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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.
Occurrence and distribution of 5-S-cysteinyl derivatives of dopamine, dopa and dopac in the brains of eight mammalian species. The 5-S-cysteinyl derivatives of dopamine, dopa (3,4-dihydroxyphenylalanine) and dopac (3,4-dihydroxyphenylacetic acid) were synthesized and used as reference compounds in high performance liquid chromatography analyses of extracts from various brain regions of eight mammalian species. All three metabolites were detected in the brains of all the species studied. The regional distribution of the metabolites was similar to that of dopamine; the metabolite concentrations ranged from less than 0.1 percent to more than 1 percent of the dopamine level, the highest ratios generally being found in sub...
Isolation and characterization of three forms of luteinizing hormone from the pituitary gland of the horse. Three isoforms of equine luteinizing hormone (eLH-A, eLH-B and eLH-C) have been isolated from horse pituitary glands. Separation was achieved on the basis of charge heterogeneity by ion-exchange chromatography. These charge differences were apparent after final purification, as determined by electrophoretic mobility on polyacrylamide disc gels (RF = 0.14, 0.19 and 0.26 for eLH-A, -B and -C, respectively). Apparent size differences were also noted between the isohormones by gel filtration on Sephadex G-100. Ve/Vo ratios for eLH-A, -B and -C were 1.72, 1.54 and 1.47, respectively. All 3 isoforms...
In vitro interference between equine herpesvirus types 1 and 2. Interference between equine herpesvirus types 1 (EHV-1) and 2 (EHV-2) was studied in equine dermis (ED) monolayer cell cultures and equine lymphocyte cultures. Cell cultures were infected with EHV-2, and after a short incubation period, the cultures were superinfected with EHV-1. At various intervals, different measurements of EHV-1 expression in dually infected cultures, compared with those in cultures infected with EHV-1 alone, were studied. In dually infected ED cell cultures, the EHV-1 cytopathic effect, EHV-1 titer, and EHV-1 enzyme-linked immunosorbent assay antigen titer were maximally ...
Direct demonstration of intrinsic follicle-stimulating hormone receptor-binding activity in acid-treated equine luteinizing hormone. After dissociating equine gonadotropins as a function of time at pH 3, we examined them by radioligand assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis under nondissociating conditions (low, 0.1% SDS). Equine follicle-stimulating hormone (FSH) rapidly lost its receptor-binding activity, and low SDS-polyacrylamide gels demonstrated dissociation into subunits. Maximum dissociation occurred after 20-30 min of pH 3 incubation. Equine luteinizing hormone (LH), however, retained most biologic activity and was largely intact after 72 h of pH 3 incubation. Dose-response curves of ac...
Two-dimensional 1H NMR studies of cytochrome c: assignment of the N-terminal helix. The 1H resonances of 11 sequential amino acids in the N-terminal helix of horse ferrocytochrome c were studied by two-dimensional nuclear magnetic resonance techniques. All the main-chain protons from Lys-5 through Ala-15 and many of the side-chain protons were assigned. J-Correlated spectroscopy (COSY) was used to distinguish protons on neighboring bonds and to recognize amino acid types. Nuclear Overhauser effect spectroscopy (NOESY) was used to define spatially contiguous protons and to determine amino acid sequence neighbors. The relayed coherence experiment (relay COSY) was used to resolv...
Molecular pathogenesis of equine coital exanthema: temperature-sensitive function(s) in cells infected with equine herpesviruses. Preliminary experiments have revealed that several laboratory and wild-type strains of the equine herpesvirus (EHV) triad were temperature-sensitive for growth when assayed at 39 degrees C. The efficiencies of plating (EOP) observed were 10(-2) for both EHV 1 and 2, and 1 X 10(-6) for EHV 3. The EOPs were determined by plaque assays which compared titrations at 34 degrees C and 39 degrees C on equine fetal dermal fibroblast cells. Growth yield experiments, assayed at 34 degrees C, reflected those EOP's, but did not indicate any difference in yields when infected cultures were incubated at 34 d...
