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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.
Determination of whole blood cholinesterase in different animal species using specific substrates. Whole blood cholinesterase was measured using acetyl-, butyryl- and propionylthiocholine as substrates in 10 healthy adult dogs, cats, horses, pigs, goats, sheep and cows, in order to determine and characterise the cholinesterase activity in whole blood of the main domestic animals. An in vitro exposure test with two anticholinesterase compounds, the organophosphate insecticide coumaphos and the carbamate insecticide imidocarb, was also performed. In whole blood of ruminants and pigs, acetylthiocholine yielded the highest cholinesterase activity and other substrates were poorly hydrolysed; in ...
Effect of homologous preovulatory follicular fluid on in vitro maturation of equine cumulus-oocyte complexes. The objective of this study was to test the hypothesis that incubating equine cumulus-oocyte complexes (COCs) in medium containing 50% or 100% homologous preovulatory follicular fluid would improve cumulus expansion and nuclear maturation. Oocytes were incubated in one of three media: 1) supplemented TCM-199 (control), 2) 50% (v/v) follicular fluid in control medium or 3) 100% follicular fluid. Cumulus expansion was evaluated subjectively, and nuclear maturation was evaluated by staining oocytes with Hoechst 33258. The hypothesis that incubating COCs in medium containing follicular fluid would...
Penetration of zona-free hamster, bovine and equine oocytes by stallion and bull spermatozoa pretreated with equine follicular fluid, dilauroylphosphatidylcholine or calcium ionophore A23187. Experiments evaluated the ability of follicular fluid (FF), dilauroylphosphatidylcholine (PC12) and the calcium ionophore A23187 (A23187) to induce capacitation in stallion and bull spermatozoa, determined by the ability of the spermatozoa to penetrate zona-free hamster, bovine and equine oocytes. Spermatozoa suspensions were incubated at 37 degrees C in one of the following treatments: 1) a modified Tyrode's medium (BGM3) alone; 2) BGM3 + FF; 3) BGM3 + PC12; 4) BGM3 + FF + PC12; 5) BGM3 + A23187; and 6) BGM3 + FF + A23187. Treated spermatozoa were incubated with zona-free hamster, bovine and ...
The(13)C-octanoic acid breath test for detection of effects of meal composition on the rate of solid-phase gastric emptying in ponies. The aim of this study was to apply the(13)C-octanoic acid breath test for detection of alterations in the rate of solid-phase gastric emptying, induced by changes in test meal composition, in ponies. After a 14 hour fast the ponies (n = 4) ingested a test meal with 0, 35 or 70 ml soya oil, and labelled with 250 mg(13)C-octanoic acid. Each pony was given each of the three test meals on three separate occasions, in a randomised order. Exhaled breath samples were collected for 12 hours after ingestion of the test meal. Breath samples were analysed by continuous flow isotope ratio mass spectrometr...
A comparison of foal and adult horse neutrophil function using flow cytometric techniques. Flow cytometric assays were used to compare phagocytic and oxidative burst activity of neutrophils from healthy foals less than 7 days of age with the activity of cells from healthy adult horses. The phagocytosis of Staphylococcus aureus by foal neutrophils was less than that observed for adult neutrophils when autologous serum was used as the source of opsonins in the assay. The use of adult serum did not significantly improve the ability of foal neutrophils to attach bacteria. The oxidative burst activity of foal neutrophils was equivalent to that of adult cells. However, when serum or plasm...
Thermal unfolding of monomeric and dimeric beta-lactoglobulins. The thermal stabilities of dimeric bovine beta-lactoglobulin and monomeric equine beta-lactoglobulin were investigated at neutral pH by means of differential scanning calorimetry, circular dichroism, tryptophan fluorescence, and by binding of an hydrophobic probe. Differential scanning calorimetry showed the presence of two structural domains with different thermal stabilities in both proteins. Thermodynamic analysis of the calorimetric signal revealed that the two domains unfold independently according to a mechanism where an equilibrium step is followed by an irreversible transition. The spe...
Construction of chimeric arteriviruses reveals that the ectodomain of the major glycoprotein is not the main determinant of equine arteritis virus tropism in cell culture. The recent development of arterivirus full-length cDNA clones makes possible the construction of chimeric arteriviruses for fundamental and applied studies. Using an equine arteritis virus (EAV) infectious cDNA clone, we have engineered chimeras in which the ectodomains of the two major envelope proteins, the glycoprotein GP(5) and the membrane protein M, were replaced by sequences from envelope proteins of related and unrelated RNA viruses. Using immunofluorescence microscopy, we monitored the transport of the hybrid GP(5) and M proteins to the Golgi complex, which depends on their heterodime...
