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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.
Rhodococcus equi pneumonia in foals: an assessment of the early diagnostic value of serum amyloid A and plasma fibrinogen concentrations in equine clinical practice. Early diagnosis and prevention of Rhodococcus equi pneumonia in foals represent important goals for equine clinicians. Recent protocols for diagnosis and treatment of Rhodococcosis in foals typically rely on a multimodal approach based on sonographic evidence suggestive of pyogranulomas, sonographic abscess scores and laboratory findings including plasma fibrinogen concentrations, blood biochemistry testing and platelet and leukocyte counts. The aim of this study was to assess the utility of weekly testing of serum amyloid A (SAA) and plasma fibrinogen concentrations in foals to achieve early ...
Purification of equine chorionic gonadotropin (eCG) using magnetic ion exchange adsorbents in combination with high-gradient magnetic separation. Current purification of the glycoprotein equine chorionic gonadotropin (eCG) from horse serum includes consecutive precipitation steps beginning with metaphosphoric acid pH fractionation, two ethanol precipitation steps, and dialysis followed by a numerous of fixed-bed chromatography steps up to the specific activity required. A promising procedure for a more economic purification procedure represents a simplified precipitation process requiring only onethird of the solvent, followed by the usage of magnetic ion exchange adsorbents employed together with a newly designed 'rotor-stator' type Hi...
Use of hypometabolic TRIS extenders and high cooling rate refrigeration for cryopreservation of stallion sperm: presence and sensitivity of 5′ AMP-activated protein kinase (AMPK). This study evaluated the effect of the use of hypometabolic TRIS extenders in the presence or the absence of AMPK activators as well as the utilization of high cooling rates in the refrigeration step on the freezability of stallion sperm. Twelve ejaculates were cryopreserved using Botucrio® as a control extender and a basic TRIS extender (HM-0) separately supplemented with 10 mM metformin, 2mM 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), 2 mM Adenosine monophosphate (AMP), 40 μM compound C AMPK inhibitor or 2 mM AMP+40 μM compound C. Our results showed that the utilization ...
Plasma concentration-dependent suppression of endogenous hydrocortisone in the horse after intramuscular administration of dexamethasone-21-isonicotinate. Detection times and screening limits (SL) are methods used to ensure that the performance of horses in equestrian sports is not altered by drugs. Drug concentration-response relationship and knowledge of concentration-time profiles in both plasma and urine are required. In this study, dexamethasone plasma and urine concentration-time profiles were investigated. Endogenous hydrocortisone plasma concentrations and their relationship to dexamethasone plasma concentrations were also explored. A single dose of dexamethasone-21-isonicotinate suspension (0.03 mg/kg) was administered intramuscularly t...
Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI. The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses ...
Development and Oviposition Preference of House Flies and Stable Flies (Diptera: Muscidae) in Six Substrates From Florida Equine Facilities. House flies, Musca domestica L., and stable flies, Stomoxys calcitrans (L.), (Diptera: Muscidae), common pests on equine facilities, were studied in the laboratory to determine the success and duration of larval development and oviposition preferences on six substrates commonly found on equine facilities. Substrates tested were hay soiled with urine and manure, fresh horse manure, pine shaving bedding soiled with urine and manure (72 h in a manure pile), builders sand bedding soiled with urine and manure aged 3 d, and soil from an overgrazed pasture mixed with urine and manure of variable age....
Selective and simultaneous determination of NSAIDs in equine plasma by HPLC with molecularly imprinted solid-phase extraction. Detection of nonsteroidal anti-inflammatory drugs (NSAIDs) in equine plasma is a significant analytical problem in veterinary anti-doping controls. Results: A new HPLC method coupled to selective extraction with molecularly imprinted polymers was developed for the simultaneous determination in equine plasma of the NSAIDs phenylbutazone, flunixin, oxyphenbutazone, ketoprofen and naproxen. The analytical performances of the method have been evaluated both in standard solutions and equine plasma samples. Recovery: Molecularly imprinted polymers solid-phase extraction for all NSAIDs was >94% with ...
Combination of an unbiased amplification method and a resequencing microarray for detecting and genotyping equine arteritis virus. This study shows that an unbiased amplification method applied to equine arteritis virus RNA significantly improves the sensitivity of the real-time reverse transcription-quantitative PCR (RT-qPCR) recommended by the World Organization for Animal Health. Twelve viral RNAs amplified using this method were hybridized on a high-density resequencing microarray for effective viral characterization.
