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.
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...
Crews HM, Dean JR, Ebdon L, Massey RC.The speciation of cadmium in retail pig kidney has been examined by size-exclusion chromatography (SEC) coupled directly to inductively coupled plasma mass spectrometry (ICP-MS). Approximately 35% of the cadmium from uncooked kidney was soluble after aqueous extraction at pH 8 and SEC - ICP-MS revealed three discrete peaks whose retention times corresponded to estimated relative molecular masses of 1.2 x 10(6), 7.0 x 10(4) and 6 x 10(3)-9 x 10(3). In the cooked kidney, 35% of the Cd was soluble and was all associated with a peak of a relative molecular mass (Mr) of 6 x 10(3)-9 x 10(3). After s...
Sletten K, Husebekk A, Husby G.The complete amino acid sequence of equine serum amyloid A (SAA) was elucidated. The protein consists of 110 amino acid residues and contains an 8-amino acid residue insertion tentatively located between positions 69 and 70, as compared with human SAA. Microheterogeneities were detected at positions 16, 44, and 59, compatible with the existence of more than one SAA gene in the horse. This corresponds to the situation in man and mouse. Pronounced homology with SAA from man and several animal species was observed, thus confirming the conserved structure of this acute phase reactant and apoprotei...
Bonnaire Y, Plou P, Pages N, Boudene C, Jouany JM.A highly sensitive procedure for GC/MS determination of etorphine in horse urine is described. This assay provides both specificity and reliability and is particularly well suited for the confirmation of radioimmunoassay screening procedures usually used for etorphine. After solvent extraction and purifications, the etorphine is characterized as a pentafluoroacetic derivative (PFAA) by using mass fragmentography. The detection limit is 0.1 ng/mL in urine; the coefficient of variation of the estimations is 10.9%. The procedure has been validated after on-field administration of 5 to 90 microgra...
Singh AK, Ashraf M, Granley K, Mishra U, Rao MM, Gordon B.A simple and reproducible column (Clean Screen-DAU, copolymeric bonded-phase silica column) extraction procedure has been described for the screening and confirmation of drugs in horse urine. The recovery of drugs by the column extraction was better than or comparable to the recovery by the liquid-liquid extraction, which is commonly used in the equine analytical laboratories. The column extraction provided broad coverage of drugs, separated extracts into three fractions (acidic/neutral, steroids, basic), produced a cleaner extract, and eliminated the need for special liquid-liquid extraction ...
Bauminger ER, Harrison PM, Nowik I, Treffry A.Ferritin stores iron within a hollow protein shell as a polynuclear Fe(III) hydrous oxide core. Although iron uptake into ferritin has been studied previously, the early stages in the creation of the core need to be clarified. These are dealt with in this paper by using Mössbauer spectroscopy, a technique that enables several types of Fe(II) and Fe(III) to be distinguished. Systematic Mössbauer studies were performed on samples prepared by adding 57Fe(II) atoms to apoferritin as a function of pH (5.6-7.0), n [the number of Fe/molecule (4-480)], and tf (the time the samples were held at room ...
Kano I, Nagai F, Satoh K, Ushiyama K, Nakao T, Kano K.Genomic DNA for Na,K-ATPase alpha 1 subunit was obtained from libraries of horse kidney genomic DNA in Charon 4A and in EMBL3 bacteriophages by screening with the full sized cDNA probe of the alpha 1 subunit of rat Na,K-ATPase as probe. The gene spans 30 kb and consists of 23 exons and 22 intervening sequences. Intron-exon boundaries were analyzed. The protein-coding nucleotide sequence encodes 1016 amino acids with an Mr of 112,264. The putative amino acid sequence of horse alpha 1 is 96-97% homologous to those of other mammalian species.
Clabough DL, Conboy HS, Roberts MC.Using radial immunodiffusion as a standard, 4 screening techniques for detection of failure of passive transfer in equine neonates were compared for sensitivity, specificity, positive and negative predictive values, efficiency, and cost. The techniques compared were latex agglutination test, membrane filter ELISA, dipstick ELISA, and glutaraldehyde coagulation (GC) test. Test results of 50 serum samples from foals 24 to 60 hours old revealed consistently highest accuracy in the GC test at IgG concentrations of 400 and 800 mg/dl, and lowest cost per test, using the GC test. Two hundred fifty-th...
