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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.
The distribution and origin of VIP in the spinal cord of six mammalian species. The distribution of VIP-immunoreactivity was studied in the spinal cord and dorsal root ganglia of 6 mammalian species. Immunoreactive fibres and cell bodies were most apparent in the dorsal horn, dorsolateral funiculus, intermediolateral cell columns and the area around the central canal. The distribution of VIP immunoreactivity was similar in all species studied, mouse, rat, guinea pig, cat, horse and the marmoset monkey. There were fewer VIP fibres in the dorsal horn of cervical and thoracic segments than in lumbosacral segments. Using radioimmunoassay this gradient increase was quantitativ...
Purification of lutropin and follitropin in high yield from horse pituitary glands. A method has been developed for the purification of equine lutropin (eLH) and equine follitropin (eFSH) from horse pituitary glands which attains high yields of both hormones in contrast to previous methods that were devoted to one or the other with inferior recovery of the hormones. Two-pass chromatography over CM-Sephadex was used to separate eLH from eFSH. Subsequent steps employing QAE (quaternary amino-ethyl)-Sephadex chromatography and gel filtration on Sephacryl S-200 produced highly purified hormone preparations. Yields of purified eLH and eFSH were 110 and 60 mg/kg of frozen pituitari...
Measurement of plasma antithrombin III activity in healthy horses. A fluorometric assay was used to determine plasma antithrombin III (AT III) activities in 15 healthy adult horses. Nearly all plasma samples had an initial value of greater than 100% thrombin inhibited, so a 1:1 dilution of the prepared samples was performed. Following dilution, the mean value of the animals was 59.17 +/- 7.4% thrombin inhibited. Mares had significantly greater AT III activity than did geldings (P less than 0.01). The results of this study indicate the horse has more AT III activity than did other domestic species in which AT III activity has been reported.
Odontodysplasia in a horse. Weight loss, poor growth and dysphagia occurred in a young stallion with some teeth missing, and soft, discolored and/or deformed deciduous incisors, premolars and molars. At necropsy, permanent teeth sites were filled with yellow, gelatinous material. Clinical signs suggested fluorosis but laboratory findings did not confirm the diagnosis.
The isolation, propagation and characterization of tissue-cultured equine rotaviruses. From 105 field cases of diarrhea in neonatal or young foals, rotavirus was detected by electron microscopy (EM) and/or by enzyme-linked immunosorbent assay (ELISA) in the feces of 65 foals on 16 different premises. ELISA was performed with Rotazyme test kits developed by Abbot and Company for the detection of rotaviruses. Twenty-four field isolates from the feces of diarrheic foals with equine rotavirus infection as ascertained by EM were placed in MA-104 cell cultures after pretreatment of the viral suspension with 10 micrograms ml-1 of trypsin and incorporation of 0.5 micrograms ml-1 or 1 mi...
Standardization of the equine infectious anemia immunodiffusion test and its application to the control of the disease in the United States. In 1972 the US Department of Agriculture (USDA) established requirements that horses which had immunodiffusion antibody against equine infectious anemia could not be transported interstate. Forty-two states had regulations requiring that horses have a negative equine infectious anemia immunodiffusion test before movement. In order to standardize immunodiffusion testing, it was stipulated in the 1972 regulations that tests must be performed in approved laboratories. The approved laboratories were required to have personnel trained in the immunodiffusion test procedure, to follow the standard pr...
The identification of C-18 neutral steroids in normal stallion urine. As part of a continuing research program associated with the detection of anabolic steroid residues in horse urine, normal samples from entire male horses have now been investigated. Isomers of three C-18 neutral steroids; 4-estren-17-ol-3-one (1), estrane-3,17-diol (2) and an unsaturated estranediol having a possible structure (3), have been identified in urine samples from two male horses aged 8 and 14 years. Of these three steroids, compound (2) was not detected in the urine of a 2.5 yr old entire male nor in the majority of post-race urine samples from entire male horses average age 3.8 yr...
Androstenedione and testosterone biosynthesis by the adrenal cortex of the horse. An homogenate from cortical tissue of mare adrenals was incubated in the presence of tritiated pregnenolone. The (3H) androstenedione and the (3H) testosterone synthesized during the incubation were extracted, purified, and co-crystallized to constant specific activity in the presence of unlabeled carriers. The rate of conversion of pregnenolone to androstenedione and testosterone was of the order of 5 and 0.15 per cent respectively. The high ratio of (3H) androstenedione to (3H) testosterone observed in this study suggests that androstenedione is the main androgen produced by mare adrenals. I...
