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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.
Agalactia in mares fed with grain contaminated with Claviceps purpurea. This article reports an outbreak of intoxication of female horses with Claviceps purpurea in southern Brazil. The outbreak affected twelve pregnant mares which were fed with black oat (Avena strigosa) during the pre-delivery period. Underdevelopment of the mammary gland in the pre-delivery period resulting in post-delivery agalactia was the most pronounced finding. These mares delivered weak and unviable foals, which showed no suckling reflex and died within a few hours of birth. Laboratory analysis of oat samples fed to the animals resulted in the identification of Claviceps purpurea scleroti...
Capacitation-like changes in equine spermatozoa throughout the cryopreservation process. Chlortetracycline (CTC) fluorescence staining analysis was used to investigate cryopreservation-induced capacitation-like changes in equine spermatozoa. Freshly ejaculated spermatozoa were found to display a high proportion of F pattern cells (uncapacitated; 93.6%) and a lower proportion of B pattern (capacitated; 5.4%) and AR pattern (acrosome-reacted; 1%) cells. Following cryopreservation in modified Kenney's medium, capacitation-like changes were observed. There was a significant increase in the proportion of spermatozoa displaying the B pattern (64.8%; P<0.001) and AR pattern (32.8%; P&...
Quantitative detection of salmeterol after inhalation in equine urine by liquid chromatography/tandem mass spectrometry. A sensitive, accurate and precise liquid chromatography/tandem mass spectrometry (LC/MS(2)) method was developed for the quantification of salmeterol in the urine of horses. The method consists of a liquid-liquid extraction with tert-butylmethyl ether and isopropanol at pH 12 after enzymatic hydrolysis. The extracts are analysed using an LC/MS system equipped with an electrospray ionisation (ESI) probe. Method validation showed excellent linearity, specificity, accuracy, precision and intra-laboratory repeatability and reproducibility. The limit of quantitative detection was 0.25 ng/mL and the...
A rapid and simple method for the separation of pure lymphocytes from horse blood. A method for the separation of pure and viable lymphocytes and granulocytes from the same blood sample in horses was reported. By centrifuging equine heparinized blood at 100 xg for 10 min at room temperature (r.t.), the resulting supernatant plasma was an almost pure (97.71 +/- 0.30%; n = 15) suspension of highly viable (98.72 +/- 0.28%) lymphocytes. When sodium citrate was used as an anticoagulant, lymphocyte suspensions collected in the same manner showed lower purity (87.89 +/- 1.59%; n = 9) and higher yields (56.56 +/- 3.89%, n = 9 versus 36.11 +/- 2.23%, n = 15). Where needed, a further ...
Quantification of clenbuterol in equine plasma, urine and tissue by liquid chromatography coupled on-line with quadrupole time-of-flight mass spectrometry. Clenbuterol (CBL) is a potent beta(2)-adrenoceptor agonist used for the management of respiratory disorders in the horse. The detection and quantification of CBL can pose a problem due to its potency, the relatively low dose administered to the horse, its slow clearance and low plasma concentrations. Thus, a sensitive method for the quantification and confirmation of CBL in racehorses is required to study its distribution and elimination. A sensitive and fast method was developed for quantification and confirmation of the presence of CBL in equine plasma, urine and tissue samples. The method i...
Method for preparing thin sections of untreated equine hoof horn for electron microscopic examination. The preparation of hard tissues such as the equine hoof horn for electron microscopic examination is very difficult. In particular the penetration of fixatives and chemicals used during fixation and embedding is a problem. The objective of this study was to find and implement an alternative method enabling the preparation of high-quality thin sections of hoof horn and other hard tissue, which maintains the hard tissue ultrastructure and can be used for immuno-labeling. Compared to commonly used fixation and embedding techniques, the preparation of thin sections from untreated material method s...
Spectroscopic and electrochemical studies of horse myoglobin in dimethyl sulfoxide. This paper reports the first report of rapid, reversible direct electron transfer between a redox protein, specifically, horse myoglobin, and a solid electrode substrate in nonaqueous media and the spectroscopic (UV-vis, fluorescence, and resonance Raman) characterization of the relevant redox forms of myoglobin (Mb) in dimethyl sulfoxide (DMSO). In DMSO, the heme active site of metmyoglobin (metMb) appears to remain six-coordinate high-spin, binding water weakly. Changes in the UV-fluorescence spectra for metMb in DMSO indicate that the protein secondary structure has been perturbed and sugge...
