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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.
Quantitative determination of betamethasone and its major metabolite in equine urine by micro-liquid chromatography-mass spectrometry. Micro-liquid chromatography-mass spectrometry (micro-LC-MS) was utilized to quantitatively determine betamethasone and its major unconjugated metabolite, 6 beta-hydroxybetamethasone, in equine plasma and urine. The advantage of micro-LC-MS over conventional gas chromatography-mass spectrometry in corticosteroid determination is illustrated and the reliable, steadfast nature of micro-LC-MS is demonstrated through example.
Mammalian ribonucleases. The absence of a glycosylated Asn-Pro-Thr sequence in horse ribonuclease and the presence of tryptophan at position 39 in horse and dromedary ribonuclease. Parts of the amino acid sequences of horse and dromedary pancreatic ribonuclease were reinvestigated. The sequence of residues 21-25 in horse ribonuclease is Ser-Asn-Pro-Thr-Tyr or Ser-Asn-Ser-Thr-Tyr. The asparagine in the latter sequence is glycosylated. Horse ribonuclease possesses four additional amino acid residues at the C-terminus, like a number of other ribonucleases. Position 39 in horse and dromedary ribonuclease is not deleted but is occupied by tryptophan.
A direct technique for the preparation of chromosomes from early equine embryos. A technique is described for the preparation of banded chromosomes from early equine embryos cultured for less than 10 h in a medium containing bromodeoxyuridine. In addition to standard Giemsa staining and C-banding, chromosomes thus prepared can also be R-banded by either the RBA or the RB-FPG methods. This technique is rapid, repeatable, and limits cell loss, making it ideal for the preparation of early embryos.
Studies on histoplasmosis farciminosi (epizootic lymphangitis) in Egypt. Isolation of Histoplasma farciminosum from cases of histoplasmosis farciminosi in horses and its morphological characteristics. Isolation of Histoplasma farciminosum from five horses, showing typical signs of histoplasmosis farciminosi (epizootic lymphangitis) was successfully attempted. The mycelial form of H. farciminosum was isolated on Sabouraud dextrose agar enriched with 2.5% glycerol, brain heart infusion (BHI) agar enriched with 10% horse blood and PPLO dextrose glycerol agar. The last medium proved to be the most effective, both for primary isolation and subculturing of the fungus. It was found that on primary isolation, the lag phase of the mycelial form of the fungus was relatively long, involving 4-8 weeks ...
Thyroid hormone binding in serum of 15 vertebrate species: isolation of thyroxine-binding globulin and prealbumin analogs. The binding of [125I]T4 to serum proteins was studied in human, monkey, cattle, sheep, goat, water buffalo, horse, swine, dog, cat, rabbit, rat, chicken, frog, and salmon. Attempts were made to isolate thyroxine-binding globulin (TBG) and thyroxine-binding prealbumin (TBPA) from serum of all species, utilizing purification methods based on the specific properties of these proteins. TBPA was found to exist in all species examined. The protein was found anodal to albumin only in human, monkey, horse, and chicken. In cattle, swine, dog, cat, rabbit, frog, and salmon, TBPA was found cathodal to al...
The amino-acid sequence of beta-lactoglobulin II from horse colostrum (Equus caballus, Perissodactyla): beta-lactoglobulins are retinol-binding proteins. beta-Lactoglobulin isolated from horse colostrum is heterogeneous and contains two components: beta-lactoglobulin I and beta-lactoglobulin II. These two proteins are monomeric and show differences in their electrophoretic mobilities, chain lengths and primary structures. The complete amino-acid sequence of beta-lactoglobulin II was determined by automated Edman degradation of the intact protein and of the peptides derived from these by digestion with trypsin or chymotrypsin and by chemical cleavage with cyanogen bromide. Unlike other beta-lactoglobulins which contain 162 amino acids, horse bet...
Phenylbutazone and its metabolites in plasma and urine of thoroughbred horses: population distributions and effects of urinary pH. A survey of plasma and urinary concentrations of phenylbutazone and its metabolites in thoroughbred horses racing in Kentucky was carried out. Post-race blood samples from more than 200 horses running at Latonia Racetrack and Keeneland in the Spring of 1983 were analysed. The modal plasma concentration of phenylbutazone was between 1 and 2 micrograms/ml, the mean concentration was 3.5 micrograms/ml and the range was up to 15 micrograms/ml. Oxyphenbutazone had a modal plasma concentration between 1 and 2 micrograms/ml, a mean concentration of 2.07 micrograms/ml and a range of up to 13 microgram...