Expression and regulation of regulator of G-protein signaling protein-2 (RGS2) in equine and bovine follicles prior to ovulation: molecular characterization of RGS2 transactivation in bovine granulosa cells. The luteinizing hormone preovulatory surge stimulates several signal pathways essential for ovulation, and the regulator of G-protein signaling protein-2 (RGS2) is thought to be involved in this process. The objectives of this study were to characterize the regulation of RGS2 transcripts in equine and bovine follicles prior to ovulation and to determine its transcriptional control in bovine granulosa cells. To assess the regulation of equine RGS2 prior to ovulation, RT-PCR was performed using total RNA extracted from equine follicles collected at various times after human chorionic gonadotropi...
Discovery and comparative analysis of a novel satellite, EC137, in horses and other equids. Centromeres are the sites of kinetochore assembly and spindle fiber attachment and consist of protein-DNA complexes in which the DNA component is typically characterized by the presence of extended arrays of tandem repeats called satellite DNA. Here, we describe the isolation and characterization of a 137-bp-long new satellite DNA sequence from the horse genome (EC137), which is also present, even if less abundant, in the domestic donkey, the Grevy's zebra and the Burchelli's zebra. We investigated the chromosomal distribution of the EC137 sequence in these 4 species. Moreover, we analyzed its...
Comparison of platelet counting technologies in equine platelet concentrates. (1) To compare the performance of 4 platelet counting technologies in equine platelet concentrates and (2) to evaluate the ability of the Magellan platelet rich plasma (PRP) system to concentrate equine platelets. Methods: Experimental study to assess method agreement. Methods: Adult mixed breed horses (n = 32). Methods: Acid citrate dextrose-A anti-coagulated whole blood was collected and PRP produced using the Magellan system according to the manufacturer's instructions. Platelets were quantified using 4 counting methods: optical scatter (Advia 2120), impedance (CellDyn 3700), hand counting,...
Leptin and leptin receptor are detectable in equine spermatozoa but are not involved in in vitro fertilisation. In human and swine, leptin (OB) has been identified in seminal plasma and leptin receptors (OB-R) on the cell surface of spermatozoa, indicating that spermatozoa are a target for OB. This hormone has also been detected in follicular fluid (FF) in women and mares, although its role requires further study. The aims of this study were to investigate the immunolocalisation and the expression of OB and OB-R in equine spermatozoa and to evaluate the involvement of OB in equine in vitro fertilisation (IVF). Since progesterone (P) and OB are both found in FF, the individual and combined effects of the...
Blastocele fluid from in vitro- and in vivo-produced equine embryos contains nuclear DNA. Normal mammalian early embryonic development involves apoptosis of blastomeres as a remodeling process during differentiation, starting at the blastocyst stage. Genomic DNA has been recently detected in the blastocele fluid of human embryos and has been amplified by real-time polymerase chain reaction (PCR) to diagnose the sex of in vitro-produced human embryos. This new approach varies from conventional preimplantation genetic diagnosis in that no cells are extracted from the embryo and only the blastocele fluid is aspirated and used as a DNA sample for diagnosis. In the present work, we inv...
Immunolocalization of insulin-like growth factor-I (IGF-I) and its receptors (IGF-IR) in the equine epididymis. Insulin-like growth factor plays a paracrine/autocrine role in regulating testicular function in the stallion, but its presence in the equine epididymis remains unknown. The aim of this study was to test the hypothesis that insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-IR) are localized in the caput, corpus, and cauda of the epididymis in an age-dependent manner. Immediately after castration, epididymal tissue was fixed, paraffin-embedded, and processed for immunohistochemistry (IHC). Western blot was also performed using equine epididymal extracts to verify the specificity of t...
Occurrence of Treponema DNA in equine hoof canker and normal hoof tissue. Equine hoof canker is a chronic pododermatitis of still unknown aetiology. Recent findings reported for 3 canker-bearing individuals are suggestive for Treponema spp. having a role in disease pathogenesis. Objective: Based on this hypothesised association, we assessed a larger number of DNA samples from hooves with canker and normal hooves for the presence of treponemal DNA. Methods: Retrospective survey of archived material. Methods: The study involved 71 archival, PCR-compatible DNA extractions purified from 59 canker samples obtained from 26 equine cases and from 12 hoof biopsies taken from...
