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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.
Furosemide, Patella vulgata beta-glucuronidase and drug analysis: conditions for enhancement of the TLC detection of apomorphine, butorphanol, hydromorphone, nalbuphine, oxymorphone and pentazocine in equine urine. We have investigated the action of five sources of beta-glucuronidase enzymes on the hydrolysis of glucuronides of apomorphine, butorphanol, hydromorphone, nalbuphine, oxymorphone and pentazocine in equine urine. For all glucuronides tested, Patella vulgata beta-glucuronidase yielded the largest thin layer chromatographic (TLC) spots. For oxymorphone, P. vulgata was the only treatment to yield detectable TLC spots under test parameters. For these six drugs, TLC spot size and chromatographic quality were compared between control horses and horses pretreated with furosemide four hours earlier. F...
Dehydroepiandrosterone synthesis by the fetal foal and its importance as an oestrogen precursor. The gonads of the fetal horse were found to be relatively devoid of 3 beta-hydroxysteroid dehydrogenase and other enzymes which metabolize dehydroepiandrosterone (DHA). In short-term in-vitro incubation experiments fetal liver converted DHA to the potential equilin precursor, 7 alpha-hydroxy DHA. DHA was converted to oestrone when incubated with extracts of horse placenta but 7 alpha-hydroxy DHA was not converted to equilin. Levels of DHA measured in peripheral blood of mares throughout pregnancy paralleled those of equilin and oestrone, and DHA concentrations fell rapidly after fetal gonadect...
Comparison of the interaction of equine LH and human chorionic gonadotrophin to equine testicular receptors. Human chorionic gonadotrophin (hCG) can be used to study horse luteinizing hormone (LH) receptors in stallion testicular tissue. hCG was more stable than horse LH during radioiodination when compared by their abilities to bind to testicular receptor sites. During incubation, neither hormone lost binding activity at 4 degrees C. Horse LH lost binding activity during incubation at 25 degrees C and both hormones lost binding activity at 37 degrees C. Both hormones bound to the same receptor sites which are specific for the hormones. The receptor sites were not degraded when incubated at 4 degrees...
Effect of PGF-2 alpha on LH receptors in the equine corpus luteum. As quantified by Scatchard analysis, a 27 000 g crude luteal membrane fraction contained a single population of unoccupied LH receptors characterized by high affinity, ka = 0.647 +/- 0.158 X 10(11) M-1 and low binding capacity, Rt = 4.91 +/- 0.78 X 10(-11) M/mg membrane fraction. Acceptable hormonal specificity, reversibility, saturability, high affinity and tissue specificity indicated that the binding protein was a physiological receptor. To ensure that the methods used for Scatchard analysis were valid, hCG was characterized for specific activity and maximum bindability, non-specific bindin...
Indirect hemagglutination test in equine infectious anemia. An indirect hemagglutination was developed for the diagnosis of equine infectious anemia using sheep red blood cells coated with group specific virus antigen which had been highly purified by affinity chromatography. The presence of indirect hemagglutination antibodies was demonstrated in horses with equine infectious anemia since the cells were specifically agglutinated by all the serum samples obtained from experimentally infected horses. Antibodies appeared within 35 days after inoculation, and development of which coincided well with that of precipitating and complement fixing antibodies. ...
Ovarian disorders: clinical and morphological observations in 30 mares. A five year prospective study of equine ovarian problems requiring surgical correction was undertaken at the Ontario Veterinary College. Thirty mares were studied, of which 14 had granulosa cell tumors, six were with anovulatory persistent follicular "structures", five had ovarian hemotoma, two presented ovarian hypoplasia and one each of ovarian dysgerminoma, teratoma and abscessation. The clinical signs manifested by the affected animals were varied. The affected ovaries were removed via flank or midline laparotomy or through colpotomy. Their morphology was studied and representative portion...
Preliminary characterization of equine interferons and their antiviral activities on bovine, ovine, and human cells. Equine dermal cells induced with poly I:C + DEAE-dextran produced low levels of interferon tentatively classified as equine interferon beta (EqIFN-beta). In contrast, dermal cells initially primed with EqIFN-beta and then superinduced with poly I:C + DEAE-dextran in the presence of cycloheximide and actinomycin D produced greater than 100-fold EqIFN-beta. Equine blood mononuclear cells induced with Newcastle disease virus and phytohemagglutinin produced high levels of interferons tentatively classified as equine interferon alpha (EqIFN-alpha) and equine interferon gamma (EqIFN-gamma), respecti...
Cibacron Blue-induced modification of neutral proteinase from horse blood leukocytes. The proteolytic activity of the elastase-like proteinase from granules of horse blood leukocytes is retained on a column of Cibacron Blue-Sepharose and can be eluted with 0.5 M KSCN. During this procedure its mol. wt. is reduced from 49000 to 30000 and isoelectric point is shifted towards higher pH. The inactive protein not adsorbed on Cibacron Blue-Sepharose is strongly acidic and shows a mol. wt. of 20000. Upon mixing this protein with the modified enzyme the native proteinase is reconstituted as shown by polyacrylamide gel electrophoresis at pH 8.3 and isoelectric focusing in a sucrose grad...
