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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.
Isolation and identification of two potent neurotoxins, aspartic acid and glutamic acid, from yellow star thistle (Centaurea solstitialis). Horses grazing for prolonged periods on yellow star thistle (YST), a plant which grows wild in western parts of the United States, develop an extrapyramidal disorder known as nigropallidal encephalomalacia (NPE). Attempts have been made to identify, isolate, and characterize the toxins responsible for the disease in animals. Using the organotypic tissue culture system on mouse cortical explants as a specific assay method for neurotoxicological evaluation, it has been possible to isolate and characterize two potent neuroexcitotoxic compounds, aspartic and glutamic acids, the former being the ma...
Genotyping of isolates of Taylorella equigenitalis from thoroughbred brood mares in Japan. Profiles of the genomic DNA of 104 strains of T. equigenitalis isolated from brood mares with contagious equine metritis in Hokkaido during the breeding seasons from 1980 to 1993, as well as those of five strains (SS28, EQ56, EQ59, EQ70 and HH139) previously isolated in Japan were examined after restriction digestion and crossed-field gel electrophoresis. These profiles were essentially identical to each other and the various isolates and strains appeared to have a common genotype, designated 'genotype J', with respect to two restriction enzymes, ApaI and NotI. These results suggest a common s...
Continuous in vitro cultivation of erythrocytic stages of Babesia equi. The protozoan parasite Babesia equi, a causative agent of equine piroplasmosis, was continuously cultivated in horse erythrocytes. The parasites were isolated from a carrier horse at a time when no parasite was detected in a thin blood smear. The culture medium consisted of modified medium 199 supplemented with 40% non-heat-inactivated horse serum in a humidified atmosphere containing 5% CO2, 2% O2, and 93% N2 at 37 degrees C. Parasites were detected after 2 days in culture. When the percentage of parasitized erythrocytes (PPE) reached 1%, the cultures were transferred into a humidified atmosp...
Equine parafilariosis in Iran. From March to September 1991, horses (n = 1567), donkeys (n = 112) and mules (n = 96) were examined for equine parafilariosis by random sampling from different areas of Iran. The clinical signs of equine parafilariosis were observed in 136 cases of two northern areas (1.4% and 41.3% infection rate) and one northeastern area (6.3% infection rate). Most of the infected cases were confirmed by laboratory findings. All infected cases were followed up to establish the epidemiological features. The results suggest that there is one enzootic area in Iran, the Caspian coast and Persian Turkman steppes...
Laboratory diagnosis of equine pituitary pars intermedia adenoma. The objective of the study was to determine the sensitivity with which an adenoma of the pars intermedia of the pituitary gland can be predicted in horses by measuring the basal concentrations of glucose, cortisol, adrenocorticotropin (ACTH), and insulin, the urinary concentration of corticoids, the urinary corticoid:creatinine ratio, and the plasma cortisol concentration after the administration of 25 IU of ACTH intravenously. The records of 24 cases of histologically confirmed equine pituitary pars intermedia adenomas were used. An adenoma of the pars intermedia of the pituitary gland in hor...
Pharmacokinetics of intravenously administration of prednisolone in the horse as determined by radioimmunoassay. A radioimmunoassay was developed for prednisolone using IgG purified from rabbit antiserum. The assay was employed to determine the pharmacokinetics of prednisolone following intravenous administration of 450 mg of prednisolone sodium succinate (Solu Delta Cortef) to five adult Thoroughbred horses. The RIA had a sensitivity of 2 ng/ml and was relatively specific. It had cross-reactivity with 21-deoxycortisol (83.3%) cortisol (27.8%), 11-beta-hydroxyprogesterone (39.2%) and 17-hydroxyprogesterone (50%). However, it did not cross-react with naturally occurring steroids (cholesterol, testosterone...