Antiviral activity of a Bacillus sp. P34 peptide against pathogenic viruses of domestic animals. P34 is an antimicrobial peptide produced by a Bacillus sp. strain isolated from the intestinal contents of a fish in the Brazilian Amazon basin with reported antibacterial activity. The aim of this work was to evaluate the peptide P34 for its in vitro antiviral properties against canine adenovirus type 2 (CAV-2), canine coronavirus (CCoV), canine distemper virus (CDV), canine parvovirus type 2 (CPV-2), equine arteritis virus (EAV), equine influenza virus (EIV), feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1). The results showed that the peptide P34 exhibited antiviral activity a...
Gene and protein expression and cellular localisation of cytochrome P450 enzymes of the 1A, 2A, 2C, 2D and 2E subfamilies in equine intestine and liver. Among the cytochrome P450 enzymes (CYP), families 1-3 constitute almost half of total CYPs in mammals and play a central role in metabolism of a wide range of pharmaceuticals. This study investigated gene and protein expression and cellular localisation of CYP1A, CYP2A, CYP2C, CYP2D and CYP2E in equine intestine and liver. Real-time polymerase chain reaction (RT-PCR) was used to analyse gene expression, western blot to examine protein expression and immunohistochemical analyses to investigate cellular localisation. Results: CYP1A and CYP2C were the CYPs with the highest gene expression in the ...
Development of a method for absolute quantification of equine acute phase proteins using concatenated peptide standards and selected reaction monitoring. The aim of this study was the development of a quantitative assay that could support future studies of a panel of acute phase proteins (APPs) in the horse. The assay was based on a quantification concatamer (QconCAT) coupled to selected reaction monitoring methodology. Thirty-two peptides, corresponding to 13 putative or confirmed APPs for the Equus caballus (equine) species were selected for the design of a QconCAT construct. The gene encoding the QconCAT was synthesized and expressed as an isotope-labeled chimaeric protein in Escherichia coli. The QconCAT tryptic peptides were analyzed on a ...
Quantitative PCR detection of Theileria equi using laboratory workflows to detect asymptomatic persistently infected horses. Equine piroplasmosis is the most important tick-borne disease of horses. Regulations on movement of horses into disease-free countries are in place to preserve international trade. Introduction of infectious disease, such as equine piroplasmosis, into non-endemic countries remains a substantial risk owing to the wide-spread distribution of vectors. Identification and restriction of movement of Theileria equi persistently infected horses is an integral part of control strategies, because persistently infected horses with low parasitaemia are an important reservoir. We used real-time PCR for dia...
The effects of sample handling and N-phenylmaleimide on concentration of adrenocorticotrophic hormone in equine plasma. Previous reports suggest that adrenocorticotrophic hormone (ACTH) degrades rapidly, limiting its use as a diagnostic test. Objective: This study quantified effects of processing delays on ACTH concentrations and investigated the addition of N-phenylmaleimide (maleimide), a protease inhibitor, as a means of reducing ACTH degradation. Methods: Experimental study. Methods: Venous blood was collected from 8 healthy horses and 8 horses with pituitary pars intermedia dysfunction (PPID) with a range of ACTH concentrations. Baseline ACTH concentrations were established immediately using a chemilumines...
Efficient transduction of equine adipose-derived mesenchymal stem cells by VSV-G pseudotyped lentiviral vectors. Equine adipose-derived mesenchymal stem cells (EADMSC) provide a unique cell-based approach for treatment of a variety of equine musculoskeletal injuries, via regeneration of diseased or damaged tissue, or the secretion of immunomodulatory molecules. These capabilities can be further enhanced by genetic modification using lentiviral vectors, which provide a safe and efficient method of gene delivery. We investigated the suitability of lentiviral vector technology for gene delivery into EADMSC, using GFP expressing lentiviral vectors pseudotyped with the G glycoprotein from the vesicular stomat...
Are horse paddocks threatening water quality through excess loading of nutrients? The Baltic Sea is one of the most eutrophied water bodies in northern Europe and more than 50% of its total anthropogenic waterborne phosphorus (P) and nitrogen (N) loads derive from agricultural sources. Sweden is the second largest contributor of waterborne N and the third largest contributor of waterborne P to the Baltic Sea. Horse farms now occupy almost 10% of Swedish agricultural land, but are not well investigated with regard to their environmental impact. In this study, potential P, N and carbon (C) leaching losses were measured from two representative horse paddock topsoils (0-20 cm; ...