Li JK, Moloney BK, Shupe JL, Gardner EJ, Leone NC, Elsner Y.Genomic DNA polymorphisms obtained by restriction fragment-length polymorphism from healthy horses and horses with hereditary multiple exostoses were analyzed. These DNA were digested by 12 restriction enzymes and were hybridized against 6 isotopically labeled oncogene probes. Hybridization was not detected with the viral oncogene, v-ras, which indicated this oncogene was absent in the equine genome. Oncogenes (c-raf-1, c-fes, c-myb, c-myc, and c-sis) were present and had similar hybridization patterns and signal intensities in DNA from healthy horses and horses with hereditary multiple exosto...
Althouse GC, Seager SW, Varner DD, Webb GW.An experiment was conducted to evaluate three commercially available test kits, the Azostix, Multistix and Uric-acid test, for the detection of urine in the equine ejaculate. Azostix, which tests for urea nitrogen, consistently detected urine in the equine ejaculate. Urine contamination was evident when a color change occurred in the reagent pad, going from yellow to green after 10 sec of exposure. The sensitivity of Azostix to urea nitrogen in contaminated samples was 39 mg/dl. The Multistix test kit also successfully detected urine in semen. In the Multistix nitrite pad the color changed fro...
Puigdemont A, Arboix M, Gaspari F, Bortolotti A, Bonati M.The protein binding of propafenone in vitro was assessed in plasma of mouse, rat, rabbit, dog, sheep, man, cow, and horse at two concentration levels. In all species and at both concentrations propafenone was found highly bound (86-99%) to plasma proteins. No significant relationship was found between free propafenone and the plasma protein fractions. A concentration-dependency was seen in plasma of mouse, sheep, man, and horse, in which the free fraction of propafenone became larger on raising the concentration. Qualitative and quantitative differences were observed in the protein plasma prof...
Saudek V, Boyd J, Williams RJ, Stefani M, Ramponi G.A complete range of two-dimensional NMR experiments was used for the assignment of the 1H-NMR spectrum of horse muscle acylphosphatase. Firstly the spin systems of some easily identifiable amino acid side chains were assigned. These side chains involved all the aromatic residues and all the leucine, valine, isoleucine, threonine, alanine, proline as well as some of the glycine residues. Analysis of nuclear Overhauser enhancement spectra in our previous work had identified the sequential and long-range patterns characteristics for secondary structure elements. This result had also provided the ...
Cook RF, Gann SJ, Mumford JA.An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of serum antibodies to equine arteritis virus (EAV). Results from this assay produced a good correlation with results from virus neutralisation tests in horses which had not been regularly vaccinated with commercially available mammalian tissue culture-derived viral vaccines. Vaccination of some horses with tissue culture-derived vaccines induced the formation of antibodies to bovine serum. These antibodies reacted with the bovine protein contaminants in the EAV ELISA antigen, producing false-positive results. Non-vir...
Archambault D, Wang ZM, Lacal JC, Gazit A, Yaniv A, Dahlberg JE, Tronick SR.To provide more sensitive and convenient methods for the detection of equine infectious anemia virus (EIAV), we developed an enzyme-linked immunosorbent assay (ELISA) employing the EIAV gag precursor (Pr55gag) produced by using recombinant DNA techniques. The antigenic reactivity of the recombinant EIAV Pr55gag was found to be equivalent to that of the virion p24gag and elicited high-titered antiserum in rabbits. When a large number of horse sera were analyzed for the presence of antibodies to EIAV by this ELISA, a radioimmunoassay for EIAV p15gag, or the standard agar gel immunodiffusion test...
Fessler JF, Bottoms GD, Coppoc GL, Gimarc S, Latshaw HS, Noble JK.Endotoxin (LPS) was quantitated in experimental subjects and in horses with naturally occurring gastrointestinal strangulation obstruction and/or septicaemic diseases to establish the fate of LPS and the clinical usefulness of the Limulus amoebocyte lysate (LAL) assay. The assay was validated for sensitivity (10 pg/ml), recovery (90 to 106 per cent), intra-assay precision (CV = 5.5 per cent) inter-assay precision (CV = 11 per cent), and stability of diluted, heat treated, frozen samples (at least 90 days). Plasma concentrations of LPS after sublethal (3 micrograms/kg) jugular or portal vein bo...
Saudek V, Wormald MR, Williams RJ, Boyd J, Stefani M, Ramponi G.Nuclear magnetic resonance spectra of acylphosphatase were searched for signs of beta-structure, i.e. characteristic nuclear Overhauser enhancement patterns displayed in the two-dimensional spectra, typical chemical shifts, coupling constants and slow 2H-H exchange. The results provided identification of the main-chain resonances of amino acid residues involved in the beta-structure. The full sequential assignment of this region was gained by identification of some amino acid spin systems and their alignment with the primary sequence. The assignment of the side-chains was virtually completed s...