Indirect haemagglutination reaction with Sarcocystis dispersa antigen. A description is given of the preparation of antigen from Sarcocystis dispersa cystozoites and the procedure of the indirect haemagglutination test (IHA). The antibodies against this antigen were detected in experimentally infected mice from day 20 p.i. (1: 640). In the following weeks the antibody titres reached the value of 1: 40,960. The sera of pigs, sheep and horses spontaneously infected with other Sarcocystis species reacted with this antigen in low titres only. The bovine sera gave negative reactions even in cases when Sarcocystis cysts were present in the muscles of the examined anima...
Cytogenetic and DNA analyses of equine abortion. Although no major structural or numerical abnormalities were found in the karyotypes of 12 aborted equine fetuses, two unrelated abortuses each carried a large polymorphism for the amount of heterochromatin in chromosome 1. In both karyotypes this chromosome was shown to be larger than its homolog. To determine the nature of the extra DNA in these chromosomes, equine DNA was isolated and characterized by buoyant density analysis. Equine mainband DNA had a buoyant density in neutral CsCl of 1.699 g/cm3, while the highly repetitive (dG+dC)-rich fraction had a buoyant density of 1.715 g/cm3. A ra...
The density profile and cholesterol concentration of serum lipoproteins in domestic and laboratory animals. 1. By means of density gradient ultracentrifugation, the density profile of the serum lipoproteins was studied in 14 species of domestic and laboratory animals: the pig, chicken, rhesus monkey, rabbit, dog, horse, sheep, cat, mouse, goat, cow, guinea-pig, trout and rat. 2. The concentration of cholesterol in whole serum and the lipoprotein fractions of these animal species was also determined. 3. There were large differences in the density profile of the serum lipoproteins among the various animals studied and the results indicate that the density limits employed for human serum lipoproteins a...
Inhibition of sperm binding to porcine ova by antibodies to equine zonae pellucidae. Sera and follicular fluid from mares previously determined as having antibodies to the zona pellucida were exposed to porcine ovarian oocytes before insemination with boar spermatozoa in vitro. There was a dramatic decline in the number of spermatozoa bound to zonae compared to treatments with sera and follicular fluid from mares negative for zona antibodies. These data suggest that antibodies reactive with the zona pellucida may be responsible for reduced or even complete infertility in some mares. In a group of 50 randomly selected mares tested for antizona antibodies, 2 pregnant mares were ...
[Gangliosides of neural and extraneural tissues of various species of mammals]. The ganglioside patterns of the forebrain, cerebellum and brain stem from horse, donkey, mule and goat have been determined by thin-layer chromatography. GM1, GD1a, GD1b and GT1b are the four major brain gangliosides. N-acetylneuraminic acid as the predominant sialic acid (congruent to 97%) and traces of N-glycolyneuraminic acid were found. The four above mentioned major gangliosides were also found in the forebrain, cerebellum and brain stem of adult rats. This pattern is not modified in rats under stress situation (at 4 degrees C for 3 months). In other extraneural organs from rats such as l...
[Sensitivity and fidelity of the rabies laboratory diagnosis]. The author describes a method for evaluating the minimal number of diagnosis failures for each animal species (this diagnosis uses the Fluorescent Rabies Antibodies Test and mouse inoculation simultaneously). The percentage of well diagnosed rabid animals on total rabid ones is called sensibility of the diagnosis: it varies according to the species of animal examined: from 99.98% for the fox, to 98.61% for the horse. The percentage of errored negative diagnosis on total negative diagnosis is called infidelity of negative responses: it varies for each species according to the sensibility of the...
Limited trypsinolysis of porcine and equine colipases. Spectroscopic and kinetic studies. Porcine and equine colipases have been submitted to mild tryptic digestion. Proteolysis occurs at the Arg5-Gly6 bond with the loss of the N-terminal pentapeptide. Studies of native and trypsin-treated colipases by circular dichroism and laser chemically induced dynamic nuclear polarization indicate that proteolysis induces conformational changes in the region of the tyrosine cluster. Experiments in the presence of phospholipid provide further evidence showing that these residues are in or close to the region of the protein interacting with aggregated lipids. Kinetic studies of the reaction of ...
Coagulation studies f plasmas from healthy domesticated animals and persons. Optimal reaction conditions for the activated partial thromboplastin time (APTT), 1-stage prothrombin time (PT), and Russell's viper venom time were studied for pooled plasmas of horses, cattle, sheep, goats, swine, dogs, cats, and persons. Changes in CaCl2 and NaCl concentrations had significant effects on the APTT and PT reactions. The APTT was more sensitive than the PT to changes in CaCl2 concentration. The CaCl2 concentration recommended by the manufacturer for the APTT was suboptimal for some of plasmas of domesticated animals in ths study. Infusorial earth (Celite, activator) concentrat...