A study on the role of evolutionarily invariant leucine 32 of cytochrome c. To investigate the role of evolutionarily invariant leucine 32 of horse cytochrome c, analogs of residues 28-38, (28-38), each containing a substituted amino acid at positions 32 or 35 were synthesized using Merrifield's method. Position 35 is leucine in horse cytochrome c but replaced by nonpolar amino acids in some species. The ability of the analogs to bind to the two-fragment complex of ferri- or ferro heme fragment (1-25)H and apofragment (39-104) was measured using gel filtration and equilibrium dialysis. Replacement of leucine 32 with isoleucine, for example, increased the dissociation ...
Continuous-flow centrifugation hemapheresis in the horse. In a continuous-flow centrifugation apheresis technique adapted for blood-component separation and collection in horses, hydroxyethyl starch was not required for erythrocyte sedimentation. The efficacy and separation characteristics of whole blood from 10 horses were evaluated at various gravitational forces (700 to 1,500 rpm), using a constant withdrawal rate (100 ml/min). Maximum leukocyte collection occurred at 700 rpm (P less than 0.01), and optimal neutrophil collection occurred at 700 to 750 rpm (P less than 0.01). Although neutrophil counts decreased and lymphocyte counts remained const...
Assessment of spermatozoal function using dual fluorescent staining and flow cytometric analyses. Spermatozoa from bulls, boars, dogs, horses, mice, and men were examined using a fluorogenic stain consisting of the membrane-permeant substrate carboxyfluorescin diacetate (CFDA) and the relatively membrane-impermeant nuclear stain propidium iodide (PI). Three distinct populations of spermatozoa were discernible in samples from each species upon microscopic examination. Individual spermatozoa, presumed to be viable because of their motility, retained products of the fluorescein chromophore throughout the cell. A second population of spermatozoa in which the nuclei stained red with PI retained...
Cytolytic activity of liposomes containing stearylamine. In order to develop the cytotoxic liposome, the cytolytic effect of polycationic liposome was examined. Upon incubation of the stearylamine-containing liposome (stearylamine-liposome) with rabbit erythrocyte, a significant extent of hemolysis was observed. Hemolytic activity of the liposome depends on the amount of stearylamine in the liposome membrane. The plots of the initial rate of hemolysis versus the concentration of stearylamine-liposome showed a sigmoidal curve, suggesting that stearylamine-liposomes act cooperatively on the erythrocyte membrane. Hemolytic activity of stearylamine-lipo...
Joint report of the Third International Workshop on Lymphocyte Alloantigens of the Horse, Kennett Square, Pennsylvania, 25-27 April 1984. The Third International Workshop on Lymphocyte Alloantigens of the Horse was held on 25-27 April 1984 in Kennett Square, Pennsylvania. Twelve laboratories from five countries participated. The principal purpose of this Workshop was to determine the phenotypic and gene frequencies of the 10 equine lymphocyte antigens (ELA) and a non-ELA lymphocyte antigen, ELY-2.1, in several breeds of horse. A total of 86 alloantisera characterized in previous workshops were tested against lymphocytes from 1179 horses. In addition, several experimental antisera were also tested against the same panel of lympho...
Comparison of the lipoprotein profiles obtained from rat, bovine, horse, dog, rabbit and pig serum by a new two-step ultracentrifugal gradient procedure. A new two-step gradient technique has been used in the separation of the different classes of lipoproteins from the serum of cows, horses, dogs, pigs, rabbits and rats. Total lipoproteins were first isolated at d 1.21 then floated through a d 1.006 to d 1.21 gradient. Collection by mean of a gradient fractionator provided directly comparable lipoprotein profiles, allowed the determination of the exact density range of each lipoprotein class and the fraction by fraction analysis of composition. Cholesterol and apo AI recoveries were high. Horse, dog, rabbit and pig exhibited three distinct lipo...
Increased acidophilia of eosinophil granules after EDTA treatment. The acidophilic reaction of eosinophil leucocyte granules from human, pig and horse blood smears was investigated by using May-Grünwald-Giemsa staining after previous treatment with EDTA and sodium citrate solutions. The same peak at 530 nm, but absorption values considerably higher than those of controls, were found in eosinophil granules after application of chelating agents, indicating that removal of metal cations could unmask basic groups in these structures.