Photomicrographic evaluation of stallion spermatozoal motility characteristics. 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.
Effects of phenylbutazone and oxyphenbutazone on basic drug detection in high performance thin layer chromatographic systems. Interference or 'masking' in thin layer chromatography occurs when the presence of one drug on a thin layer plate physically obscures or interferes with the detection of another drug. We investigated the ability of phenylbutazone and oxyphenbutazone to mask or interfere with the detection by high performance thin layer chromatography (HPTLC) of basic drugs used illegally in horse racing. Of fifty-five basic drugs called 'positive' since 1981 by laboratories affiliated with the Association of Official Racing Chemists (AORC), forty did not comigrate with phenylbutazone or oxyphenbutazone and cou...
The sequence of equine muscle carbonic anhydrase. The sequence of equine muscle carbonic anhydrase (CA-III) has been determined. The 2 reactive cysteines of the 5 such residues have been localized. A strong sequence homology to other mammalian carbonic anhydrases exists, and 91% of the residues in the equine and bovine muscle forms are identical.
Equine cytogenetics: role in equine veterinary practice. The prognostic use of karyotyping in equine breeding has been recommended since 1976. Specimens used and laboratory methods are described. The system of karyotype evaluation is explained together with a glossary of terms. The principal aberrations in horses are defined. The present state of knowledge in cytogenetics in horses is reviewed dealing with infertility in the mare, where sex chromosomes give clear indication of problems; the situation with respect to anomalies found in stallions and hermaphroditism is explained coupled with recent concepts of chromosomal roles in reproduction. The fu...
Rapid species identification of group C streptococci isolated from horses. Two commercial systems, the API 20S (Analytab Products, Plainview, N.Y.) and the Rapid Strep (API System S.A., Montalieu-Vercieu, France), were evaluated for ease of use and accuracy in the rapid identification of group C streptococci isolated from horses. A total of 85 Streptococcus isolates were tested, including S. equi (67 isolates), S. zooepidemicus (13 isolates), and S. equisimilis (5 isolates). All S. equi and S. zooepidemicus isolates were correctly identified within 24 h by the Rapid Strep system. Specific grouping sera was necessary to distinguish between S. equisimilis and group G o...
Nitroblue tetrazolium reduction test and serum lysozyme assay in newborn Arabian foals and mares.
Summary:
Serum lysozyme (LZM) activity and nitroblue tetrazolium (NBT) reduction capacity of circulating phagocytes were determined by the lysoplate technique and the microquantitative method, respectively. As blood donors 47 clinically normal Arabian foals ranging in age from 6 hours to 22 days and their 40 dams were used.
A significantly (p < 0.05) lower serum LZM activity was found in foals older than 24 hours, compared with the mares (40.84 U/ml ± 15.32 and 48,96 U/ml ± 17.9, respectively), but the LZM/phagocyte ratio was similar in foals and mares.
Circulating phagocytes from...
Two techniques for detection of antibodies against Corynebacterium (Rhodococcus) equi in horse sera. Two techniques were developed to detect antibodies against the exosubstance of C. equi called equi-factor. In the first technique serum samples are tested against native equi-factor produced by the growth of C. equi on agar medium. A positive result is manifested by the development of precipitation lines. The second test is based on neutralization of prepurified equi-factor by antibody, resulting in the inhibition of its hemolytic synergism with staphylococcal beta toxin. Sera (125 samples) from horses of different ages, kept in localities with a history of C. equi infections, were examined. T...
Glycogen depletion patterns in horses competing in day 2 of a three day event. Muscle biopsy samples were collected from the left middle gluteal muscle of the horses participating in day 2 (speed and endurance test) of a three day event. Six Thoroughbred horses were biopsied the day before and within 30 minutes of completion of the speed and endurance test. Serial muscle sections were reacted histochemically for myosin adenosine triphosphatase activity after acid pre-incubation to demonstrate Type I, IIA and IIB fibers and the glycogen content in the individual fibers was assessed using the periodic acid Schiff (PAS) reaction. Total glycogen in muscle was measured fluoro...
