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.
Tanaka T, Aramaki S, Momose A.A thin-layer chromatographic screening procedure for some basic, neutral and acidic drugs was developed using 3 ml of horse plasma. Chloroform-2-propanol (95:5, v/v) was used as the extraction solvent. The drugs were identified by a high-performance thin-layer chromatographic plate and spraying successively with some detection reagents. In this study, the extraction recovery rates and the detection limits were determined at the same time.
Singh AK, McArdle C, Gordon B, Ashraf M, Granley K.A high performance liquid chromatographic method is described for the simultaneous determination of furosemide and bumetanide in horse plasma. The C8 (3 microns) reversed phase column (4.8 x 150 mm) provided clear separation of furosemide and bumetanide with other components present in the horse plasma. The detection limit for both the drugs was 10 ng/mL. Both drugs were stable in plasma (at natural or acidic pH) for up to 24 h. The method is sufficiently sensitive to detect furosemide levels in plasma obtained from horses receiving a therapeutic dose of furosemide.
Pemberton AD, Slater JS, Milne EM.A method for flunixin detection in equine serum extracts involving thin layer chromatography, spraying the chromatogram with alkaline sodium hypochlorite solution and heating with a detection limit of 50 ng ml-1 is described.
Sosnicki AA, Lutz GJ, Rome LC, Goble DO.Until now, there has been no reliable method for histochemical determination of fiber types of single skinned muscle fibers. The major problem arises from the fact that most histochemical techniques use cross-sections of a large group of fibers and compare a given fiber with those surrounding it. This is not possible with a single skinned fiber which has been separated from a bundle to be used for mechanical analysis. A further problem is that the skinning procedure itself may alter the staining pattern. We have developed a procedure by which multiple cross-sections of single skinned fibers ca...
Dintinger T, Gaillard JL, Moslemi S, Zwain I, Silberzahn P.The ability of equine and human placental microsomes to aromatize testosterone and 19-nortestosterone was studied. When 3 microM [1 beta,2 beta-3H]testosterone was used as substrate, the specific activity of equine placental microsomal aromatase was 2.5 times higher than that of the human microsomal enzyme. Although 19-nortestosterone was aromatized 67 times more rapidly by equine than by human aromatase, we found that equine aromatase exhibited a markedly weaker affinity for this substrate than did the human enzyme. Competitive inhibition of testosterone aromatization by 19-nortestosterone oc...
Tang PW, Crone DL.A new method for hydrolyzing steroid conjugates (both sulfates and glucuronides conjugates) that is efficient, effective, and inexpensive is described. This method comprises incubation of the conjugates--after salting-out into ethyl acetate or elution from a C18 cartridge--with anhydrous methanolic hydrogen chloride (methanolysis) for 10 min. It has been successfully applied to our routine radioimmunoassay screening and GC/MS confirmation studies of steroids in prerace and postrace equine urine samples. Comparative GC/MS studies on entire (male horse) urine samples showed that methanolysis gav...
Desmet J, Van Dael H, Van Cauwelaert F, Nitta K, Sugai S.The enthalpy change of the binding of Ca2+ and Mn2+ to equine lysozyme was measured at 25 degrees C and pH 7.5 by batch microcalorimetry: delta H degrees Ca2+ = -76 +/- 5 kJ mol-1, delta H degrees Mn2+ = -21 +/- 10 kJ mol-1. Binding constants, log KCa2+ = 6.5 +/- 0.2 and log KMn2+ = 4.1 +/- 0.5, were calculated from the calorimetric data. Therefore, delta S degrees Ca2+ = -131 +/- 20 JK-1 mol-1 and delta S degrees Mn2+ = 8 +/- 44 JK-1 mol-1. Removal of Ca2+ induces small but significant changes in the circular dichroism spectrum, indicating the existence of a partially unfolded apo-conformatio...
Mück WM, Henion JD.The performance of microbore high-performance liquid chromatography and capillary zone electrophoresis, both equipped with on-line tandem mass spectrometric detection capability, was evaluated critically for the determination of endogenous amounts of leucine enkephalin and methionine enkephalin in equine cerebrospinal fluid. Using an identical sample clean-up and enrichment procedure, capillary zone electrophoresis-mass spectrometry is limited in its concentration detection capacity owing to its much smaller injection volume. Leucine enkephalin was identified in post-mortem equine cerebrospina...