An activator protein-1 complex mediates epidermal growth factor regulation of equine glycoprotein alpha subunit expression in trophoblast cells. Equids and primates are the only species known to express the placental hormone chorionic gonadotropin (CG). CG is a member of the heterodimeric glycoprotein family and is composed of an alpha subunit linked to a hormone-specific beta subunit. Previously, we have reported that epidermal growth factor (EGF) regulates the equine glycoprotein hormone alpha subunit promoter through a protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) signal transduction pathway in trophoblasts. The current study investigates the regulatory element/factors involved in the induction of equine glycoprotei...
Development of a multiplex real-time reverse transcriptase-polymerase chain reaction for equine infectious anemia virus (EIAV). A single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) using a fluorogenic real-time PCR detection method is described for the quantitation of equine infectious anemia virus (EIAV) RNA in the plasma of equids. To compensate for variations inherent in sample preparation a multiplex real-time RT-PCR system was developed that permitted the simultaneous calculation of the nucleic acid recovery rate along with the copy number of viral RNA molecules. Detection of EIAV RNA was linear from 10(9) to 10(1) molecules with intra- and inter-assay variability of less than 1% at 10(8), 10(6),...
Evaluation of microbial culture techniques for the isolation of Pythium insidiosum from equine tissues. The purpose of this study was to evaluate the effects of sample handling, storage, and culture techniques on the isolation of Pythium insidiosum from infected equine tissues. Tissue and kunker samples obtained immediately posteuthanasia from a horse with subcutaneous pythiosis were used to assess the effects of sample type (kunkers vs. tissues), media type (selective vs. nonselective), storage technique, and storage time on P. insidiosum isolation rate. Overall, isolation rates were higher from fresh kunkers (94.6%) and stored kunkers (76.4%) than from fresh tissues (8.3%) or stored tissues (4...
Effect of extraction time and acid concentration on the separation of proglycogen and macroglycogen in horse muscle samples. The objective of this study was to determine whether the concentrations of proglycogen (PG) and macroglycogen (MG) in biopsy samples of horse muscle are influenced by extraction time or perchloric acid (PCA) concentration. In study 1, individual muscle-biopsy samples from 10 horses were divided into 4 parts each and then randomly subjected to 4 periods of extraction (10, 20, 60, or 120 min) with 1.5 M PCA. In study 2, individual muscle-biopsy samples from 6 horses were divided into 24 pieces each and then randomly subjected to 12 combinations of extraction time (10, 20, 30, or 40 min) and PCA ...
Flow cytometry: clinical applications in equine medicine. The use of flow cytometry in veterinary diagnostics is becoming a valuable clinical tool with a broad range of applications. Physical characteristics of cells can be determined by the flow cytometer laser and electronics through the measurement of changes in light scatter properties. Other components and functions of cells can be defined through the application of fluorochrome dyes that have an affinity for cellular components. Traditionally, common clinical applications are immunophenotyping of cells of the hematopoietic system with fluorescent-labeled antibodies raised against specific cell ...
A PCR-RFLP for KIT associated with tobiano spotting pattern in horses. An MspI polymorphism was identified in intron 13 of the equine homologue of proto-oncogene c-kit (KIT) by comparing DNA sequences from horses with solid coat colour and horses homozygous for the tobiano spotting (To) gene. The allele associated with solid coat colour was designated KM0, while the allele associated with the tobiano pattern created an additional MspI restriction site and was designated KM1. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) studies using DNA from hair follicles demonstrated that all 129 of 129 tobiano patterned horses possessed the KM1...
Expression of interleukin-1 (IL-1) system genes in equine cumulus-oocyte complexes and influence of IL-1beta during in vitro maturation. A growing body of evidence suggests that the ovary is a site of inflammatory reactions, and thus, ovarian cells could represent sources and targets of the interleukin-1 (IL-1) system. The aim of the present work was to investigate the expression of IL-1alpha, IL-1beta, IL-1ra, IL-1R1, and IL-1R2 genes in equine cumulus cells and oocytes. Moreover, the influence of IL-1beta on in vitro maturation of cumulus-oocytes complexes (COCs) was examined. COCs were collected using ultrasound-guided follicular puncture in vivo. Oocytes and cumulus cells were isolated from preovulatory and subordinate foll...
Determination of the chondroitin sulfate disaccharides in dog and horse plasma by HPLC using chondroitinase digestion, precolumn derivatization, and fluorescence detection. A sensitive and selective HPLC method for the determination of the disaccharides of chondroitin sulfate in horse and dog plasma was validated. Chondroitin sulfate is degraded by chondroitinase ABC to three primary unsaturated disaccharides, (1) 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose, (2) 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose, and (3) 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose, when treated with chondroitinase. Plasma samples (0.5 ml) were treated with 50 mU of chon...