Identification of equine herpesviruses 1 and 4 by polymerase chain reaction. To develop and validate specific, sensitive and rapid (< 8 hour) diagnostic tests using polymerase chain reaction (PCR) for the diagnosis of abortion and respiratory disease caused by equine herpesvirus 1 (EHV1; equine abortion virus) and EHV4 (equine rhinopneumonitis virus). Methods: Primer sets based on nucleotide sequences encoding glycoprotein H (gH) of EHV1 and gB of EHV4 were designed and used in single round and second round (seminested) PCRs, and in a multiplex PCR for the diagnosis of EHV1 and EHV4 infections. Methods: Oligonucleotide primers were designed for each virus, PCR condi...
Toxicokinetics of ergovaline in the horse after an intravenous administration. The toxicokinetics of ergovaline (an ergopeptine mycotoxin present in some grasses infected with endophytic fungus of the genus Neotyphodium) were studied after intravenous administration of a single dose of 15 microg/kg bwt in four gelding horses. Plasma ergovaline concentrations were measured by high performance liquid chromatography, and the kinetic data were described by a three-compartment model. The elimination half-life and the total clearance of ergovaline were found to be 56.83 +/- 13.48 min and 0.020 +/- 0.004 L/min x kg, respectively. According to the toxicological data previously r...
Nucleotide sequence and restriction fragment length polymorphisms of the equine Cvarepsilon gene. IgE is the dominant immunoglobulin isotype involved in type I hypersensitivities in mammals. The heavy chain constant region domains of equine IgE are encoded by a single gene, the Cvarepsilon gene. By restriction analysis of cDNA from 15 unrelated horses, we have now identified two Cvarepsilon alleles, characterised by a Sma I restriction fragment length polymorphism, which we designated Cvarepsilon(a) and Cvarepsilon(b). Sequence analysis of both, Cvarepsilon(a) and Cvarepsilon(b) cDNA, showed in addition two single base exchanges resulting in two amino acid substitutions. Both sequences hav...
Detection of North American West Nile virus in animal tissue by a reverse transcription-nested polymerase chain reaction assay. A traditional single-stage reverse transcription-polymerase chain reaction (RT-PCR) procedure is effective in determining West Nile (WN) virus in avian tissue and infected cell cultures. However, the procedure lacks the sensitivity to detect WN virus in equine tissue. We describe an RT-nested PCR (RT-nPCR) procedure that identifies the North American strain of WN virus directly in equine and avian tissues.
The use of a neutralizing monoclonal antibody to detect infections of equine herpesvirus type 2 (EHV-2). A blocking enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to equine herpesvirus 2 in serum samples of horses. By measuring the binding to a single epitope, this blocking ELISA gives a good picture of the antibody status in the animal. The test is based on a monoclonal antibody with neutralizing activity and had a sensitivity of 94% and a specificity of 100%. Antibodies due to newly acquired infection in foals were successfully detected with this blocking ELISA.
Sequence conservation and antigenic variation of the structural proteins of equine rhinitis A virus. The nucleotide and deduced amino acid sequences of the P1 region of the genomes of 10 independent equine rhinitis A virus (ERAV) isolates were determined and found to be very closely related. A panel of seven monoclonal antibodies to the prototype virus ERAV.393/76 that bound to nonneutralization epitopes conserved among all 10 isolates was raised. In serum neutralization assays, rabbit polyclonal sera and sera from naturally and experimentally infected horses reacted in a consistent and discriminating manner with the 10 isolates, which indicated the existence of variation in the neutralizatio...
Mapping the sequences that mediate interaction of the equine herpesvirus 1 immediate-early protein and human TFIIB. The sole immediate-early (IE) gene of equine herpesvirus 1 encodes a 1,487-amino-acid (aa) regulatory phosphoprotein that independently activates expression of early viral genes. Coimmunoprecipitation assays demonstrated that the IE protein physically interacts with the general transcription factor TFIIB. Using a variety of protein-binding assays that employed a panel of IE truncation and deletion mutants expressed as in vitro-synthesized or glutathione S-transferase fusion proteins, we mapped a TFIIB-binding domain to aa 407 to 757 of the IE protein. IE mutants carrying internal deletions of ...
Influence of epidermal growth factor on mammalian oocyte maturation via tyrosine-kinase pathway. Epidermal growth factor (EGF) has been reported to promote different functions in mammalian ovaries, including oocyte maturation. The aim of the present study was to establish: that EGF influences oocyte maturation in ovine and equine, that a tyrosine kinase-dependent intracellular mechanism mediates EGF effect and, that EGF-R receptor is detectable in ovarian follicles by immunohistochemistry methods. Selected ovine and equine oocytes were aspirated from 2-5 mm (ovine) or 25 mm (equine) follicles and cultured in TCM 199 for 22 (ovine) or 36 hours (equine). They are then subjected to culture w...