Opiate-like and adrenocorticotrophin-like materials in equine pancreas. Equine pancreatic acetone powder was extracted with an acetone-water-HCl mixture. An acid acetone powder resulted by adding a copious volume of acetone to the extract. The powder was subjected to salt fractionation, gel filtration and chromatography on CM-cellulose. Steroidogenic activity, ACTH-like immunoreactivity and opiate receptor binding activity were distributed among the CM-cellulose chromatographic fractions derived from material unretarded as well as from material retarded on Sephadex G-25. The data indicates a separation of steroidogenic and opiate receptor binding activities, and t...
Preparation of Clostridium septicum antigen for hyperimmunization of horses using a dialyzed culture. The preparation of toxic cultures of Clostridium septicum is described, using an apparatus with a straight dialysis tubing, where the medium is filled both into the nutrition and cultivation space of the apparatus. Using the cultivation to nutrition volume rate 1:2, mean titre of lethal antigen in filtrates 3.86 limes mortis per mL and 300 dosis lethalis minima per Lm was obtained in comparison with the values of 2.22 and 150 respectively in flask filtrates. Native filtrates of dialyzed cultures were better antigens for hyperimmunization of horses than the culture filtrates from flasks.
Single-radial-immunodiffusion potency tests of inactivated influenza vaccines for use in man and animals. Single-radial-immunodiffusion (SRD) provides a sensitive and reproducible in vitro assay for haemagglutinin (HA) concentration in inactivated influenza vaccines. The use of SRD for human influenza vaccine standardization and application for equine and avian influenza vaccines is discussed. In clinical trials, vaccine HA concentration measured by SRD has been shown to be directly related to antibody responses and to protection against challenge. The use of SRD may considerably reduce the usage of animals for potency testing of veterinary influenza vaccines.
Comparison of antiproteolytic activities of alpha-1-proteinase inhibitors from the plasma of some mammalian species. Alpha-1-proteinase inhibitors isolated from plasmas of horse, ox, pig, rabbit and man were used for determination of some kinetic parameters of interaction with three horse leucocyte proteinases and bovine pancreatic trypsin and chymotrypsin. Effective molar ratio of enzyme-to-inhibitor, inactivation rate constant and inhibition constant were measured. In horse, ox, pig and rabbit two principal electrophoretic forms of alpha 1-PI could be distinguished. Both forms effectively inhibited trypsin but usually only one form reacted promptly and stoichiometrically with chymotrypsin and leucocyte ela...
Electrophoretic polymorphism and molecular structure of equine C3. Plasma or serum samples from 12 Arabian and 181 standardbred horses have been typed using an immunofixation technique to determine electrophoretic polymorphism of equine third complement component (C3). Six distinctly different electrophoretic patterns of equine C3 have been recognized thus far. SDS PAGE analysis of equine C3/anti C3 complexes revealed that the submolecular structure comprised an alpha chain and beta chain of molecular weights approximately 118,000 and 63,000 daltons respectively. The molecular weights of the alpha and beta chains were similar in all electrophoretic variants t...
A soluble class I molecule analogous to mouse Q10 in the horse and related species. Horse serum is shown to contain a soluble class I molecule analogous to the secreted Q10 molecule in the mouse. This molecule has several similarities to the recently described mouse Q10 molecule: it is smaller than membrane-bound equine class I molecules; it occurs in a high molecular mass complex of 200-300 kd in serum; and the serum levels of the equine molecule are similar to that of the Q10 molecule (about 30 micrograms/ml). A soluble molecule is also detected in the sera of species related to the horse; it has in fact been found in all the wild members of the order Perissodactyla so far ...
Comparison of specificity of human and horse leucocyte proteinases with synthetic peptide substrates. Highly purified horse leucocyte proteinases 1, 2A and 2B hydrolyze synthetic substrates which are decomposed also by human leucocyte elastase but they are unable to hydrolyze typical substrates of cathepsin G. Thus in distinction to other mammalian species horse leucocytes are devoid of cathepsin G and contain only elastases.