Molecular characterization of the equine AEG1 locus. Acidic epididymal glycoprotein 1 (AEG1), also called cysteine-rich secretory protein 1 (CRISP1), is a member of the CRISP protein family which is characterized by 16 conserved cysteine residues at the C-terminus. The CRISP proteins are expressed in the male genital tract and are thought to be involved in sperm-egg fusion. Therefore, their genes are of interest as candidate genes for inherited male fertility dysfunctions and as putative quantitative trait loci for male fertility traits. In this report, the cloning and DNA sequence of 90 kb of horse genomic DNA from equine chromosome 20q22 conta...
Expression of a chemokine by ciliary body epithelium in horses with naturally occurring recurrent uveitis and in cultured ciliary body epithelial cells. To determine whether a chemokine (RANTES)-like protein expressed by ciliary epithelium plays a role in uveitis. Methods: 3 clinically normal horses intradermal, 5 eyes from 5 horses with recurrent uveitis, and 10 normal eyes from 5 age- and sex-matched horses. Methods: Cross-reactivity and sensitivity of recombinant human (rh)-regulated upon activation, normal T-cell expressed and secreted (RANTES) protein were evaluated in horses by use of intradermal hypersensitivity reactions and a chemotaxis assay. Aqueous humor and ciliary body of eyes from clinically normal horses and horses with uveitis...
Contribution of heme-propionate side chains to structure and function of myoglobin: chemical approach by artificially created prosthetic groups. Horse heart myoglobin was reconstituted with mesohemin derivatives methylated at the 6- or 7-position to evaluate the role of the heme-6-propionate or heme-7-propionate side chain in the protein. The association and dissociation of the O(2) binding for the deoxymyoglobin with 6-methyl-7-propionate mesoheme are clearly accelerated. Furthermore, the myoglobin with 6-methyl-7-propionate mesoheme shows fast autoxidation from oxymyoglobin to metmyoglobin compared to the myoglobin with 6-propionate-7-methyl heme and the reference protein. These results indicate the 6-propionate plays an important ph...
Two bi-allelic single nucleotide polymorphisms within the promoter region of the horse tumour necrosis factor alpha gene. Primers based on GenBank sequences within the 5' untranslated region (UTR) of the human and horse tumour necrosis factor alpha (TNF-alpha) genes were designed and used to amplify a 522-bp product. Sequencing of five clones derived from five independent PCRs obtained from three different animals of three different breeds (Old Kladruber, Akhal-Teke and Shetland Pony) revealed a high level of sequence identity to the TNF-alpha promoter regions of other species. The existing GenBank horse sequences were confirmed and extended upstream by 230 nucleotides. Based on the sequence obtained, a new horse...
Equine oocyte maturation with epidermal growth factor. Epidermal growth factor (EGF) has been shown to have a positive effect during oocyte in vitro maturation in several species. This study was performed to establish the capacity of equine oocytes to undergo nuclear maturation in the presence of EGF and to localise its receptor in the equine ovary by immunohistochemical methods. Oocytes were obtained by aspiration and subsequent scraping from equine follicles (15-25 mm diameter) and cultured in 3 different treatment groups for 36 h: control Group (modified TCM 199 with 0.003% BSA), EGF Group (TCM-199 supplemented with 50 ng/ml EGF) and EMS Group ...
Analysis of protein ions in the range 3000-12000 Th under partial (no discharge) atmospheric pressure chemical ionization conditions using ion trap mass spectrometry. A new approach, based on the use of atmospheric pressure chemical ionization ion trap mass spectrometry (APCI-ITMS), but without a corona discharge, was investigated for application to creating and monitoring protein ions. It must be emphasized that APCI is not usually used in protein analysis. In order to verify the applicability of the proposed method to the analysis of proteins, two standard proteins (horse cytochrome c and horse myoglobin) were analyzed. A mixture of the two proteins was also analyzed showing that this novel approach, based on the use of APCI, can be used in the analysis o...
Cytological analysis of equine bronchoalveolar lavage fluid. Part 2: Comparison of smear and cytocentrifuged preparations. The aim of this study was to develop a diagnostically useful smear method for preparation of equine bronchoalveolar lavage fluid (BALF) for use by practitioners. A smear method for equine BALF preparation which included the addition of serum was developed, and cell morphology, differential cell counts (DCC) and repeatability of counting DCC compared with those of cytocentrifuged BALF preparations. BALF samples (n = 21) were collected from 5 control horses and 5 heaves-susceptible horses. Smear preparations of BALF produced smaller, darker, staining cells, making cytological identification more...
