has a potential for unbiased detection of infectious agents, diagnosis of polymicrobial infections and discovery of emerging pathogens. Herein, next generation sequencing (NGS)-based metagenomic approach was used to investigate the cause of illness in a subset of horses recruited for a tick-borne disease surveillance study during 2017-2019. Blood samples collected from 10 horses with suspected tick-borne infection and five apparently healthy horses were subjected to metagenomic analysis. Total genomic DNA extracted from the blood samples ...
Pre-analytical stability of sorbitol dehydrogenase in equine heparinized plasma. Sorbitol dehydrogenase (SDH) activity is one of the most sensitive and specific markers for hepatocellular injury in horses, but its reported lability makes it impractical for use in many clinical settings. To date, stability of SDH in equine samples has only been evaluated in a limited number of studies in serum samples of horses with activities within reference intervals. The objective of the study was to determine pre-analytical stability of equine SDH activity in heparinized plasma stored at different temperatures for up to 72 h. Twenty client-owned horses admitted to a veterinary teaching...
Diagnosing Strongylus vulgaris in pooled fecal samples. Strongylus vulgaris is the most pathogenic intestinal helminth parasite infecting horses. The migrating larvae in the mesenteric blood vessels can cause non-strangulating intestinal infarctions, which have a guarded prognosis for survival. Infections are typically diagnosed by coproculture, but a PCR test is available in some countries. While it is ideal to test horses individually, many veterinarians and clients wish to pool samples to reduce workload and cost of the diagnostic method. The purpose of this study was to determine if pooling of fecal samples would negatively impact diagnostic pe...
Artificial Long Estrus Protocols Administered Prior to Progesterone Increase Endometrial Uterocalin Expression in Anestrous Mares. Uterocalin (P19) is a lipocalin protein that has binding activity for the provisioning of the embryo with lipids, including those essential for morphogenesis and pattern formation. Therefore, it is thought that P19 plays an important role in supporting the survival of the early embryo. Previous research has shown that the endometrium from acyclic mares expressed more P19 following a simulated long estrus: treatment of 8 mg of estradiol benzoate (EB) split over five consecutive days, followed by long-acting progesterone administration (LA-P4) 72 hours later, when compared to a single 2.5 mg EB ...
Establishment and assessment of an amplicon sequencing method targeting the 16S-ITS-23S rRNA operon for analysis of the equine gut microbiome. Microbial communities are commonly studied by using amplicon sequencing of part of the 16S rRNA gene. Sequencing of the full-length 16S rRNA gene can provide higher taxonomic resolution and accuracy. To obtain even higher taxonomic resolution, with as few false-positives as possible, we assessed a method using long amplicon sequencing targeting the rRNA operon combined with a CCMetagen pipeline. Taxonomic assignment had > 90% accuracy at the species level in a mock sample and at the family level in equine fecal samples, generating similar taxonomic composition as shotgun sequencing. The...