Lieto LD, Cothran EG.A cDNA library was built using RNA extracted from the skin tissue of an adult horse. The library was primed with oligo (dT) and sequences were directionally inserted in order to produce an expression library. The library has 5.8X 10(5) plaque forming units with 99.6% recombinant phage. The average insert size is 1.3 Kbp. Three hundred and thirteen expressed sequence tags (ESTs) were generated from sequencing of the 5 prime end of randomly selected skin cDNA clones. The ESTs were sequenced on an ABI 377 using Big-Dye chemistry. A similarity search was performed on each EST using the NCBI non-re...
Hatzipanagiotou A, Rieland E, Enbergs H.It was the aim of this project to investigate the changes of the lysozyme activity in the milk of mares during the lactation period. Further on the influence of race, date of conception and foaling, age and number of lactations on the lysozyme activities in milk was analysed. Milk samples were collected from 44 mares (trotters, warmblood, quarter horses) from eight farms between the 1st and 90th day p. p. The activity of the lysozyme was measured by a turbidometric method. Summarizing the following results are obtained: Lysozyme activities in mare milk of the 1st and 3rd day p. p. were higher ...
Richardson DW, Dodge GR.To clone the entire coding sequence of equine matrix metalloproteinase-3 (MMP-3, stromelysin) and tissue inhibitor of metalloproteinase-1 (TIMP-1) and compare their nucleotide and amino acid sequences with those of MMP-3 and TIMP-1 from other species. Methods: Articular cartilage harvested from the joints of 4 foals, 2 yearlings, and 3 adult horses. Methods: A cDNA library was constructed from mRNA extracted from equine chondrocytes. The library was screened and clones selected that contained the cDNA for MMP-3 and TIMP-1. The cDNA was sequenced and the nucleotide and deduced amino acid sequen...
Moodley Y, Baumgarten I, Harley EH.We investigated the applicability of microsatellite primers, designed in horses, for use in plains and mountain zebras. Fifteen of the 20 tested horse-isolated primer pairs reliably amplified polymorphic loci in two wild equid species. We used this information to assess whether levels of genetic variation and repeat size differed in species from which microsatellites were isolated and in closely related target species. Target equid species exhibited similar levels of genetic variation to the horse, the species from which primers were originally isolated. We show that ascertainment bias results...
Hagedorn HW, Schulz R.The use of diuretics in horses subject to doping control is prohibited. Thus, a sensitive screening procedure is required to identify the chemically different diuretics. We communicate here a method to detect three commonly employed acidic diuretics: bumetanide, ethacrynic acid, and furosemide. A liquid-liquid extraction on Extrelut 3 was performed at weak acidic and basic conditions using ethyl acetate as organic solvent. For analysis by GC, the diuretics were methylated on-column in the presence of MSTFA/TMAH, avoiding the commonly employed highly toxic derivatizing agent methyl iodide. For ...
Younkin JT, Santschi EM, Kukanich B, Lubbers BV, Warner M.Penicillin is administered intravenously (IV) or intramuscularly (IM) to horses for the prevention and treatment of infections, and both routes have disadvantages. To minimize these shortcomings, a 24-hr hybrid administration protocol (HPP) was developed. Our objective was to determine penicillin plasma concentrations in horses administered via HPP. Venous blood was collected from seven healthy horses administered IV potassium penicillin G at 0 and 6 hr and IM procaine penicillin G at 12 hr. Blood was collected at 2-hr intervals from 0 to 20 hr and at 24 hr. Plasma penicillin concentration...
Province CA, Amann RP, Pickett BW, Squires EL.Two experiments were conducted to evaluate the effects of six extenders and three glycerol levels on the motility of sperm stored at 5 degrees C. Using a split-ejaculated design, semen from 10 dogs and 12 stallions was extended with egg-yolk-tris (EYT), egg-yolk-bicarbonate (EGB), Beltsville F-3 (BF-3), Cornell University (CUE), caprogen (CAP) and heated skim milk (SM) extenders. After cooling to 5 degrees C, additional extender containing 0% to 12% glycerol was added to provide a final concentration of 0%, 3% or 6% glycerol. Regardless of glycerol level, a higher (P<0.05) percentage of can...
