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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.
[Monoclonal antibodies directed against equine blood group antigens]. The chief application of blood typing in domestic animals is in the verification of parentage. However, the acquisition of good standardized reagents in sufficient quantity remains an obstacle for the development of this work. The production of monoclonal antibodies directed against blood group determinants offers an attractive means of improving both the quality and quantity of serological reagents, and could facilitate the definition of new specificities. Fusions between a mouse myeloma line and splenocytes from mice immunized with horse red cells have resulted in four hybridomas producing a...
Quantitation of serum phospholipase A2 by enzyme-diffusion in lecithin agar gels. A comparative study in man and animals. A sensitive gel-diffusion assay for determination of phospholipase A was developed. PLA standards, serum, faecal and pancreas homogenate samples with PLA-activity were allowed to diffuse from wells into agar-gels containing lecithin-membranes. The turbidity cleared radially upon PLA-activity. The diameters of the cleared zones showed a linear relationship with the log of the enzyme concentration. Serum samples resulted in some turbidity within the cleared zones. This interference originating from serum lipoproteins could be abolished by hydrophobic absorption. The gel-diffusion method was comp...
The mechanism of Na+-L-lactate cotransport by brush-border membrane vesicles from horse kidney. Analysis by isotopic exchange kinetics of a sequential model and stoichiometry. The present study determines the characteristics of isotopic Na and lactate exchange under equilibrium conditions in horse kidney brush-border membrane vesicles. The influence of one solute (Na+ or lactate) on the isotopic exchange of the co-transported species (lactate or Na) was analyzed in detail. Analysis of the data suggests that Na and lactate interact sequentially with the carrier. The observed apparent symmetry between the activating effect of low Na concentrations and the inhibiting effect of high Na concentrations on the lactate exchange process suggests that the carrier functions ac...
A distinct environment for iron (III) in the complex with horse spleen apoferritin observed by x-ray absorption spectroscopy. Cell-specific variations in apoferritin structure correlate with variations in iron metabolism that suggest functional specificity of the protein shell. Using EPR spectroscopy, we previously showed that vanadyl binds to specific sites on apoferritin, and that VO2+ binding is reduced by Fe(II) and Fe(III) (the natural substrates) and by metals known to influence iron storage (Chasteen, N. D., and Theil, E. C. (1982) J. Biol. Chem. 257, 7672-7677). Such observations suggest that the metal-binding site is important to apoferritin function and may define a location where the influence of cell-spec...
Horse lymphocyte cell synchronization: improved technique for chromosome analysis. A method using methotrexate for horse lymphocyte cell synchronization and thymidine for incorporation into DNA replication is described. This method provides a powerful technique for the study of chromosomal abnormalities and detailed analysis of chromosomal replication patterns. The determination of horse karyotypes with many similar chromosomes needs a special method which reveals the numerous and informative stages of the cell cycle. Horse lymphocyte cell cultures treated with colcemid (20 min) and harvested 6 hours after the release of the 17 hour-block with methotrexate show the best resu...
Studies on prolactin: conformational comparison of human, equine, and porcine pituitary prolactins. The conformations of human, equine, and porcine pituitary prolactins, as evidenced by various optical properties, have been compared. The alpha-helix contents of all three proteins are essentially identical to each other (60 +/- 5%), as well as to prolactins isolated from other mammalian species. Direct absorption (zero and second-order), difference absorption, fluorescence emission, and circular dichroism spectra suggest that the majority of tyrosine and tryptophan side chains in these three proteins exist in very similar microenvironments within the folded forms of the hormones. Thus, the ge...
Saprophytic fungi isolated from the hair of domestic and laboratory animals with suspected dermatophytosis. Hair samples from domestic and laboratory animals with suspected dermatophytosis were examined for the presence of saprophytic fungi. A nutritionally poor base medium, developed by the author, was used in the isolation and identification of the saprophytes. Three hundred and ninety-four specimens were examined of which 246 were from dogs, 75 from cats, 30 from horses, 19 from cows, 12 from guinea pigs, 5 from rats, 2 from parakeets, 2 from chinchillas and one each from a goat, a mink and a lesser panda (Ailurus fulgens). Moulds classified in 32 genera were isolated. The commonest in order of f...
Changes in plasma cortisol concentrations during the ovulatory cycle of the mare. Daily blood samples from four mares were assayed for cortisol through a total of eight ovulatory cycles. Mean cortisol concentrations on days -14, -13, -10, -9 and -8 before ovulation (dioestrus) were greater than on days -5 to -1 (oestrus). The highest mean (+/- S.E.M) value of cortisol occurred on day -10 (260 +/-28 nmol/l) and the lowest on day -2 (142 +/- 14 nmol/l). A single episode on a day in late dioestrus characterized the maximum cortisol value per cycle for five of eight cycles. Extraction of plasma samples with petroleum ether or chromatography before assay, to eliminate interferen...
Phloroglucinol microassay for plasma xylose in dogs and horses. The phloroglucinol microassay technique for measuring plasma concentrations of xylose was compared with the more tedious orcinolferric chloride technique. Sequential blood samples were collected from 5 dogs and 6 horses every 30 minutes after oral administration of 0.5 g of D-xylose/kg of body weight. Comparison of the results by regression analysis shows a highly significant (P less than 0.01) positive linear correlation for both dogs (r = 0.95) and horses (r = 0.77). These results indicate that xylose in canine and equine plasma can be accurately measured by the phloroglucinol technique.