Antibiotic susceptibilities of Salmonella species isolated at a large animal veterinary medical center: a three year study. The antibiograms of 408 Salmonella species isolated from large animals were collected during a three year study from 1981 through 1983. The predominant Salmonella serogroup among these isolates was group B. A consistently high percentage of all isolates were resistant to ampicillin and tetracycline. A pattern of increasing resistance to chloramphenicol and gentamicin was documented for serogroup B isolates while the susceptibility of the isolates to neomycin increased. There was a decrease in the incidence of susceptibility to sulfamethoxazole-trimethoprim among the group E isolates. These cha...
Antigenic determinants of acylphosphatase from porcine skeletal muscle. Analysis of the quantitative precipitin reaction of acylphosphatase from porcine skeletal muscle with rabbit antiserum indicated the presence of at least two antigenic determinants on the porcine enzyme molecule. Immunological cross-reactivities of acylphosphatases from equine and rabbit skeletal muscles were examined. In double immunodiffusion with the antiserum, the precipitin lines of the porcine and equine enzymes completely fused, while the rabbit enzyme gave no precipitin line. The reaction between the 125I-labeled porcine enzyme and its antibody was inhibited to the same extent by the p...
Hybrids from equine LH: alpha enhances, beta diminishes activity. LH hybrids were prepared by combining eLH alpha and eLH beta with the corresponding subunits of oLH, pLH and hCG. Recombinants were isolated by gel filtration and assessed by SDS-polyacrylamide gel electrophoresis under both dissociating and non-dissociating conditions. All combinations of subunits produced hybrid LH molecules. Hybrids prepared by combining eLH beta with oLH alpha, pLH alpha or hCG alpha were very inactive in rat radioligand and Leydig cell in vitro bioassays. Hybrids prepared with eLH alpha were very active in both assays. The greatest potentiating activity was observed when ...
[Hyalurodinase activity of beta-hemolytic streptococci of the Lancefield group C]. A total of 110 strains of beta-hemolytic streptococci, belonging to serogroup C (Lancefield), isolated from horses (71 S. zooepidemicus, 27 S. equisimilis and 12 S. equi) as well as 5 reference strains were tested for their ability to produce hyaluronidase. The determinations were carried out in a culture test on agarose gel and in a liquid test system (turbidity test according to DiFerrante). The results of both methods used showed that the three Streptococcus species could be differentiated by the relative quantitative determination of hyaluronidase activity. S. equisimilis strains produce 5...
Plasma and serum concentrations of phenylbutazone and oxyphenbutazone in racing Thoroughbreds 24 hours after treatment with various dosage regimens. The plasma and serum concentrations of phenylbutazone (PBZ) and oxyphenbutazone were measured in 158 Thoroughbred horses after various doses of PBZ wer given. All horses were competing or training at racetracks in various parts of the country. All horses used in the study had not been given PBZ 24 hours before they were placed on a specific dosage schedule. Samples were collected 24 hours after the last PBZ administration. Four grams of PBZ were given daily by stomach tube, paste, or tablet for 3 days. On day 4, 24 hours before sample collection, an IV dose of 2 g of PBZ was given, regardless ...
Morphology of three strains of contagious equine metritis organism. Examination of recently isolated cultures of three strains of Contagious Equine Metritis Organism grown on specially formulated, serum-free, clear typing medium revealed the presence of numerous colonial opacity variants. These colonies were prepared by a number of fixation and staining techniques and examined by scanning and transmission electron microscopy. Opaque and transparent phenotypes produced copious amounts of extracellular material compared with intermediate-opacity phenotypes which produced little or none. Also unique to intermediate colonies were numerous thin intercellular strand...
Estrogen metabolites in equine ovarian follicles: gas chromatographic-mass spectrometric determinations in relation to follicular ultrastructure and progestin content. Equine follicular fluid was aspirated at various developmental stages (viable, preovulatory and atretic) determined by ultrastructural study. Estrogens and progestins were analyzed by gas chromatography-mass spectrometry associated with stable isotope dilution. Progesterone and 17-hydroxyprogesterone were the principal progestins of the preovulatory and viable follicles. Among the catechol estrogens, 2-hydroxy-estradiol was particularly abundant in the preovulatory follicle and its definitive identification was made by the scan of a full mass spectrum.