Influence of epidermal growth factor on mammalian oocyte maturation via tyrosine-kinase pathway. Epidermal growth factor (EGF) has been reported to promote different functions in mammalian ovaries, including oocyte maturation. The aim of the present study was to establish: that EGF influences oocyte maturation in ovine and equine, that a tyrosine kinase-dependent intracellular mechanism mediates EGF effect and, that EGF-R receptor is detectable in ovarian follicles by immunohistochemistry methods. Selected ovine and equine oocytes were aspirated from 2-5 mm (ovine) or 25 mm (equine) follicles and cultured in TCM 199 for 22 (ovine) or 36 hours (equine). They are then subjected to culture w...
Immortalization of equine trophoblast cell lines of chorionic girdle cell lineage by simian virus-40 large T antigen. Immortalized cell lines have many potential experimental applications including the analysis of molecular mechanisms underlying cell-specific gene expression. We have utilized a recombinant retrovirus encoding the simian virus-40 (SV-40) large T antigen to construct several immortalized cell lines of equine chorionic girdle cell lineage - the progenitor cells that differentiate into the equine chorionic gonadotropin (eCG) producing endometrial cups. Morphologically, the immortalized cell lines appear similar to normal chorionic girdle cells. Derivation of the immortalized cell lines from a cho...
A reduction in interstitial cells of Cajal in horses with equine dysautonomia (grass sickness). Equine dysautonomia (grass sickness) is a common, frequently fatal disease of horses characterised by dysfunction of the gastrointestinal tract. Interstitial cells of Cajal are the c-Kit-immunoreactive cells responsible for the generation of pacemaker activity in gastrointestinal smooth muscle. Impairment of this pacemaker action has been implicated in several motility disorders in humans and laboratory mammals. The aim of this study was to test the hypothesis that changes in interstitial cells of Cajal may be involved in the pathophysiology of the intestinal dysfunction observed in equine gra...
Reversed-phase liquid chromatographic method for estrogen determination in equine biological samples. Equine unsaturated estrogens are the main components of brand formulations indicated for hormonal replacement therapy in both hypogonadic and postmenopausal women. These hormones are produced by the fetoplacental unit during equine gestation. A method is described for the quantitative determination of equilenin (EL), equilin (EQ), 17alpha-dihydroequilin (17dEQ), and estrone (El) in the plasma of a pregnant mare. Blood samples are obtained weekly during pregnancy by jugular venipuncture using sodium ethylenediaminetetracetic as the anticoagulant. For the quantitation of these estrogens, plasma ...
A partially unfolded state of equine beta-lactoglobulin at pH 8.7. The urea-induced unfolding transition of equine beta-lactoglobulin was studied at pH 8.7 using circular dichroism (CD), ultracentrifugation, and gel filtration chromatography. The unfolding transition curves showed that at least one intermediate accumulates at moderate concentrations of urea. Furthermore, analytical ultracentrifugation experiments indicated that the intermediate forms a dimer. Thus, the urea-induced unfolding transition was measured by CD at various protein concentrations and was analyzed by a model assuming the four conformational states (the native, intermediate, dimeric int...
Mechanism of oxidation of oxymyoglobin by copper ions: comparison of sperm whale, horse, and pig myoglobins. The influence of Cu2+ concentration, pH, and ionic strength of the solution as well as redox-inactive zinc ions on the rate of oxidation of sperm whale, horse, and pig oxymyoglobins (oxy-Mb) by copper ions has been studied. These myoglobins have homologous spatial structures and equal redox potentials but differ in the number of histidines located on the surface of the proteins. It was shown that oxy-Mb can be oxidized in the presence of Cu2+ through two distinct pathways depending on which histidine binds the reagent and how stable the complex is. A slow pH-dependent catalytic process is obse...
Characterization of alpha-adrenoceptor subtypes in smooth muscle of equine ileum. To determine the concentration and binding characteristics of alpha-adrenoceptor subtypes in smooth muscle cell membranes of equine ileum. Methods: Segments of longitudinal and circular smooth muscle from the ileum of 8 male and 8 female adult horses. Methods: Distribution of alpha-adrenoceptor subtypes was assessed by use of radioligand binding assays incorporating [3H]-prazosin and [3H]-rauwolscine, highly selective alpha1- and alpha2-adrenoceptor antagonists, respectively. Characterization of adrenoceptor subtypes was performed by use of binding inhibition assays. Results: On the basis of b...