Acylation and carbamylation of equine muscle carbonic anhydrase (CA-III) upon reaction with p-nitrophenyl esters and carbamoyl phosphate. Equine muscle carbonic anhydrase (CA-III) behaves like ubiquitin in undergoing extensive acylation of N epsilon-lysine residues upon reacting with p-nitrophenyl esters. The enzyme undergoes extensive carbamoylation of lysine residues when reacted with carbamoyl phosphate. The modification of from 6 to 7 lysine residues results in the production of a series of more anodic electrophoretic components. The derivatization of the lysine residues leads to a marked decrease in the enzyme's ability to hydrate CO2. The equine CA-III possesses both acid and alkaline phosphatase activities in contrast to ...
Polymorphic restriction sites in the horse beta-globin gene cluster. Horse DNA samples digested with PstI and probed with the rabbit beta 1 globin gene show three phenotypes determined by one fragment of variable length (about 5.1 or 3.3 kb). Family data demonstrate that these fragments segregate as Mendelian alleles. The frequencies of the two alleles are 0.66 for the 3.3-kb fragment and 0.34 for the 5.1-kb one. Another polymorphism has been detected with BamHI. Again three phenotypes determined by two alleles (fragments of 7.5 and 3.8 kb) have been observed. Allelic frequencies of the 7.5- and 3.8-kb fragments are 0.24 and 0.76 respectively. The two polymorph...
Removing formaldehyde from embalmed cadavers by percolating the body cavities with dilute ethanol. Formaldehyde was removed from embalmed animal cadavers by pumping ethanol (20%) through the pleural and peritoneal cavities of 4 goats, 4 cows and 4 horses. The goats were percolated intermittently for 7 days and the large animals continuously for 72 h. Just after opening the body cavities, samples of air close to the organs were collected and analyzed for formaldehyde using a spectrofluorimetric method. The concentration of formaldehyde in the air samples was in goats 0.45 +/- 0.44 microgram/l (mean +/- SD), cows 0.42 +/- 0.29 microgram/l and horses 0.43 +/- 0.25 microgram/l.
[Chaetotaxy of Gastrodiscus aegyptiacus cercaria (Trematoda, Paramphistomoidea) a horse parasite]. Cercarial chaetotaxy of Gastrodiscus aegyptiacus (Paramphistomoidea), parasite of the Horse, is described. Cercariae were shed by experimentally infected Bulinus forskalii. This description is the first one of a Gastrodiscidae. The cercarial chaetotaxy of the Gastrodiscidae is quite different from those of Paramphistomoidea and Diplodiscidae.
DNA polymorphism in the major histocompatibility complex of man and various farm animals. In the past few years it has been possible by combining enzymatic cleavage of genomic DNA and the Southern blot hybridization technique to explore the endonuclease recognition site polymorphism of the MHC. HLA class I and DR and DQ alpha and beta class II specific probes as well as human C4 and Bf class III probes were used. All these probes were shown to cross-hybridize with DNA from pigs, cattle, sheep and horses. Hybridization of human genomic DNA with a class I probe showed 15-25 bands per genome depending on the enzyme used. Distinct endonucleases generated clusters of restriction fragmen...
Studies on the sequence of variable antigen types in ponies infected with a clone of Trypanosoma evansi. The sequential appearance of variable antigen types (VATs) of a clone of Trypanosoma evansi was studied in four ponies. Using luminol-dependent chemiluminescence, VAT populations which had been isolated from parasitemic peaks of single ponies, were tested for specificity with serum samples collected from other ponies. When antibody activity was demonstrated in a combination of trypanosomes and serum, it was concluded that a major VAT appeared in common. In the serum of all animals antibody activity was demonstrated to all VAT populations isolated from the other ponies during the first 4 weeks ...
Identification of betamethasone and a major metabolite in equine urine. Betamethasone and its major unconjugated metabolite, 6-beta-hydroxybetamethasone, were detected in equine urine by thin-layer chromatography and characterized by micro-liquid chromatography/mass spectrometry (micro-LC/MS). Their structures were confirmed by a combination of infrared spectroscopy and nuclear magnetic resonance spectroscopy.