Singh AK, Gordon B, Hewetson D, Granley K, Ashraf M, Mishra U, Dombrovskis D.Gas chromatography with chemical ionization mass spectrometry and selected-ion monitoring provided a sensitive method for the screening and confirmation of steroids in horse urine and plasma. Chemical ionization mass spectrometry was more sensitive than the electron impact ionization mass spectrometry for most of the steroids except for testosterone, prednisone-metabolite-2 and prednisolone-metabolite-2. The chromatographic conditions used in this study provided clean separation of different natural and synthetic steroids. Approximately 75-85% of the steroids added to plasma and approximately ...
Watanabe K, Sohara T, Takeda M, Ueno K, Suzumura N, Rokurouda Y, Rokurouda I, Yamamoto S.Each of five genetic variants of horse serum transferrin (Tf), D, F, H, O, and R, was separated into two bands by polyacrylamide gel isoelectric focusing (PAGIEF). The more acidic band, termed component a, was more abundant than the other one, termed component b, in all variants. Components a and b of TFO variant were immunologically indistinguishable from each other by double immunodiffusion test. Determination of the content ratio of component a to component b in each variant revealed that the variants were classified into two groups: one group (D, F, and H) had a relatively high ratio withi...
Roser JF, Lofstedt RM.Blood and urine samples collected from 12 mares at frequent intervals from 25 to 210 d of pregnancy were analyzed for equine chorionic gonadotropin (eCG). Blood and urine samples were collected daily through two consecutive ovulatory periods from five cyclic mares for comparative purposes. Separate radioimmunoassays (RIA) were developed to detect eCG in the urine and plasma. A simple and quick commercial dipstick enzyme-linked immunospecific assay (ELISA), developed for eCG in the blood, was also utilized in this study to detect eCG in the urine. In the 12 pregnant mares, eCG concentrations in...
Bochsler PN, Slauson DO, Chandler SK, Suyemoto MM.The use of cultured tissue has not yet become widespread in research involving equine disease, and this may be attributable in part to the scarcity of published reports concerning tissue culture methods for this species. We report here the isolation of equine microvascular endothelium (EMVE) from fresh omental tissue of horses and ponies. Fresh donor tissue was minced, subjected to collagenase digestion, and filtered. Cells were layered on 5% bovine serum albumin for gravity sedimentation, the bottom layer was collected, and the cells were plated onto fibronectin-coated flasks. Medium consiste...
Takehana K, Abe M, Iwasa K, Hiraga T.Complex carbohydrates were examined in glandular cells of the horse duodenal gland by using lectin histochemical techniques. In the horse, the duodenal gland was distributed in the area from the uppermost part of the small intestine to a point about 6m caudal to the pylorus. It consisted of two types of cells, mucous and serous cells. The former was found in glands distributed almost all over this part, but the latter was present in glands distributed restrictedly to the uppermost part of the small intestine at a point about 10 cm caudal to the pylorus. The cytoplasm of the mucous cell contain...
Nappert G, Vrins A, Breton L, Beauregard M.A retrospective study of 19 ataxic horses admitted to the College of Veterinary Medicine of the University of Montreal during the period of January 1985 to December 1988 is presented. There were 11 cases of cervical vertebral malformation, four of equine degenerative myeloencephalopathy, two of equine protozoal myeloencephalitis, one each of vertebral osteomyelitis and intervertebral disc protrusion. The clinical diagnosis of ataxia in horses requires neurological, radiographic, myelographic, and laboratory examinations.
Olson LE, Perkowski SZ, Mason DE, Muir WW.Strips of trachealis muscle were dissected from the mid-cervical portion of the trachea from horses that were free of respiratory tract disease. The epithelium and mucosa were removed from one group of tissues and were left intact in a second group of tissues. Each tissue was suspended in a bath filled with Krebs-bicarbonate solution that was aerated with 5% CO2 in oxygen and maintained at 37 degrees C. Isometric tension was continuously recorded. The contractile response to square-wave electrical stimulations increased as frequency (3, 5, 10, 15, 20, 25, and 30 Hz), voltage (10, 15, 18, and 2...