Characterization of a density-corrected ultrasonic pneumotachometer for horses. A density-corrected ultrasonic pneumotachometer designed specifically for horses (UF202) was evaluated and characterized with the aid of a custom-built apparatus. UF202 provided voltage outputs for airflow through and gas density within the flowhead. Baseline stability for flow channel output (VUF202) was or = 0.9976). Under optimal conditions, VUF202 accuracy was determined to be +/- 1.00% FS and repeatability was +/- 0.78% FS. VUF202 resolution was 24 ml/s. The rise time for VUF202 was 18 ms, and the -3-dB point was 18 Hz; digital compensation provided a flat frequency response to 32 Hz. VU...
D-dimer improves the prognostic value of combined clinical and laboratory data in equine gastrointestinal colic. The discriminating ability of 15 parameters alone or in combinations, including results from analysis of plasma endotoxin, the Nycomed plasma D-Dimer test and phospholipase A2, were analyzed to predict morbidity and mortality in equine gastrointestinal colic. Endotoxaemia was a characteristic feature of the colic horses. The problem of adequately predicting nonsurvivors among colic horses required several parameters to be included in the logistic model: if the "classical parameters", (heart rate, respiratory rate, PCV, anion gap) were included in the model, addition of plasma D-dimer, phosphol...
The hemostatic profile of equine ovarian follicular fluid. The coagulation factors VII and X and fibrinogen were detected in equine ovarian follicular fluid. The amounts of fibrinogen and factor X were approximately 40 percent of that found in normal equine plasma while the level of factor VII was lower, at approximately 14 percent. The addition of human recombinant tissue factor caused fibrin formation in the follicular fluid. The thrombin generating activity appears to be confined to the tissue factor pathway since no activity associated with factors VIII:C, IX or IX was detected. Fibrinolytic activity, at higher levels than that found in plasma, wa...
The nucleotide sequence of asinine herpesvirus 3 glycoprotein G indicates that the donkey virus is closely related to equine herpesvirus 1. The nucleotide sequence of the glycoprotein G (gG) homologue of asinine herpesvirus 3 (AHV3), a respiratory alphaherpesvirus of donkeys, was determined. The AHV3 gG gene consists of 1233 base pairs (bp) and codes for a predicted protein of 411 amino acids. This is identical in size to the equine herpesvirus 1 (EHV1) gG gene and 6 amino acids longer than the equine herpesvirus 4 (EHV4) gG gene. The predicted amino acid sequence of AHV3 gG has characteristics of a class 1 membrane protein. The amino acid sequence of AHV3 gG shows 92% and 60% identity to EHV1 gG and EHV4 gG respectively. Two regi...
Detection of equine arteritis virus (EAV) by polymerase chain reaction (PCR) and differentiation of EAV strains by restriction enzyme analysis of PCR products. A polymerase chain reaction (PCR) based assay capable of detecting and differentiating seven strains of equine arteritis virus (EAV) from around the world was developed. The primers for the PCR were chosen from the ORF6 gene encoding the unglycosylated membrane protein (M). Viral RNA from cell culture fluids infected with each of the seven EAV strains and RNA from the live vaccine, Arvac, was detected by PCR using four sets of primers. The sensitivity of detection was increased from 100 to 1,000 times by performing nested PCR enabling the detection of RNA at a level of 0.5-5 PFU. Differentiati...
Enzyme-linked immunosorbent assay for myosin heavy chains in the horse. The content in slow and fast myosin heavy chains (MHC 1 and MHC 2) of 5 equine muscles was determined using an enzyme-linked immunosorbent assay. The results obtained with this immunoenzymatic method were compared with complementary techniques: electrophoresis and immunohistochemistry. Slices of masseter, diaphragm, tensor faciae latae, semitendinosus and cutaneus trunci were obtained from a 12-year-old saddle horse after slaughter. Muscular proteins were specifically extracted to be analysed by ELISA. The technique used 2 complimentary monoclonal antibodies (MAb). MAb 1 was prepared from a hu...