Cytological analysis of equine bronchoalveolar lavage fluid. Part 3: The effect of time, temperature and fixatives. Bronchoalveolar lavage fluid (BALF) samples are often subject to time delays, possibly with temperature fluctuations, between collection and processing. The aim of this study was to evaluate the effects of time, temperature and 2 different fixatives on equine BALF cytology, in order to develop guidelines for optimal equine BALF storage conditions. Total nucleated cell count (TCC), differential cell counts (DCC), absolute cell counts (ACC), cell viability, cell morphology and bacterial growth of BALF samples stored at 4, 18 (+/- addition of formalin- or alcohol-based fixatives) and 38 degrees C...
IgG antibody subclass response against equine herpesvirus type 4 in horses. In this study, IgG subclass responses against equine herpesvirus type 4 (EHV-4) were examined by enzyme-linked immunosorbent assay (ELISA) using a type-specific region of EHV-4 glycoprotein G (gG). ELISA using sera collected from horses experimentally infected with EHV-4 revealed that IgGa and IgGb antibodies were detected at high level, but IgGc and IgG(T) antibody responses were detected at low level or were undetectable. The IgGa antibody response reached its peak on day 10 post-infection, and then dropped. The IgGb antibody response reached its maximum level on day 12 post-infection, and t...
Lead poisoning of horses in the vicinity of a battery recycling plant. The diagnosis of lead poisoning in horses living on farmland in the vicinity of a battery recycling plant was based on clinical signs as well as on laboratory findings. Chemical analysis of six surface soils (0-15 cm) and herbage samples taken at different distances to the recycling plant showed very high total lead levels in the closest sites to the facility and a clear decrease with distance. Total lead levels in soil samples ranged from 127 to 5657 mg kg(-1), with more than 70% of lead extractable by EDTA in the most polluted soils. Lead levels in the aerial part of herbage samples were in ...
Complete coding sequence of equine inducible nitric oxide synthase. THE gene encoding equine inducible nitric oxide synthase (iNOS) was cloned and sequenced. The strategy consisted of the amplification by PCR of message isolated from primary cultures of equine articular chondrocytes (Frean et al 1997) treated with 100 mg mlÿ1 LPS (Escherichia coli serotype O127:B8). PCR primer pairs were generated by comparison of cross-species published iNOS nucleotide sequences. The sequences of the 30 and 50 termini were derived by the generation of PCR products spanning those regions. The assembly of sequenced PCR fragments revealed a 3930 complete nucleotide sequence wit...
Analysis of feline, canine and equine hemograms using the QBC VetAutoread. Blood samples form 120 consecutive clinical cases (40 cats, 40 dogs and 40 horses) were analyzed on the QBC VetAutoread analyzer and the results compared with those obtained by a Baker 9000 electronic resistance cell counter and a 100-cell manual differential leukocyte (WBC) count. Packed cell volume (PCV), hemoglobin (Hb) concentration, mean cell hemoglobin concentration (MCHC), and platelet, total WBC, granulocytes, and lymphocyte plus monocyte (L+M) counts were determined. Indistinct separation of red blood cell and granulocytes layers on the QBC VetAutoread was observed in samples from fiv...
Artifactually increased serum bicarbonate values in two horses and a calf with severe rhabdomyolysis. Extremely high bicarbonate (HCO3-) and anion gap values were measured in two horses and a calf using the Hitachi 911 automated serum biochemistry analyzer. All three animals had severe muscle disease as evidenced by markedly increased aspartate aminotransferase and creatine kinase activities. Laboratory error was suspected as the source of the increased HCO3- because values calculated from blood gas analysis were normal. It was hypothesized that increased serum lactate dehydrogenase (LDH) activity and pyruvate concentration overwhelmed the oxamate LDH inhibitor in the enzymatic HCO3- assay, re...
[A multicenter study of a new Helicobacter pylori selective medium. Columbia horse blood agar HP]. We conducted a study for the growth of and selectivity for the desired microorganisms using a newly developed selective culture medium for Helicobacter pylori, Columbia horse blood agar HP (CHBHP), at three different Japanese clinical laboratories, Hokkaido, Kanto and Kyusyu. When standard strains and clinical isolates of H. pylori were examined, the recovery of the organism on the CHBHP media was comparable to that of conventional selective and nonselective media. However, colonies were obviously larger on the CHBHP media. These media yielded the highest H. pylori positive rate for clinical s...
A novel model for equine recurrent airway obstruction. Equine recurrent airway obstruction (RAO; a term combining both chronic obstructive pulmonary disease (COPD) and summer pasture associated obstructive pulmonary disease (SPAOPD)) is one of the most common equine respiratory diseases with up to 50% of horses affected worldwide. The etiopathogenesis of RAO is unknown although pulmonary hypersensitivity to inhaled mold antigens may be involved. Recent work in our laboratory demonstrating elevated levels of IL-4 and IL-13 mRNA in the airways and peripheral blood of horses with RAO is consistent with an atopic component to RAO. Little is known rega...