Purification and characterization of epimeric estradiol dehydrogenases (17 alpha and 17 beta) from equine placenta. Estradiol 17 alpha-dehydrogenase (EC 1.1.1.148) and estradiol 17 beta-dehydrogenase (EC 1.1.1.62) from horse placenta have been purified to homogeneity. Both enzymes are localized in the microsomal fraction and are solubilized in 1.5% sodium cholate. The 17 alpha- and 17 beta-dehydrogenases are separated by selective elution from hydroxylapatite with 0.5 and 1.0 M potassium phosphate, respectively. Subsequent purification is achieved by two affinity-absorption steps using reactive blue 2-agarose and estriol hemisuccinate-Sepharose. Homogeneous estradiol 17 alpha-dehydrogenase has a specific ac...
GuHC1 induced unfolding-folding transition of a hinge-bending protein: horse muscle phosphoglycerate kinase. The unfolding-folding transition of phosphoglycerate kinase induced by GuHC1 was studied at equilibrium. Various signals were used to follow the transition: fluorescence emission, difference spectra, circular dichroism and enzymatic activity. The non-coincidence of transition curves obtained from different structural parameters indicate a deviation from a two-state process. The view that structural domains behave as independent "folding units" is critically discussed.
Cholesteric organization of DNA in the stallion sperm head. The fine structure of chromatin in sperm heads was investigated by different microscopic techniques: in vivo examinations in the polarizing microscope, thin sections and freeze-fracture replicas observed by transmission electron microscopy. The freeze-fractured chromatin appears to be formed of superimposed lamellae, each one 330 A thick. These lamellae are parallel to the flattening plane of the sperm head. This situation was already described in other mammal spermatozoa and in particular in the bull and the rabbit. This work presents a new interpretation of this lamellated aspect. The chroma...
Hyaluronic acid concentration in synovial fluid from normal and arthritic joints of horses. A method previously described was used to determine the hyaluronic acid concentration in synovia from normal and arthritic horse joints. The concentration of hyaluronic acid in the synovia from arthritic joints was found to be significantly lower than the concentration in fluid from normal joints.
Structures of the dienoic lactones of horse sebum. The C34, C36, and C38 dienoic omega-lactones were isolated from sebum of the horse (Equus caballus) and the double bond positions were determined by stepwise chemical dissection and analysis of the fragments. The structures found could be formed by delta 9-desaturation at the C18-stage of fatty acid biosynthesis followed by a second delta 9-desaturation when the chains reached C24, C26, C28, C30, or C32 and then addition of one to seven 2-carbon units. These findings provide insight into the dimensions and organization of the endoplasmic reticulum in cells of the sebaceous glands.
Determination of flunixin in equine plasma by reversed-phase liquid chromatography. Flunixin is determined in equine plasma by liquid chromatography on LiChrosorb RP-18 with 70% methanol in phosphate buffer pH 3.1 as the eluent, with detection at 284 nm. Plasma is deproteinized with methanol and the supernatant is then injected directly into the system. With a short pre-column (5 x 3 mm i.d.), which is replaced after 25-40 injections of sample, 420 plasma samples could be analysed on one analytical column. The detection limit in plasma is 0.30 micromol/l (89 ng/ml) and the method can be used in pharmacokinetic studies.
Inhibition of equine S-adenohomocysteine hydrolase by 2′-deoxyadenosine. 2'-Deoxyadenosine and 9-beta-D-arabinofuranosyladenine (ARA) are apparent suicide inhibitors for equine S-adenosylhomocysteine hydrolase. In initial velocity studies of the synthetic reaction converting adenosine and homocysteine to S-adenosylhomocysteine, adenine, adenosine 5'-triphosphate, and 9-beta-D-arabinofuranosyladenine were found to be competitive inhibitors with Kis of 3.8 microM, 1.1 mM, and 30 microM, respectively. In contrast, linear mixed inhibition was observed for 2'-deoxyadenosine, indicating that 2'-deoxyadenosine must bind in more than one fashion to the enzyme.
The plasma protease inhibitor system (Pi) of Standardbred horses. The plasma protease inhibitor system (Pi) of Standardbred horses was studied by thin-layer, high-voltage, acid polyacrylamide gel electrophoresis (pH 4.6) followed by protein staining and staining for trypsin and chymotrypsin inhibition. In addition to the eight Thoroughbred alleles (PiF, G, I, L, N, S1, S2, U), another 10 alleles, designated PiH, J, K, O, P, Q, R, V, X, Z, were postulated to account for the 98 Pi types which were observed in Standardbreds. Detailed inhibitory spectra of the 'new' alleles were determined and further exceptions to the Pi1, Pi2 classification of Juneja et al. (1...