A type-specific serological test to distinguish antibodies to equine herpesviruses 4 and 1. We describe a type-specific ELISA, which distinguishes antibody to equine herpesvirus 4 (EHV4; equine rhinopneumonitis) and EHV1 (equine abortion virus) thereby identifying horses that have been infected with either or both of these antigenically related viruses. The antigens used are parts of the EHV4 and EHV1 glycoprotein G (gG) homologues expressed in E. coli as fusion proteins [Crabb and Studdert, 1993: J Virol 67: 6332-6338). The expressed proteins comprise corresponding regions of the gG molecules that are highly divergent and encompass strong, typespecific epitopes. Plasma samples from ...
Prejunctional muscarinic autoreceptors on horse airway cholinergic nerves. Muscarinic autoreceptors on horse airway cholinergic nerves were studied by examining the effects of muscarinic receptor antagonists on electrical field stimulation (EFS)-induced acetylcholine (ACh) release in trachealis preparations. All the antagonists including atropine (non-selective), pirenzepine (M1-selective), AF-DX 116 (M2-selective), and hexahydrosiladifenidol (M3-selective) augmented ACh release concentration-dependently. The augmentation was not due to displacement of ACh molecules from tissue receptors into the bath liquid because incubation with atropine after EFS had no influence...
Large equine blastocysts are damaged by vitrification procedures. Viability following vitrification of equine blastocysts with different sizes was investigated in vitro. Twenty-four blastocysts were classified into three groups according to their diameters ( 300 microns; n = 8 each). The solution used for vitrification was defined as EFS and contained 40% ethylene glycol, 18% Ficoll and 0.3 M sucrose in modified-phosphate-buffered saline (m-PBS). During pretreatment with 20% ethylene glycol in m-PBS for 20 min, the larger blastocysts responded to the osmotic pressure caused by 20% ethylene glycol more slowly than the smaller blastocysts. Single blastocysts w...
Capacitation in vitro of stallion spermatozoa: comparison of progesterone-induced acrosome reactions in fertile and subfertile males. Mammalian sperm that have completed capacitation are capable of undergoing the acrosome reaction in response to a number of biological and chemical stimuli. In the present report, we have investigated the ability of progesterone to stimulate acrosome reactions of stallion sperm capacitated in vitro. Motile sperm were selected by a two-layer Percoll gradient centrifugation and were incubated in TALP medium modified by the 1:1 (v/v) addition of TEST-yolk medium for 5 hours at 39 degrees C, under 5% CO2 in humidified air. Sperm incubated in vitro in TALP-TEST medium had a higher percentage of acr...
Crystalline composition of equine urinary sabulous deposits. The composition and crystal morphology of 141 equine sabulous deposits were determined by infrared spectroscopy (IR), scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX). The IR analysis revealed that all investigated deposits contained calcium carbonates (calcite, CaCO3, and/or vaterite, CaCO3) as major constituents; 42 samples were composed of calcite and vaterite, 33 of calcite, 18 of calcite/vaterite and calcium oxalate, and 17 of vaterite. The remaining specimens contained calcite/vaterite and other compounds (calcium phosphates, sulphate and/or oxalates and/or s...
Major histocompatibility complex class I-restricted cytotoxic T-lymphocyte responses in horses infected with equine herpesvirus 1. An experimental system that permits sensitive and reproducible detection of equine herpesvirus 1 (EHV-1)-specific cytotoxic T-lymphocyte (CTL) activity in the horse was developed. Peripheral blood mononuclear cells (PBMC) collected from immune horses were restimulated in vitro by culture with live EHV-1. Cytotoxic activity against virus-infected, pokeweed mitogen-stimulated lymphoblast targets was assessed in a 4-h 51Cr release assay. The optimal conditions for in vitro stimulation of equine memory CTLs and for preparation of EHV-1-infected target cells expressing viral antigens were systemati...