Jungeblut CW.1. A flocculation reaction has been described which occurs between alcoholic extracts of pneumococci and antipneumococcus serum. 2. The reaction appears to be species-specific. It is not strictly type-specific, as slight or moderate cross-reactions occurred between Type I serums and Type II and Type III extracts. 3. The flocculating power of the serum from five horses undergoing immunization with pneumococcus, Type I, did not develop to any extent before the end of the 4th or 5th month. 4. In the case of two of these horses in which it was possible to carry out parallel tests on a larger numbe...
Whalley JM, Love DN, Tewari D, Field HJ.A series of recombinant baculoviruses containing genes for glycoproteins C, D, H and L of equine herpesvirus 1 (EHV-1) have been constructed, and the EHV-1 products characterised by gel electrophoresis and immunoblotting. The EHV-1 glycoproteins expressed in insect cells were similar but not identical in apparent sizes to those expressed in EHV-1 infected mammalian cells. Each of the EHV-1 products was recognised by convalescent equine sera, indicating that they were all targets for an equine immune response. Mice immunised with baculovirus-expressed EHV-1 gD and gC acquired an enhanced abilit...
Hernández-Jáuregui P, Sosa A, González-Angulo A.Equine spermatozoa were obtained from ejaculates of young stallions. The seminal plasma was removed and the sperm pellets washed three times with 0-15 M-NaCl solution before final centrifugation at 4500 g for 15 min. The pellets were fixed in a mixture of 2-5% glutaraldehyde in 0-1 M-cacodylate buffer, pH 7-4, with 0-5% Alcian blue and post-fixed in 1% osmium tetroxide with 1% lanthanum nitrate; other samples were treated with ruthenium red. All samples were dehydrated in ascending concentrations of ethanol, embedded in araldite and thin sections examined in an electron microscope. Electron de...
Wahdati A, Lindner A, Sommer H.The influence of temperature and duration of storage on glutathione peroxidase (GSH-Px) activity in plasma and whole blood samples of horses was investigated. Furthermore the relationships between the GSH-Px activities in plasma and whole blood as well as the GSH-Px values related to hematocrit and the hemoglobin content of the blood samples of 93 different horses were calculated. At 20-22 degrees C, the GSH-Px activity in plasma and whole blood samples remained stable over at least three days while it was reduced by 38% and 65% after 2 and 3 days, if the whole blood samples were kept stored a...
Van Huffel XM, Varner DD, Hinrichs K, Garcia MC, Strzemienski PJ, Kenney RM.A photomicrographic method for evaluation of stallion spermatozoal motility was developed, and spermatozoal image and velocity characteristics were defined. The photomicrographic method was compared with visual estimation of motility in the same semen sample over time. Using photomicrography, velocities and percentages of individual spermatozoal image characteristics were determined. Although there was a high correlation between results of the 2 methods, results of the photomicrographic method were more repeatable than were those of the visual method.
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...
Ramponi G, Manao G, Camici G, White GF.It has been shown that horse muscle acylphosphatase is inhibited by pyridoxal 5'-phosphate and that the inhibition is pH dependent, reversible and competitive with respect to substrate binding. Spectral analysis on the EI complex demonstrates the presence of a Schiff base. Reduction of the pyridoxal 5'-phosphate-inhibited enzyme with sodium borohydride, followed by amino acid analysis, produces a diminution of the free lysine peak and the appearance of a new peak corresponding to epsilon-pyridoxyllysine. The results suggest that there is at least one NH2-lysyl residue of horse muscle acylphosp...
Current drug metabolismDecember 19, 2020
Volume 22, Issue 3 215-223 doi: 10.2174/1389200221666201217141025
Hu SX, Ernst K, Benner CP, Feenstra KL.Pharmacokinetic and pharmacodynamic assessment of ester-containing drugs can be impacted by hydrolysis of the drugs in plasma samples post blood collection. The impact is different in the plasma of different species. Objective: This study evaluated the stability of a prodrug, ketoprofen methylester (KME), in commercially purchased and freshly collected plasma of mouse, rat, dog, cat, pig, sheep, cattle and horse. Methods: KME hydrolysis was determined following its incubation in commercially purchased and freshly collected plasma of those species. Different esterase inhibitors were evaluated f...