Synovial fluid gelatinase concentrations and matrix metalloproteinase and cytokine expression in naturally occurring joint disease in horses. To determine concentrations of matrix metalloproteinase (MMP)-2 and -9 in synovial fluid; and mRNA expression of MMP-1, -13, and -3; interleukin[IL]-1alpha and beta; and tumor necrosis factor (TNF)-alpha in synovial membrane and articular cartilage from horses with naturally occurring joint disease. Methods: Synovial fluid (n = 76), synovial membrane (59), and articular cartilage (45) from 5 clinically normal horses and 55 horses with joint disease categorized as traumatic (acute [AT] or chronic [CT]), osteochondritis dissecans (OCD), or septic (S). Methods: Synovial fluid gelatinase concentra...
Reactivity of serum samples of dogs and horses tested by use of class-specific recombinant-based enzyme-linked immunosorbent assays for detection of granulocytic ehrlichiosis. To test serum samples of dogs and horses by use of class-specific recombinant-based ELISA for establishing a diagnosis of granulocytic ehrlichiosis attributable to infection with organisms from the Ehrlichia phagocytophila genogroup. Methods: Serum samples from 43 client-owned dogs and 131 horses (81 with signs of acute illness and 50 without signs of disease). Methods: Serum samples were analyzed, using ELISA with a recombinant 44-kd protein antigen for IgM and IgG antibodies to the human granulocytic ehrlichiosis (HGE) agent (NCH-1 strain). Western blot analyses, using infected human promyel...
Plasma pharmacokinetics and faecal excretion of ivermectin, doramectin and moxidectin following oral administration in horses. The present study was carried out to investigate whether the pharmacokinetics of avermectins or a milbemycin could explain their known or predicted efficacy in the horse. The avermectins, ivermectin (IVM) and doramectin (DRM), and the milbemycin, moxidectin (MXD), were each administered orally to horses at 200 microg/kg bwt. Blood and faecal samples were collected at predetermined times over 80 days (197 days for MXD) and 30 days, respectively, and plasma pharmacokinetics and faecal excretion determined. Maximum plasma concentrations (Cmax) (IVM: 21.4 ng/ml; DRM: 21.3 ng/ml; MXD: 30.1 ng/ml) w...
Identification and characterisation of beta-adrenoceptors on intact equine peripheral blood lymphocytes with the radioligand (-)-[125I]-iodocyanopindolol. In this study, beta-adrenoceptors of intact equine lymphocytes were identified and subclassified by (-)-[125I]-iodocyanopindolol (ICYP) binding. ICYP binding to intact equine lymphocytes was rapid, saturable (maximal number of binding sites 320 +/- 20 ICYP binding sites/cell, n = 12) and of high affinity (KD value for ICYP 14.4 +/- 1.7 pmol/l, n = 12). Binding was stereospecific as shown by the 10 times greater potency of (-)-propranolol to inhibit binding than its (+)-isomer. Beta-adrenoceptor agonists inhibited ICYP binding with an order of potency: (-)-isoprenaline >(-)-adrenaline >(-...
Influence of oocyte collection technique on initial chromatin configuration, meiotic competence, and male pronucleus formation after intracytoplasmic sperm injection (ICSI) of equine oocytes. There is a great variability in the success of horse oocyte maturation and fertilization among laboratories. This study was conducted to determine if the meiotic and developmental competence of horse oocytes could be dependent on the method of oocyte collection, i.e., aspiration of follicular fluid with a vacuum apparatus, or opening follicles and scraping the granulosa layer. Horse oocytes were recovered from abattoir ovaries by aspiration or scraping and classified as having compact (Cp), expanded (Ex), or partial (P) cumuli. In Experiment 1 (Part A in May and Part B in October), oocytes wer...
Serological diagnosis of equine influenza using the hemagglutinin protein produced in a baculovirus expression system. The hemagglutinin (HA) protein of an equine influenza strain, A/equine/La Plata/1/93 (LP/93), was produced using a baculovirus expression system. Silkworm larvae inoculated with recombinant baculovirus expressed high quantities of the HA protein which was then purified to greater than 95% purity by fetuin-affinity chromatography. Purified HA protein was used subsequently in an ELISA for detection of antibodies in horse sera. Two hundred serum samples from vaccinated racehorses were reacted on ELISA plates coated with 40.0 ng/ml of purified HA protein. Subsequent optical density (OD) levels rev...
Evidence that Equine rhinitis A virus VP1 is a target of neutralizing antibodies and participates directly in receptor binding. Equine rhinitis A virus (ERAV) is a respiratory pathogen of horses and is classified as an Aphthovirus, the only non-Foot-and-mouth disease virus (FMDV) member of this genus. In FMDV, virion protein 1 (VP1) is a major target of protective antibodies and is responsible for viral attachment to permissive cells via an RGD motif located in a distal surface loop. Although both viruses share considerable sequence identity, ERAV VP1 does not contain an RGD motif. To investigate antibody and receptor-binding properties of ERAV VP1, we have expressed full-length ERAV VP1 in Escherichia coli as a glutat...