Houghton E, Ginn A, Teale P, Dumasia MC, Copsey J.The stereochemistry of an isomer of 5-estrane-3,17 alpha-diol, the major metabolite of 19-nortestosterone in horse urine has been established by the use of methylene unit (MU) values. The empirical MU values of the bis-trimethylsilyl (TMS) derivatives of the eight available isomers of 5-androstane-3,17-diol and four isomers of 5-estrane-3,17 beta-diol were determined by capillary gas chromatography using three different columns. From this data the theoretical MU values for the bis-TMS derivatives of the four 5-estrane-3,17 alpha-diol isomers were predicted. Comparison of the experimentally det...
Freeman DE, Ferrante PL, Kronfeld DS, Chalupa W.A D-xylose absorption test was conducted on 4 healthy mares deprived of food for 12, 36, 72, and 96 hours before the test, with a 13- to 15-day adjustment period between each test. Maximal plasma concentrations after 72 and 96 hours of food deprivation were approximately 36% lower than those obtained after the 12- and 36-hour periods (P = 0.0001). Absorption curves were flatter and the decrease in plasma concentration was slower after the 72- and 96-hour periods of food deprivation. The rate of D-xylose absorption (P = 0.0108) and the initial rate of urinary excretion (P = 0.0117) were slower ...
Watson ED, Sertich PL.Embryos, endometrial biopsies, and uterine lavage fluid were collected from pregnant and non-pregnant mares 14 days after ovulation. Embryos were cultured for 20.5 h with and without endometrial tissue from pregnant mares, and endometrial tissue was cultured alone. Endometrial content of PGF tended to be higher (P = 0.06) in non-pregnant than in pregnant mares, but the amount of PGF released from tissue during culture was similar for pregnant and non-pregnant mares. Lavage fluid from non-pregnant mares also tended (P = 0.08) to contain higher concentrations of PGF. Coincubation of embryos with...
Mason DE, Muir WW, Olson LE.Smooth muscle strips from the midcervical portion of the trachea and bronchial smooth muscle strips from third-generation airways of horses were placed in tissue baths, and isometric contractile force was measured. Active force was measured in response to electrical stimulation and exogenous acetylcholine. Square-wave electrical stimuli were applied at various voltages (10, 12, 15, 18, 20, 25 V), frequencies (3, 5, 10, 15, 20, 25, 30 Hz), and pulse durations (0.2, 0.5, 1.0, 1.5, 2.0 ms). Isometric contractile force increased as voltage, frequency, and pulse duration increased. Maximal contract...
Messier PE, Drouin R, Richer CL.We present an immunochemical technique for the detection of 5-bromo-2'-deoxyuridine (BrdU) incorporated discontinuously into the chromosomal DNA. A monoclonal anti-BrdU antibody and a protein A-gold complex were used to produce chromosome banding of human and equine chromosomes, specific for electron microscopy (EM). Well-defined bands, symmetry of sister chromatids, concordance between homologues, and band patterns similar to those observed by light microscopy facilitate chromosome identification and karyotyping. From prophase to late metaphase, chromosomes condense and bands appear to fuse. ...
Ball BA, Altschul M, Freeman KP, Hillman RB.Trophoblastic vesicles have been used to study early embryonic development and maternal recognition of pregnancy in domestic animals. The purpose of this study was to characterize the formation of trophoblastic vesicles from Day-12 to Day-16 equine conceptuses. Conceptuses (n = 19) were collected nonsurgically from mares, the capsule was removed, and the conceptus (trophoblast and inner cell mass) was dissected into 2- to 4-mm fragments. Conceptus fragments were cultured in either Ham's F10 (HF10) or Minimum Essential Media (MEM) with 10% fetal bovine serum (FBS) in 24-well plates. Plates were...
Grundy FJ, Baumann RP, O'Callaghan DJ.The immediate early (IE) proteins of herpesviruses are important regulatory factors which control the expression of genes at the transcriptional level. We report the DNA sequence of the immediate early gene of the alphaherpesvirus equine herpesvirus type 1 (EHV-1). This sequence is shown to be extremely rich in guanine and cytosine, resulting in a highly biased codon usage. The IE gene region possesses 38 open reading frames (ORFs) greater than 300 bp in length, 11 of which have coding regions of at least 100 amino acids (aa) following potential translation initiator codons. The largest ORF co...