Fock U, Jockusch BM, Schubert WD, Hinssen H.The actin-binding protein gelsolin is highly conserved in vertebrates and exists in two isoforms, a cytoplasmic and an extracellular variant, generated by alternative splicing. In mammals, these isoforms differ only by an N-terminal extension in plasma gelsolin, a short sequence of up to 25 amino acids. Cells and tissues may contain both variants, as plasma gelsolin is secreted by many cell types. The tertiary structure of equine plasma gelsolin has been elucidated, but without any information on the N-terminal extension. In this paper, we present topographical data on the N-terminal extension...
Ho EN, Leung DK, Leung GN, Wan TS, Wong HN, Xu X, Yeung JH.Mesterolone (1alpha-methyl-5alpha-androstan-17beta-ol-3-one) is a synthetic anabolic androgenic steroid (AAS) with reported abuses in human sports. As for other AAS, mesterolone is also a potential doping agent in equine sports. Metabolic studies on mesterolone have been reported for humans, whereas little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of mesterolone in racehorses with an objective to identify the most appropriate target metabolites for detecting mesterolone administration. In vitro biotransformation st...
Blackall PJ, Christensen JP, Bisgaard M.The objective of this work was to examine the diversity within Australian isolates of Actinobacillus equuli and related organisms by the genotypic method of ribotyping. Methods: Ribotyping, performed using the enzyme HaeIII, was used to examine the diversity in 12 field isolates of A equuli (five being capable of fermenting L-arabinose), one field isolate of Pasteurella caballi and two unclassifiable field isolates. Isolates were obtained from Australian horses, except for three isolates of A equuli (one L-arabinose positive and two L-arabinose negative) which were obtained from horses and a p...
Lechmann J, Schoster A, Ernstberger M, Fouché N, Fraefel C, Bachofen C.Equid alphaherpesvirus 1 (EHV-1) infections can have a major impact on the horse industry and equine welfare by causing abortion or respiratory or neurologic disease. A single nucleotide polymorphism (A→G) in open reading frame (ORF) 30, encoding the catalytic subunit of the DNA polymerase, has been shown to be a strong predictive marker for neuropathogenicity. Given that a previously established real-time PCR (rtPCR) protocol yielded unsatisfactory results concerning determination of the EHV-1 genotype, we developed and evaluated a new conventional PCR protocol enabling identification of th...
Ghanem YM, Ibrahim el-SM, Yamada S, Matsumura T, Osterrieder N, Yamaguchi T, Fukushi H.The pathogenicities of RacL11 and Kentucky D strains of equine herpesvirus 1 in the hamster infection model are different from those of Ab4p and the Japanese isolates. Virus genome restriction fragment length polymorphism analysis and sequence comparison of an intergenic region, glycoproteins and tegument genes showed higher conservation but with some strain-specific differences. These results indicate that point nucleotide differences in RacL11 and Kentucky D might be responsible for their pathogenicity in rodent models.
Puppione DL, Fischer WH, Park M, Whitelegge JP, Schumaker VN, Golfeiz S, MacDonald MH.Apolipoprotein A-II, the second major apolipoprotein of human HDL, also has been observed in a variety of mammals; however, it is either present in trace amounts or absent in other mammals. In humans and chimpanzee, and probably in other great apes, apoA-II with a cysteine at residue 6 is able to form a homodimer. In other primates as well as other mammals, apoA-II, lacking a cysteine residue, is monomeric. However, horse HDL has been reported to contain dimeric apoA-II that following reduction forms monomers. In this report, we extend these observations by reporting on the first complete sequ...
Guan F, Robinson MA, Soma LR.Etanercept is a protein-based medication for the treatment of human patients with rheumatoid arthritis and other autoimmune-based diseases; its pharmacological action is to inhibit and antagonize tumour necrosis factor alpha. Etanercept was rumoured to be used in horse racing in North America. To detect such use, the aim of this study was to develop a liquid chromatography-mass spectrometry (LC-MS) method for confirmation of etanercept in equine plasma. Etanercept was extracted from plasma by anti-human IgG antibody linked to magnetic beads. The analyte was reduced and alkylated, and then dige...
Van Der Vekens N, van Dievoet MA, De Puydt H, Decloedt A, Ven S, De Clercq D, Deprez P, van Loon G.Although cardiac troponin T (cTnT) assays have been used to detect myocardial damage in horses, a cTnT assay has not been analytically validated, to our knowledge. The aims of this study were to estimate the precision of a high-sensitivity cTnT assay in horses and determine the effect of hemolysis on the measured cTnT concentration. Serum samples from horses were mixed in 3 different pools. Pool 1 consisted of samples from 3 healthy horses, pool 2 from 6 horses with heart failure or atypical myopathy, and pool 3 from 10 horses with atypical myopathy. The within- and between-run coefficients of...