The biosynthesis of 3 beta-hydroxy-5,7-androstadien-17-one by the horse fetal gonad. Horse fetal gonadal tissue was incubated with 3 beta-hydroxy-5,7-pregnadien-20-one and 5,7-cholestadien-3 beta-ol and it was shown that both substrates were converted to 3 beta-hydroxy-5,7-androstadien-17-one. These findings support the proposal that in this tissue there is a 5,7-diene pathway producing 3 beta-hydroxy-5,7-androstadien-17-one, the putative precursor of equilin in the placenta.
Iron deposition in apoferritin. Evidence for the formation of a mixed valence binuclear iron complex. A preliminary EPR investigation of iron accumulation in apoferritin has identified paramagnetic species generated during the early stage of iron deposition within the apoprotein shell. A featureless resonance at g' = 4.3, attributable to solitary high spin Fe3+ ions bound to the protein, is generated when Fe(II) is added to apoferritin at a level of 0.5 Fe/subunit (12 Fe/molecule) followed by air oxidation. This resonance accounts for 36% of the added iron. The remainder is EPR-silent and is probably present as oligomeric Fe3+ species. The intensity of the g' = 4.3 signal is reduced 3-fold upo...
Laboratory evaluation of aqueous humor in the healthy dog, cat, horse, and cow. Using routinely available clinical laboratory methods, aqueous humor samples were evaluated from 12 healthy dogs, 15 healthy cats, 7 healthy horses, and 6 healthy cows. Aqueous humor was almost acellular; cells that were present had degenerated beyond recognition. Protein concentration was low; only albumin was detectable on electrophoresis. Creatine phosphokinase and lactate dehydrogenase isoenzymes were not detected. Artifacts induced by sampling were insignificant compared with alterations in aqueous humor composition that occur with ocular diseases.
Turbidimetric measurement of IgG(T) in the serum of healthy Thoroughbreds and ponies. The turbidimetric analysis of IgG(T) in the serum of horses is described. Reference values are provided for 'worm-free' ponies (2.6 +/- 0.7 g/litre), stabled Thoroughbreds two years old and over (4.1 +/- 1.3 g/litre), grazing Thoroughbred broodmares (7.1 +/- 2.4 g/litre) and regularly wormed adult and young ponies grazing pasture contaminated with intestinal parasite eggs and larvae.
Antifungal sensitivity testing for equine keratomycosis. We evaluated 31 fungal specimens obtained from equine corneas over a 10-year period, 1973 to 1983. More than half were received in late summer and early autumn, and the number tended to increase in frequency during the 1980s. These isolates included 13 different genera and 20 different species. The prevalent genus was Aspergillus (35%). On the basis of examinations for tube-dilution minimal inhibitory concentrations and minimal fungicidal concentrations of 16 fungal isolates, the imidazole antibiotics such as miconazole and ketoconazole consistently showed the lowest geometric mean titers for ...
[Comparative characteristics of the vitreous body proteins in vertebrates]. Using disc-electrophoresis in polyacrylamide gel and immunochemical methods, studies have been made on proteins from the vitreous body of mammals (albino mouse, rat, guinea pig, pig, dog, cat), birds (hen), amphibians (the frog Rana ridibunda) and fish (the perch Perca fluviatilis). It was found that vitreous body proteins in man and animals include both the specific proteins and those of the blood serum. During evolution, specific antigens of the vitreous body attained strict species specificity, although some of them preserved the initial properties.
Variant specific opsonization of Trypanosoma evansi measured by luminol-dependent chemiluminescence. Using luminol-dependent chemiluminescence (LCL), the specificity of antibodies to variable antigen type (VAT)-populations of Trypanosoma evansi was studied in four infected ponies. Trypanosomes of each wave of parasitemia were isolated and multiplied in irradiated mice. Their opsonization by serum collected during the infection was investigated with LCL and results for isolated VAT-populations are shown in the paper. Antibodies specific to each VAT-population were first found three days after the maximum of a parasitemic wave. There was no cross reactivity between different VAT-populations. LC...