Hamada T, Watanabe G, Kokuho T, Taya K, Sasamoto S, Hasegawa Y, Miyamoto K, Igarashi M.A sensitive radioimmunoassay (RIA) for the determination of inhibin in peripheral plasma and tissue homogenates of different species has been developed using antisera to partially purified bovine follicular fluid (bFF) inhibin and 125I-labelled bFF 32 kDa inhibin. Antisera were produced by immunization of rabbits with partially purified bFF inhibin prepared by immunoaffinity chromatography. Increasing doses of a high titre antiserum could neutralize the suppressing effect of bFF, porcine follicular fluid and rat ovarian homogenate on FSH secretion from rat anterior pituitary cells in culture. ...
Gray WL, Yalamanchili R, Raengsakulrach B, Baumann RP, Staczek J, O'Callaghan DJ.Equine herpesvirus type 1 (EHV-1) preparations enriched in defective interfering particles (DIPs) have previously been demonstrated to mediate the coestablishment of persistent infection and oncogenic transformation in primary hamster embryo fibroblasts. In this study, it was demonstrated that infection of a rabbit kidney (RK) cell line with EHV-1 DIP-enriched preparations also results in the establishment of persistent infection. Viral transcription was characterized in RK cells infected with DIP-enriched stocks and compared to viral transcription in RK cells infected with standard (STD) EHV-...
Higashi T, Harasawa R.The three equine adenovirus strains isolated in different locations showed a similar cleavage pattern with HindIII and the DNA homology among the strains was confirmed by Southern blot hybridization. The three strains revealed differences in cleavage patterns with BamHI, EcoRI and PstI, suggesting the presence of DNA polymorphisms among equine adenoviruses.
Townson DH, Pierson RA, Ginther OJ.Plasma progesterone concentrations in mares were determined in two experiments during the time that the luteal glands were detectable by transrectal ultrasonography. In both experiments, corpora lutea were classified into two types of morphologies based on their ultrasonic appearance: centrally nonechogenic luteal glands (fluid-filled) and uniformly echogenic luteal glands (non-fluid-filled). In Experiment 1, daily blood samples were taken from horse mares during August through October and May through July. There were no significant effects of season or luteal morphology on progesterone concen...
McManus DP, Rishi AK.A segment of the ribosomal RNA gene of Schistosoma mansoni and a DNA fragment specific to Echinococcus granulosus, cloned in plasmids, have been used as DNA probes to assess the extent of genetic variability within E. granulosus and some distinct strains have been identified. The DNA analysis, involving restriction endonuclease digestion and Southern blot hybridization with the probes, did not demonstrate any significant genetic variation within the U.K. horse/dog or sheep/dog strains but confirmed the distinctiveness of the two strains shown in previous studies. The sheep/dog strain was shown...
Morris DD, Harmon BG, Moore JN.A study was performed to determine whether equine antiserum to core lipopolysaccharide (LPS) would enhance phagocytosis of smooth gram-negative (GN) organisms by equine macrophages. Five healthy adult horses (group A) were immunized with a bacterin prepared from the J-5 mutant of Escherichia coli 0111:B4 and Salmonella minnesota R595 to produce antibodies to core LPS. Five horses (group B) served as nonimmunized controls and were given physiologic saline solution instead of the rough mutant bacterin. Serum antibody titers to core LPS and to smooth E coli 0111:B4 were determined by indirect ELI...
Scudamore CL, Pemberton AD, Miller HR, McDonnell AM, Thomson SR, Dawson A, Watson ED.An enzyme linked immunosorbent assay (ELISA) was developed and used to estimate the concentrations of the serine proteinase inhibitor, alpha-1 proteinase inhibitor (API), in uterine flushings recovered from mares at different stages of the oestrous cycle and before and after the induction of experimental endometritis. There was a significant increase in the concentrations of API and albumin relative to total protein in flushings recovered during oestrus compared with dioestrus but no difference was observed in the concentrations of these proteins relative to total protein before and after the ...
Richards SL, Cawley AT, Raftery MJ.Dermorphin, a hepta-peptide with potent analgesic properties, is classified as a doping agent in equine racing. Since its discovery, a number of biologically active structural analogs have been synthesized and made commercially available so there is a need for reliable methods of detection. Results: A sensitive detection method was developed for dermorphin and six analogs in equine urine. Peptide enrichment was achieved using weak cation exchange with subsequent separation and detection by nano-UHPLC-MS/MS. Method validation parameters included: specificity, linearity (5-10000 pg/ml), recovery...