Gas/liquid chromatographic analysis of pemoline in biological fluids using electron capture detection. An analytical gas/liquid chromatographic (GLC) protocol is described for the quantitation of pemoline in biological fluids of the horse. Plasma samples containing known quantities of pemoline and its analog as an internal standard (IS) were deproteinized with 5-sulfosalicylic acid, heated at 80 degree C, and centrifuged. 5-Phenyl-2,4-oxazolidinedione, the hydrolytic product of pemoline in acid medium, was extracted with dichloromethane (DCM). The organic layer was in turn re-extracted with 1% NaHCO3. The aqueous layer was acidified with HCI, and re-extracted with DCM, which was evaporated to d...
Diagnostic methods in infectious respiratory disease. For laboratory diagnosis of respiratory disease it is of overwhelming importance that the specimens taken are adequate, taken from the correct site and at the correct time. The lower regions of the respiratory tract are particularly difficult to sample but are more likely to yield the causative agent of a pneumonia. Infections involving the upper respiratory tract are much easier to sample and appropriate aspiration apparatus can be used. Consideration must be given to the timing of sample collection in relation to the life cycle of the causative micro-organism. Sampling of several animals is ...
Serologic and molecular comparisons of several equine herpesvirus type 1 strains. The molecular and serologic relatedness of 2 recent respiratory tract isolates of equine herpesvirus type 1, designated T1 and T2, were compared with the Army 183, Kentucky-A hamster-adapted (KyA-ha), and L-M cell-adapted (KyA-LM) strains. Electrophoresis in polyacrylamide gels revealed differences in virion structural proteins among 4 purified strains. Seven envelope glycoproteins (molecular weight of 93,000, 65,000, 62,000, 60,000, 36,000, 20,000, and 18,000) corresponding to virion proteins 13, 16, 17, 18, 23, 25, and 26a, respectively, found in both the Army 183 and KyA-ha strains had slig...
Analysis of the genome of equine herpesvirus type 1: arrangement of cleavage sites for restriction endonucleases EcoRI, BglII and BamHI. The genome of an Australian isolate of equine herpesvirus type 1 (equine abortion virus) has been analysed using the restriction endonucleases EcoRI, BglII and BamHI, and a physical map constructed. Terminal fragments were identified by exonuclease treatments, and linkage of fragments was deduced by a combination of single- and double-digest experiments and cross-blot hybridizations. The genome has a mol. wt. of 100 x 10(6) and is comprised of a short unique region bounded by repetitive sequences, which is present in both orientations in approximately equal amounts in the DNA population, and a...
Plasma oxytocin concentrations in cyclic mares and sexually aroused stallions. An experiment was conducted to measure plasma oxytocin concentrations at 4 different stages of the estrous cycle in 11 pony mares. Plasma oxytocin concentrations (muU/ml +/- SE) were found to be higher (P<.01) on day 2 of estrous (39.8 +/- 12.5) and day 5 post-ovulation (33.1 +/- 12.0) than on day 10 (2.3 +/- 1.6) and day 15 post-ovulation (6.8 +/- 4.1). A second experiment was conducted to measure jugular plasma oxytocin concentrations before and after sexual arousal in six pony stallions. Oxytocin concentrations (muU/ml +/- SE) were higher (P<0.06) after sexual arousal (50.5 +/- 8.9) than be...
Concentrations of progesterone, 17 alpha-hydroxyprogesterone and 20 alpha-dihydroprogesterone in the plasma of mares during pregnancy and at parturition. Plasma concentrations of progesterone and 17 alpha-hydroxyprogesterone were high in the 2nd and 3rd months of gestation, but 20 alpha-dihydroprogesterone increased from a level of 2 ng/ml, during the first 3 months, to 10-15 ng/ml during months 5-10, to reach 80-120 ng/ml during the last 30 days before foaling.
Collection and cultivation in vitro of equine mammary macrophages. Equine macrophages were obtained from female Shetland ponies by injection of Escherichia coli lipopolysaccharide through the lactiferous ducts of the mammary gland. After 6 to 11 days, balanced salt solution was injected into the mammary gland to wash out accumulated cells. Harvested cells contained a mixture of macrophages, lymphocytes, and neutrophils, with the majority of the cells of mononuclear type. In culture, cells adherent after 24 hours were characterized as macrophages by morphologic features, nonspecific esterase staining, and by the presence of complement and immunoglobulin recept...
Molecular cloning of equine herpesvirus type 1 DNA: analysis of standard and defective viral genomes and viral sequences in oncogenically transformed cells. Genomic DNA sequences of equine herpesvirus type 1 (EHV-1) have been cloned as BamHI and EcoRI restriction fragments into the plasmid pBR322 and propagated in Escherichia coli. With the exception of two EcoRI restriction fragments that reside in the S region of the viral genome, all of the cloned fragments demonstrated the same electrophoretic mobilities, restriction cleavage sites, and blot-hybridization patterns as did the parent fragments produced by BamHI or EcoRI digestion of virion DNA. The EcoRI J fragment and the BamHI E fragment of the L-region terminus were cloned after the addition ...