Levionnois OL, Mevissen M, Thormann W, Spadavecchia C.The objective of this study was to assess a pharmacokinetic algorithm to predict ketamine plasma concentration and drive a target-controlled infusion (TCI) in ponies. Firstly, the algorithm was used to simulate the course of ketamine enantiomers plasma concentrations after the administration of an intravenous bolus in six ponies based on individual pharmacokinetic parameters obtained from a previous experiment. Using the same pharmacokinetic parameters, a TCI of S-ketamine was then performed over 120 min to maintain a concentration of 1 microg/mL in plasma. The actual plasma concentrations of ...
Brankston G, Rossi TM, O'Sullivan TL, Greer AL.This retrospective study describes testing patterns and the incidence of subsp. in Ontario to assess the utility of laboratory data for surveillance purposes. Laboratory records for equine infectious disease test submissions were extracted from the Animal Health Laboratory (AHL) at the University of Guelph for the years 2008 to 2018. Yearly and seasonal trends in testing and the proportion of tests that returned positive results were assessed. The number of samples submitted for testing decreased over the 11-year period (odds ratio = 0.96, 95% confidence interval: 0.92 to 0.999; = 0.04). ...
Delbeke FT, Debackere M.A high performance liquid chromatographic method to measure plasma and urine fenoprofen levels in equine biofluids is described. Liquid-liquid extraction with diethylether was used to isolate the drug from plasma and urine. The accuracy and reproducibility of the method were within acceptable limits over the concentration range 0-10 micrograms/mL and 0-20 micrograms/mL respectively from plasma and urine. Detection limits were 0.05 microgram/mL (2 mL plasma) and 0.2 microgram/mL (0.5 mL urine). This procedure was applied to ascertain the pharmacokinetics of a 3 g dose of fenoprofen calcium in a...
Dumasia MC, Houghton E.1. Isomers of 3,17-dihydroxyandrostan-16-one, 3,16-dihydroxyandrostan-17-one and androstane-3,16,17-triol have been identified as urinary metabolites of testosterone in the horse. 2. Following XAD-2 extraction of urine samples, Sephadex LH-20 chromatography was used to separate the extract into conjugate groups. Metabolites obtained after hydrolysis of the conjugates have been investigated by g.l.c.-mass spectrometry. 3. Testosterone, 3,17-dihydroxyandrostan-16-one and 3,16-dihydroxyandrostan-17-one were found only in the sulphate fraction. 5 alpha-Androstane-3 beta,17 beta-diol, and two isome...
Kong N, Yi R, Zhao S, Sandhu J, Lam G, Loganathan D, Morrissey B.A sensitive hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method was developed and validated for the simultaneous detection and quantification of etilefrine and oxilofrine in equine blood plasma and urine. The method is highly sensitive and specific with good precision and accuracy. In plasma the limit of detection and limit of quantification are 0.03 and 0.1 ng/mL, respectively, for both analytes. In urine the limit of detection and limit of quantification are 0.3 and 1 ng/mL, respectively, for both analytes. The suitability of the method for doping con...
Bannai H, Nemoto M, Tsujimura K, Yamanaka T, Kondo T, Matsumura T.Non-specific hemolysis has often been observed during complement-fixation (CF) tests for equine herpesvirus type-1 (EHV-1), even when the sera have virus-specific CF antibodies. This phenomenon has also been reported in CF tests for various infectious diseases of swine. We found that the sera from 22 of 85 field horses (25.9%) showed non-specific hemolysis during conventional CF testing for EHV-1. Because pretreatment of swine sera with potassium periodate (KIO4) improves the CF test for swine influenza, we applied this method to horse sera. As we expected, horse sera treated with KIO4 did not...
Siegel RC, Cathou RE.Complexes of IgM equine anti-dansyl antibodies and different dansyl substituted carriers were tested for their ability to fix complement (C). Only dansyl92-Ficoll and dansyl12-poly-L-lysine were found to be effective. Dansyl13-bovine serum albumin, dansyl127-keyhole limpet hemocyanin, and reduced and alkylated dansyl10-ribonuclease were all ineffective. Lack of C fixation by the dansyl-ribonuclease was not due to lack of antibody-antigen complex formation, since binding at the concentrations employed for C fixation was established. However, in contrast, polymerized dansyl-ribonuclease (polydis...