Diagnosis of equine gammaherpesvirus 2 and 5 infections by polymerase chain reaction. Nested polymerase chain reaction (PCR) assays were developed for the detection of equine herpesvirus 2 (EHV2) and equine herpesvirus 5 (EHV5) using the nucleotide sequences from the glycoprotein B (gB) gene of EHV2 and the thymidine kinase (TK) gene of EHV5. The simultaneous use of EHV2 specific and EHV5 specific primers in one nested amplification assay (multiplex PCR) enabled a rapid, specific and sensitive diagnosis for each virus. PCR was found to be 10(3) times more sensitive than virus isolation by cell culture for EHV2 and 10(6) for EHV5. In separate PCR assays, the routine detection li...
Flow-cytometric studies of the phagocytic capacities of equine neutrophils. Methodological aspects of flow-cytometric evaluation of the phagocytic properties of equine neutrophils were elucidated. The kinetics of attachment and ingestion were studied, and the phagocytic process was more rapidly completed when serum-opsonized yeast cells were used than with use of IgG-opsonized yeast cells. Trypan blue was successfully used to quench fluorescence of non-ingested yeast cells. There were only minor differences in the kinetics of phagocytosis between quenched and unquenched samples, indicating that attachment is rapidly followed by ingestion. Trypan blue quenching caused ...
Unidirectional fluxes of short-chain fatty acids across segments of the large intestine in pig, sheep and pony compared with guinea pig. Unidirectional fluxes of short-chain fatty acids across pig, sheep and pony caecum, proximal and distal colon were studied under short-circuit current conditions in Ussing chambers. Findings are compared with results from guinea pig. Marked species differences are apparent; highest mucosal-to-serosal fluxes of acetate, propionate and butyrate were seen in guinea pig, lower values in pig and smallest fluxes in sheep and pony. Segmental differences between caecum, proximal and distal colon exist mainly in guinea pig and are less developed in pig, sheep and pony. Inhibition of Na+/H+ exchange by ...
The identification of polymorphic microsatellite loci in the horse and their use in thoroughbred parentage testing. Six new horse microsatellite loci were identified by sequencing M13 clones containing horse genomic inserts which gave positive signals when probed with a CA/GT repeat probe. Oligonucleotide primer pairs were synthesized for these loci and for two previously described horse microsatellites, HTG4 and HTG6. Polymerase chain reaction assays were then carried out on a panel of 20 different unrelated Thoroughbred horse DNAs. DNAs from eight cases of double covering which could not be solved by conventional blood typing were also examined. Several of the loci amplified were found to be polymorphic a...
Horse plasma corticotrophin-releasing hormone (CRH): characterisation and lack of a late gestational rise or a plasma CRH-binding protein. Immunoreactive corticotrophin-releasing hormone (irCRH) was present in methanolic extracts of equine peripheral blood and showed no elevation in maternal peripheral serum in late gestation (0.54 +/- 0.25 pmol/l; mean +/- S.D.) compared with control horses (0.41 +/- 0.15 pmol/l). The irCRH of methanolic extracts of pituitary venous plasma had a similar elution position following reverse-phase HPLC to synthetic human CRH(1-41) and to irCRH released from horse stalk-median eminence tissue incubated in vitro. Gel chromatographic studies showed no evidence for a plasma CRH-binding protein (CRHBP) a...
Preliminary study of the metabolism of 17 alpha-methyltestosterone in horses utilizing gas chromatography-mass spectrometric techniques. Little is known about the metabolism of 17 alpha-alkyl anabolic steroids in horses. In this study, the metabolism of 17 alpha-methyltestosterone is investigated by oral administration of a (1 + 1) mixture of the steroid and its deuteriated analogue. Both compounds were synthesized from dehydroisoandrosterone (DHA), using a Grignard reaction followed by an Oppenauer oxidation. Post-administration urine extracts were analysed by gas chromatography--mass spectrometry (GC-MS) using both electron impact (IE) and chemical ionization (CI). Interpretation of the data was facilitated by observation of ...