Outbreaks of Vesicular stomatitis Alagoas virus in horses and cattle in northeastern Brazil. The current article describes outbreaks of vesicular stomatitis (VS) in horses and cattle in Paraiba and Rio Grande do Norte states, northeastern Brazil, between June and August 2013. The reported cases affected 15-20 horses and 6 cattle distributed over 6 small farms in 4 municipalities, but additional data indicated the involvement of a large number of animals on several farms. The disease was characterized by blisters; eruptive lesions in coronary bands, lips, mouth, and muzzle; salivation; claudication and loss of condition. Swollen lower limbs and lips, and ulcerated and erosive areas in ...
UTF1, a putative marker for spermatogonial stem cells in stallions. Spermatogonial stem cells (SSCs) continuously undergo self-renewal and differentiation to sustain spermatogenesis throughout adulthood in males. In stallions, SSCs may be used for the production of progeny from geldings after cryopreservation and therapy for infertile and subfertile stallions. Undifferentiated cell transcription factor 1 (UTF1) is a putative marker for undifferentiated spermatogonia in humans and rats. The main purposes of this study are to determine the following: 1) changes in the expression pattern of UTF1 at various reproductive stages of stallions, 2) subpopulations of sp...
Development and validation of a hydrophilic interaction liquid chromatography with tandem mass spectrometry method for the simultaneous detection and quantification of etilefrine and oxilofrine in equine blood plasma and urine. A sensitive hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method was developed and validated for the simultaneous detection and quantification of etilefrine and oxilofrine in equine blood plasma and urine. The method is highly sensitive and specific with good precision and accuracy. In plasma the limit of detection and limit of quantification are 0.03 and 0.1 ng/mL, respectively, for both analytes. In urine the limit of detection and limit of quantification are 0.3 and 1 ng/mL, respectively, for both analytes. The suitability of the method for doping con...
A specific adenylyl cyclase inhibitor (DDA) and a cyclic AMP-dependent protein kinase inhibitor (H-89) block the action of equine growth hormone on in vitro maturation of equine oocytes. The objectives of this study were firstly to determine whether the stimulatory function of equine growth hormone (eGH) on equine oocyte maturation in vitro is mediated via cyclic adenosine monophosphate (cAMP); and secondly if the addition of eGH in vitro influences oocyte nuclear maturation and if this effect is removed when GH inhibitors are added to the culture. Cumulus-oocyte complexes (COCs) were recovered from follicles <25 mm in diameter and randomly allocated as follows: (i) control (no additives); and (ii) 400 ng/ml of eGH. A specific inhibitor against cyclic AMP-dependent protein ...
Flow cytometric detection of circulating platelet-derived microparticles in healthy adult horses. To develop a flow cytometric assay to quantify platelet-derived microparticles (PMPs) in equine whole blood and plasma. Methods: Citrate-anticoagulated whole blood from 30 healthy adult horses. Methods: Platelet-poor plasma (PPP) was prepared from fresh whole blood by sequential low-speed centrifugation (twice at 2,500 × g). Samples of fresh whole blood and PPP were removed and stored at 4° and 24°C for 24 hours. Platelet-derived microparticles were characterized in fresh and stored samples on the basis of the forward scatter threshold (log forward scatter < 10(1)) and labeling with anne...
Changes in platelet morphology and function during 24 hours of storage. For in vitro studies assessing the interaction of platelets with implant materials, common and standardized protocols for the preparation of platelet rich plasma (PRP) are lacking, which may lead to non-matching results due to the diversity of applied protocols. Particularly, the aging of platelets during prolonged preparation and storage times is discussed to lead to an underestimation of the material thrombogenicity. Here, we study the influence of whole blood- and PRP-storage times on changes in platelet morphology and function. Blood from apparently healthy subjects was collected according...
Isolation of equine endothelial cells and life cell angiogenesis assay. Arterial or venous thromboses are frequent clinical complications with the risk of fatal progression. Recent studies suggest the disruption of angiogenesis in the course of thrombus resolution as the underlying pathomechanism. Very similar to the situation in human patients, equine vessels have been described to be particularly susceptible to thrombosis. In contrast to humans, equine donors are readily available to obtain organs and tissues for isolation of endothelial cells. Objective of this study was to isolate equine endothelial cells and develop an angiogenesis assay from primary cultures...