Hunyadi L, Datta P, Rewers-Felkins K, Sundman E, Hale T, Fajt V, Wagner S.The objective of this study was to determine the pharmacokinetic parameters of oclacitinib maleate as a top dress given to adult horses. Six adult horses with a mean weight of 528Â kg were administered a single dose of 0.5Â mg/kg oclacitinib maleate. Blood was collected prior to drug administration and at 15Â min, 30Â min, 45Â min, 1, 2, 4, 6, 8, 12, 24, 48, and 72Â h after treatment. Oclacitinib maleate plasma concentrations were measured by liquid chromatography/mass spectrometry. Pharmacokinetic parameters were found best to fit a one-compartment model. Mean C was 486Â ng/ml (range 423-549Â...
Wertz PW, Downing DT.The lipids of horse hoof have been analyzed by quantitative thin-layer chromatography. The major components include cholesterol (37-40%), six groups of ceramides (10-15%), and cholesteryl sulfate (15-20%). Free fatty acids are abundant (15.8%) in the outer fully keratinized hoof, but are present at only low levels (3.1%) in the softer hyponychium. The material identified as cholesteryl sulfate was isolated by preparative thin-layer chromatography and characterized by a combination of chemical, chromatographic, and spectroscopic methods. The infrared spectrum of the isolated material had absorp...
You Y, Uboh CE, Soma LR, Guan F, Li X, Rudy JA, Chen J.A rapid and sensitive method was developed for the screening, quantification and confirmation of ethyl glucuronide (EG) and ethyl sulfate (ES) as biomarkers for alcohol administration to racehorses using liquid chromatography coupled on-line with triple quadrupole tandem mass spectrometry. Urine sample aliquots (0.1 mL) were pre-treated by protein precipitation. Separation of EG and ES was achieved on an Ultra PFP column. Isocratic elution with a flush step was performed using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Analysis was performed by negative electrospra...
Eckersall PD, Beeley JG, Snow DH, Thomas A.The two major polypeptides H (Mr 49,000) and L (Mr 33,000) of equine sweat have been purified by gel filtration and characterised by gel electrophoresis and compositional analysis. Both H and L are glycoproteins containing sialic acid, neutral sugars, N-acetylglucosamine and N-acetylgalactosamine, but the two polypeptides differ considerably in the extent of glycosylation. H and L also differ in amino acid composition, but both contain only low levels of sulphur containing amino acids and histidine. These glycoproteins may behave as surfactants.
Siwińska N, Żak A, Słowikowska M, Kubiak K, Jaworski Z, Niedzwiedź A.Hair is a more biologically stable material than other tissues and contains a relatively constant level of minerals, drugs or toxins. The content of essential elements in hair depends on dietary and non-dietary factors. The aim of this study was to assess and compare the effect of diet on the elemental composition and morphological properties of the hair of healthy Polish Konik horses. Mane hair was collected from 19 horses living in The National Park in Popielno in Poland. Six of these horses were free-ranged all year with permanent access to pasture, while 13 horses were kept in the stable a...
Reubel GH, Studdert MJ.Based on sequence homology with human adenovirus 2 (HAdV2), the hexon gene of equine adenovirus 1 (EAdV1) was identified. HindIII restriction fragments containing the hexon and other viral genes were cloned into the plasmids pUC19 and pBlueScript SK(-) and sequenced. The nucleotide sequence of the hexon gene was completely determined and partial sequence data were obtained for seven other EAdV1 genes. Amino acid (aa) sequence comparison with published adenovirus (AdV) proteins identified the genes for the IIIa, penton, pVII, PVI, 23K proteinase, DNA binding and 100K proteins. The eight EAdV1 g...
Patterson SD, Bell K.The carbohydrate side chains of the major plasma serpins of the horse and wallaby have been characterized by lectin analyses of protein blots from two-dimensional gels using the major human plasma serpin, alpha 1-protease inhibitor, as a control. Eight lectins were used in the characterization in conjunction with enzymatic deglycosylation of complex and high mannose side chains, chemical desialylation and defucosylation, and one round of 'Smith degradation', all being performed on the nitrocellulose blots. Assuming a standard complex side chain structure, the results of the 21 lectin/treatment...