Generic immunoassay of corticosteroids with minimum pre-treatment of urine samples. A generic, rapid and sensitive enzyme linked immunosorbent assay (ELISA) test has been developed which allows large-scale simultaneous testing of synthetic corticosteroids viz., flumethasone, dexamethasone and betamethasone. This assay can be directly applied to diluted urine samples (1 + 9) without hydrolysis of glucuronide or sulfate conjugates or any other treatment of samples. The polyclonal antibody was obtained by immunizing sheep with a flumethasone derivative linked to human serum albumin. This polyclonal antibody displayed high-reactivity with several synthetic corticosteroids whilst ...
Determination of succinonitrile in horse urine by gas chromatography-nitrogen-phosphorus detector and gas chromatography-mass spectrometry. A chromatographic method was developed to detect and confirm the presence of succinonitrile (SDN) in horse urine samples, for antidoping control. The urine samples (5 ml) were extracted with diethyl ether and screened by gas chromatography-nitrogen-phosphorus detector and the confirmation of the drug's presence was accomplished by using gas chromatography-mass selective detection. The recovery of extraction was 78 and 81% for 1.0 and 2.0 micrograms ml-1 (relative standard deviation, < 10%), respectively. Urine samples collected after the administration of Energisan were positive for SDN (1-30 ...
Screening of non-steroidal anti-inflammatory drugs, barbiturates and methyl xanthines in equine urine by gas chromatography-mass spectrometry. A gas chromatographic-mass spectrometric (GC-MS) screening procedure for 23 acidic drugs in equine urine is described. With the GC-MS method fifteen anti-inflammatory drugs, five barbiturates and three methyl xanthines can be detected with good sensitivity and selectivity. The method consists of alkaline hydrolysis, extraction with organic solvent using salting-out, clean-up extraction, methylation and screening with GC-MS in selected-ion monitoring mode. The limit of detection is 10 micrograms 1(-1) or lower, for most drugs.
Release of lipid from the equine placenta during in vitro incubation. An in vitro incubation technique was used to examine release of lipids from the equine placenta. Placental tissue was obtained at term (n = 5, term = 320-365 days) and earlier in gestation (n = 8, mean = 266 days). Term placentae were incubated at two temperatures, 4 degrees C (control) and 37 degrees C for 2 h. Pre-term placentae were incubated at 37 degrees C with two different concentrations of fatty acid in the medium. Tissues and media were analysed for their lipid concentrations. Term and pre-term placentae released free fatty acid (FFA) and phospholipid into the incubation medium during...
Monoclonal antibodies to the equine CD2 T lymphocyte marker, to a pan-granulocyte/monocyte marker and to a unique pan-B lymphocyte marker. Murine monoclonal antibodies, HB88A, B29A and DH59B separately identify the CD2 T lymphocyte molecule, a unique pan-B lymphocyte surface marker and a pan-granulocyte/monocyte surface molecule, respectively, in the horse. Specificity was shown by two-color immunofluorescent flow cytometry and immunofluorescent microscopy. MAb HB88A reacted with a 52 kDa pan-T lymphocyte molecule present on 75% +/- 7 of peripheral blood lymphocytes (PBL) (n = 15 horses). It also reacted with lymphocytes restricted to T lymphocyte dependent areas of lymph node and spleen. Specificity of mAb HB88A to CD2 was demon...
Pentoxifylline inhibits mediator synthesis in an equine in vitro whole blood model of endotoxemia. Whole blood from 10 healthy horses was aseptically collected into heparin or citrate anticoagulant and incubated in vitro for 6 hr in the absence (saline control) or presence of 1 ng endotoxin/ml blood. Pentoxifylline (0.1, 1, 10, or 100 micrograms/ml blood) was added 1 hr before, at the same time, or 1 hr after endotoxin. As compared to saline controls, pentoxifylline alone had no effect on mediator production, with the exception of significantly increasing 6-ketoprostaglandin F1 alpha concentration. Pentoxifylline inhibited endotoxin-induced increases in tumor necrosis factor (TNF) and inter...