Borna disease of horses. An immunohistological and virological study of naturally infected animals. The brains of eight horses that had suffered from natural Borna disease were examined with virologic, immunohistological, and electron-microscopic methods. All brains harbored infectious virus as shown by inoculation of experimental animals. Regional assessment of the infectivity exhibited the highest titers in the hippocampus and piriform cortex and the lowest in the cerebellum. Conventional histology yielded pathologic alterations very similar to those of the classical description of the disease. Immunohistology demonstrated the highest amounts of Borna disease virus-specific antigen in the ...
[Origin of the FSH + LH double activity of equine chorionic gonadotropin (eCG/PMSG)]. The LH and FSH activities of equine choriogonadotropin (eCG) have been compared in several species with those of the highly purified homologous pituitary gonadotropins. The molar FSH/LH activity ratio of eCG determined by RRA is 0.20 in the pig, 0.25 in the rat and 0 in the horse. These data demonstrate the LH monospecificity of eCG in its own species as it is the case for hCG. We have also shown that equine LH exhibited a FSH-activity similar to that of eCG in the pig and in the rat but not in the horse. In the female rat, the binding activity to FSH receptors and the in vitro FSH activity of...
Two-dimensional electrophoresis of horse serum proteins: genetic polymorphism of ceruloplasmin and two other serum proteins. Two-dimensional agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis of horse serum proteins revealed genetic polymorphism of ceruloplasmin (Cp) and two unidentified serum proteins tentatively designated serum protein 1 (SP1) and serum protein 2 (SP2). Family data were consistent with the hypothesis that the observed Cp and SP1 phenotypes were each controlled by two codominant, autosomal alleles. The three common SP2 phenotypes were shown to be controlled by two codominant, autosomal alleles. Population data and limited family data indicated the occurrence of two additio...
Detection of equine infectious anemia virus in horse leukocyte cultures derived from horses in various stages of equine infectious anemia viral infection. The enzyme-linked immunosorbent assay (ELISA) antigen-positive and agar-gel immunodiffusion test (AGID)-negative horses do not have infective equine infectious anemia (EIA) virus. The ELISA testing of horse leukocyte culture (HLC) supernatants did detect EIA virus in a HLC that was infected with the Wyoming strain of EIA virus and in HLC derived from horses in febrile, acute, or subacute stages of EIA infection. In supernatants of HLC derived from chronic and inapparent carrier horses, EIA virus was not detected with ELISA. Direct fluorescent antibody tests detected EIA virus in HLC infected w...
Heterogeneity of horse transferrin: the role of carbohydrate moiety. Homozygous horse transferrin (Tf O) is highly heterogeneous. In starch gel electrophoresis it gives at least 9 zones. Two main components (2a and 4b) were purified by rivanol and ammonium sulphate precipitation, DEAE-Sephadex chromatography and SP-Sephadex chromatography. Molecular weights of 75 200 and 80 500 for components 2a and 4b, respectively, were determined by sedimentation equilibrium ultracentrifugation. Amino acid compositions of the two components were similar, and there were no differences in the N-terminus (glutamic acid followed by glutamine) and the C-terminus (valine). Differe...
Structural proteins of equine infectious anemia virus and their antigenic activity. Using purified equine infectious anemia (EIA) virus labeled with 3H-glucosamine or 14C-protein hydrolysate, structural proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As a result, 2 glycoproteins and 10 proteins with molecular weights (mol wt) ranging from 12,000 to 115,000 daltons were demonstrated. Of 12 structural proteins, 3 proteins, namely a glycoprotein with mol wt of 76,000 (gp76) and 2 proteins with mol wt of 25,000 (p25) and 12,000 (p12), respectively, had distinct antigenic activity from one another in immunodiffusion. Development of antibodies a...
[An attempt at demonstrating the participation of autoaggressive processes in the pathogenesis of periodic eye inflammation in horses]. In the studies attempts were to demonstrate the occurrence of immunological reactivity against antigens of the lens and tunica vascularis of the eye in periodical inflammation of eyes in horses. For this purpose antigens from the lens and tunica vascularis of the eye, prepared in our laboratory, were used in the experiments. The reactivity of horses with monthly symptoms of blindness against the above antigens was determined in vivo (skin tests and PCA) and in vitro (ID reaction). The results obtained mainly in skin tests account for its occurrence in some percentage of diseased animals, becau...