Tetens J, Venugopal CS, Holmes EP, Koch CE, Hosgood G, Moore RM.To evaluate the in vitro effects of adenosine tryphosphate (ATP) on vasomotor tone of equine colonic vasculature. Methods: Arteries and veins from the left ventral colon of 14 mixed-breed horses euthanatized for reasons unrelated to cardiovascular or gastrointestinal tract disease. Methods: Endothelium-intact and -denuded arterial and venous rings were precontracted with 10(-7) and 1.8 x 10(-8) M endothelin-1, respectively. In 1 trial, endothelium-intact rings were also incubated with 10(-4) M N omega-nitro-L-arginine methyl ester (L-NAME) to inhibit nitric oxide (NO) production. Adenosine tri...
Goehring LS, Kessels BG, van Maanen C, Voorbij HA, Sloet van Oldruitenborgh-Oosterbaan MM.The measurement of albumin concentrations in cerebrospinal fluid (CSF) and serum for albumin quotient (AQ) calculations in normal horses was performed by 2 methods: 1) total protein measurement, followed by electrophoresis of the samples to obtain an albumin percentage; and 2) albumin immunoprecipitation quantitated by nephelometry. The results of both methods correlated well, and nephelometry was chosen to determine the albumin concentrations in CSF samples obtained from an indwelling subarachnoidal catheter for daily sampling. Because the use of an indwelling catheter to collect repetitive C...
Dunnett M, Richardson DW, Lees P.Hair analysis to detect drug administration has not been studied extensively in horses. This study aimed to (a) develop an analytical method for enrofloxacin and its metabolite ciprofloxacin in mane and tail hair, (b) relate measured values to doses, routes of administration, hair colour, and (c) demonstrate long-term detectability. Samples were extracted in trifluoroacetic acid at 70 degrees C. Extracts were cleaned-up by solid-phase extraction and analysed by high-performance liquid chromatography with UV-diode array detection. Analyte recoveries were > 87%. Horses were sampled after therape...
Mancheño JM, Jayne S, Kerfelec B, Chapus C, Crenon I, Hermoso JA.Horse pancreatic lipase-related proteins PLRP1 and PLRP2 are produced by the pancreas together with pancreatic lipase (PL). Sequence-comparison analyses reveal that the three proteins possess the same two-domain organization: an N-terminal catalytic domain and a C-terminal domain, which in PL is involved in colipase binding. Nevertheless, despite the high level of sequence identity found, they exhibit distinct enzymatic properties. The intrinsic sensitivity of the peptide bond between Ser245 and Thr246 within the flap region of PLRP2 to proteolytic cleavage probably complicates PLRP2 crystalli...
Bailly-Chouriberry L, Garcia P, Cormant F, Loup B, Popot MA, Bonnaire Y.Cobra (Naja naja kaouthia) venom contains a toxin called α-cobratoxin (α-Cbtx) containing 71 amino acids (MW 7821 Da) with a reported analgesic power greater than morphine. In 2013, the first analytical method for the detection of α-Cbtx in equine plasma was developed by Bailly-Chouriberry et al, allowing the confirmation of the presence of α-Cbtx at low concentrations (1-5 ng/mL or 130-640 fmol/mL) in plasma samples. To increase the method sensitivity and therefore to improve the detection of α-Cbtx in post-administration plasma samples, a nano-liquid chromatography-mass spectrometry/hi...
Sandbaumhüter FA, Theurillat R, Bettschart-Wolfensberger R, Thormann W.The combination of ketamine and an α -receptor agonist is often used in veterinary medicine. Four different α -receptor agonists, medetomidine, detomidine, xylazine, and romifidine, which differ in their chemical structure and thus in selectivity for the α -receptor and in the sedative and analgesic potency, are typically employed during surgery of equines. Recovery following anesthesia with ketamine and an α -receptor agonist is dependent on the α -receptor agonist. This prompted us to investigate (i) the inhibition characteristics for the N-demethylation of ketamine to norketamine and (...
Hu WG, Alvi AZ, Fulton RE, Suresh MR, Nagata LP.A recombinant gene encoding a single-chain variable fragment (scFv) antibody against Venezuelan equine encephalitis virus (VEE) was cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. A streptavidin-binding peptide gene fused to a 6His tag was attached downstream to the scFv gene. The recombinant fusion protein was expressed in bacteria as inclusion bodies that were subsequently solubilized with 8 M urea and renatured by an arginine system. Purification of the fusion protein was achieved by immobilized metal affinity chromatography. Enzyme-linked immunosorbent assay (ELISA...
Naef A, Damerau B, Keller HU.The relationship between the short transient intracellular increase in cAMP levels on the one hand and chemotaxis or crawling movements on the other hand was investigated using human and equine granulocytes. C5ades arg, f-met-leu-phe, human serum albumin and immunoglobulin were used as stimulating agents. There was no strict correlation between the induction of crawling movements or of chemokinesis in general and the generation of the cAMP peak. But there was so far a strict parallelism between the occurrence of the chemotactic response and the cAMP peak. However, the magnitude of the peak was...
Mawhinney I, Davis N, Carson T, Torrens N, Wales A.The study examined and compared the sensitivity of culture and a quantitative PCR assay for screening equine semen for the presence of Taylorella equigenitalis (CEMO). Chilled semen samples, both raw and treated with extender, from two stallions were spiked with the organism at seven or 23 days postejaculation and prepared in serial dilutions. Culture of the 7-day raw semen readily detected CEMO at all dilutions, but extended semen yielded counts that were two log cycles lower at equivalent dilutions, with the organism being nearly undetectable at the maximal dilutions. By contrast, PCR sensit...
Sayers DE, Theil EC, Rennick FJ.Cell-specific variations in apoferritin structure correlate with variations in iron metabolism that suggest functional specificity of the protein shell. Using EPR spectroscopy, we previously showed that vanadyl binds to specific sites on apoferritin, and that VO2+ binding is reduced by Fe(II) and Fe(III) (the natural substrates) and by metals known to influence iron storage (Chasteen, N. D., and Theil, E. C. (1982) J. Biol. Chem. 257, 7672-7677). Such observations suggest that the metal-binding site is important to apoferritin function and may define a location where the influence of cell-spec...
Bakala A, Karlik W, Wiechetek M.In this study a detailed description of the equine hepatocyte isolation procedure is presented. Livers were obtained from horses slaughtered at the local slaughterhouse. For blood removal and liver preservation the following steps are suggested: perfusion with the oxygenated HBSS (0-2 degrees C, with continuous flow of 500-800 ml/min for 3-6 min), protection from ischemia injury by flushing with ice-cold University of Wisconsin Solution (UW, flow rate of 500-800 ml/min), and finally immersion of the liver lobe in UW solution (2 degrees C) during its transport to the laboratory. For equine isol...
Agnihotri K, Pease B, Oakey J, Campbell G.In 2011, a 2-year-old horse in northern Queensland, Australia, was reported to have developed mild neurologic signs, and a blood sample was submitted for laboratory investigation. Virus isolation was performed using the blood sample, and an orbivirus was isolated. This was confirmed to be a strain of Elsey virus (ELSV) after transmission electron microscopy and nucleotide sequencing. The nucleotide sequence was compared with those in GenBank, and had 100% identity with ELSV previously reported from the Northern Territory, Australia. ELSV is taxonomically closely related to Peruvian horse sickn...
Hermanson JW, Hegemann-Monachelli MT, Daaod MJ, LaFramboise WA.The biceps brachii of horses is subdivided into a lateral and medial head. Electrophoresis of samples from the lateral head revealed three slow-migrating native myosin isoforms, including one that does not correspond to slow myosin isoforms described for other mammalian muscles. In contrast, the medial head contained a single slow isoform. Both the lateral and medial heads contained three fast-migrating isoforms corresponding with the FM-2, FM-3 and FM-4 isoforms reported for other mammalian fast-twitch muscle fibers. Electrophoresis of myosin heavy chains (MHCs) revealed only two MHC bands, o...
Waelchli RO, Jaworski T, Ruddock WD, Betteridge KJ.Equine blastocyst fluid has a lower [Na+], a higher [K+] and a lower osmolality than does normal blood serum. Based on the assumptions that the sodium pump is primarily responsible for fluid accumulation and that ions transported actively into the blastocyst increase blastocyst osmolality above that of the external medium, we hypothesized that the [Na+] and the osmolality of mare uterine fluid are lower than those of blastocyst fluid. Microdialysis and ion chromatography were used to estimate [Na+] and [K+] of uterine fluid. Mares (n=10) were used for in vivo measurements at different stages o...
Haikerwal A, Barrera MD, Bhalla N, Zhou W, Boghdeh N, Anderson C, Alem F, Narayanan A.Acutely infectious new world alphaviruses such as Venezuelan Equine Encephalitis Virus (VEEV) pose important challenges to the human population due to a lack of effective therapeutic intervention strategies. Small interfering RNAs that can selectively target the viral genome (vsiRNAs) has been observed to offer survival advantages in several in vitro and in vivo models of acute virus infections, including alphaviruses such as Chikungunya virus and filoviruses such as Ebola virus. In this study, novel vsiRNAs that targeted conserved regions in the nonstructural and structural genes of the VEEV ...
Minguet G, de la Rebière G, Franck T, Joris J, Serteyn D, Sandersen C.To investigate the effects of the volatile anaesthetic sevoflurane on the release of total and active myeloperoxidase (MPO) by non-stimulated and stimulated polymorphonuclear neutrophils (PMNs) in whole blood from healthy horses. Methods: In vitro experimental study. Methods: Adult healthy horses. Methods: Samples of whole venous blood were collected and incubated in air or in air plus 2.3% or 4.6% sevoflurane for 1 hour. PMNs were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP), with a combination of cytochalasin B (CB) and fMLP or with phorbol myristate acetate (PMA). Total an...
Aßmann AD, Ohlerth S, Suárez Sánchez-Andráde J, Torgerson PR, Bischofberger AS.To determine the diagnostic performance of computed tomographic arthrography (CTA) and 3 Tesla magnetic resonance imaging (MRI) for detecting artificial meniscal, meniscotibial ligament (MTL) lesions and cruciate ligament (CL) lesions in horses. Methods: Ex vivo controlled laboratory study. Methods: Nineteen stifles from adult horses. Methods: Stablike defects (n = 84) (16 mm long, 10 mm deep) were created in the menisci (n = 35), CLs (n = 24), and MTLs (n = 25) via arthroscopy prior to MRI and CTA (80 mL contrast at 85 mg/mL per joint). Two radiologists, unaware of the lesions...
Her GR, Watson JT.Sensitive and specific methodology based on capillary column gas chromatography-electron capture-negative chemical ionization-mass spectrometry has been developed for the quantitative analysis of corticosteroids from biological fluids. The feasibility of this method is demonstrated in the quantitative analysis of dexamethasone in horse urine following administration of the drug. A structurally similar compound, 6 alpha-methylprednisolone, is added to the urine as an internal standard. The free dexamethasone and the internal standard are extracted and oxidized to high-electron-affinity 1,4-andr...
Marr KA, Lees P, Cunningham FM.Adherence to vascular endothelium and extracellular matrix proteins is a pre-requisite for neutrophil accumulation at sites of inflammation. In this study, equine neutrophil adherence to fibronectin and autologous serum-coated plastic in response to PAF, hrIL-8, hrC5a and PMA has been measured. In addition, the mechanisms involved have been investigated using monoclonal antibodies (MoAbs) against the beta2 integrin CD18. PAF and hrC5a caused similar, concentration dependent, increases in adherence to fibronectin- and serum-coated plastic (maximum responses 19 +/- 4% and 19 +/- 3% for PAF and 1...
Ferreira-Silva JC, Basto SRL, Moura MT, Rocha JM, Freitas Neto LM, Santos Filho JP, Silva Filho ML, Oliveira MAL.The work described here aimed to verify the efficiency of different extenders for cryopreservation of equine semen using sperm motility and acrosin activity as spermatic parameters. The semen was fractioned into two equal parts and resuspended in an 11% lactose solution in a 1:1 proportion, where it remained for 20 minutes at room temperature. The semen was centrifuged at 600 g for 10 minutes, and after the second centrifugation, each pellet received the freezing extender (Merck or Zorlesco) and was loaded into 4 mL straws. Each straw was placed in liquid nitrogen vapor steam for 15 minutes ...