Horse red blood cells frozen with 20% (w/v) glycerol and stored at -150 C for five years. When equine RBC were frozen with 20% (w/v) glycerol and stored at -150 C for as long as 5 years, there were no adverse effects on freeze-thaw or freeze-thaw-wash recovery or oxygen transport function. The manner in which the glycerol was added to, and removed from, the equine RBC was shown to be an important consideration in ensuring optimal freeze-thaw-wash recovery values.
The use of heterologous radioimmunoassays for the measurement of follicle-stimulating hormone and luteinizing hormone concentrations in horse and donkey serum. Heterologous double-antibody radioimmunoassay were developed for the measurement of FSH and LH concentrations in the serum of both horses and donkeys. The FSH assay employed a rabbit anti-ovine FSH serum which showed a complete lack of cross-reaction with equine chorionic gonadotrophin (eCG) and negligible cross-reaction with equine LH. The LH assay utilized an antiserum raised against highly purified eCG. This similarly showed negligible cross-reaction with equine FSH but its high cross-reactivity with eCG prevented the measurement of equine LH concentrations in serum when eCG was also presen...
Affinity chromatographic purification of horse muscle acylphosphatase: evidence of the existence of multiple molecular forms. Acylphosphatase was purified from horse muscle by a new procedure involving an affinity chromatography step and subsequent ion-exchange chromatography. This procedure was considerably milder than the preceding one, gave an overall yield of about 60% of activity and permitted isolation of three molecular forms with acylphosphatase activity. All these enzymatic forms are tightly bound to Sepharose 4B-linked anti-horse muscle acylphosphatase antibodies. Two of these forms (Ho1 and Ho3) are present in larger amounts: Ho1 corresponds to the enzyme purified according to the older procedure; this enz...
Identification of 3 beta-hydroxy-5,7-pregnadien-20-one and 3 beta-hydroxy-5,7-androstadien-17-one as endogenous steroids in the fetal horse gonad. The 5,7-dienes, 3 beta-hydroxy-5,7-pregnadien-20-one and 3 beta-hydroxy-5,7-androstadien-17-one were extracted from fetal horse gonads and purified by solvent partition, thin-layer chromatography and high performance liquid chromatography. The isolated steroids were identified by comparison with the synthetic steroids using ultraviolet and mass spectroscopy and by gas chromatography-mass spectroscopy. The identification of these compounds as endogenous steroids, together with the data on their biosynthesis reported previously, support the proposal that in the fetal horse gonad there is a 5,7-d...
Standard antisera produced in ponies for the identification of bovine mycoplasmas: comparative growth-inhibition results from six laboratories. Antisera to 10 mycoplasma species of bovine origin were produced in 10 ponies and were distributed for evaluation in growth-inhibition tests at 6 laboratories in Australia, England, Denmark, France, and the United States. Except for a few failures with some antigens produced at the 6 laboratories, the antisera induced large zones of growth inhibition in homologous, but not heterologous, systems. These antisera may be useful as standard reagents for the identification of the bovine mycoplasmas.
Comparison of pyrimidine 5’nucleotidase activity in erythrocytes of sheep, dogs, cats, horses, calves, and Mongolian gerbils. Pyrimidine 5'nucleotidase (P5N) activities of erythrocytes for Mongolian gerbils, cats, dogs, sheep, horses, and calves were measured, using a radiometric technique with [14C]cytidine monophosphate as the substrate. Erythrocytes of gerbils had the highest activity [1,177.1 +/- 133.6 mU/g of hemoglobin (Hb)]. Feline erythrocytes had 327.4 +/- 204.4 mU/g of Hb. Canine erythrocytes had 148.0 +/- 19.8 mU/g of Hb. Ovine erythrocytes (44.3 +/- 20.9 mU/g of Hb), equine erythrocytes (30.0 +/- 15.9 mU/g of Hb), and bovine erythrocytes (14.1 +/- 6.9) had relatively low P5N activity. The P5N activity was...
Evaluation of a series of testing procedures to predict neonatal isoerythrolysis in the foal. A series of modified (field) tests were compared to a crossmatch between mare and foal for their reliability in predicting neonatal isoerythrolysis (NI) in eight foals born to experimentally alloimmunized mares. In the field tests, mare's serum, plasma and colostrum were combined with foal erythrocytes washed by a modified procedure to determine which combination was the best predictor of impending NI. A consistent grading system for agglutination and hemolysis was employed. The field tests using mare's plasma demonstrated less agglutination and hemolysis than tests where serum was employed. I...
High-performance liquid affinity chromatography on silica-bound alcohol dehydrogenase. Horse liver alcohol dehydrogenase was immobilized on glycerylpropyl-silica (10 micron, 1000-A pores) activated with 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride). The coupling and activity yield was almost 100%. The coenzyme-binding sites were equivalent and virtually unaffected by the immobilization process, as judged from Scatchard plots and active-site titrations. The silica-bound enzyme, packed in steel columns, was integrated with HPLC equipment and then successfully used for chromatography of adenine nucleosides, adenine nucleotides, and triazine dyes. Dissociation constants w...
Enzymatic trimethylation of lysine-72 in cytochrome c. The present observations are the continuation of our earlier study on the physicochemical mechanism of protein-lysine methylation. In this paper the electrophoretic behaviour (pI values) of two chemically modified horse heart cytochromes c at lysine-72 with trifluoromethylphenylcarbamoyl (neutral group) or carboxydinitrophenyl (acidic group) is compared with the enzymatically methylated cytochrome c. The results indicate that although both chemically modified cytochromes c have lower pI values than the unmodified cytochrome c, the enzymatic methylation appears to be much more efficient in lowe...
Measurement of superoxide dismutase, diamine oxidase and caeruloplasmin oxidase in the blood of thoroughbreds. Methods were developed for the measurement of superoxide dismutase (SOD), diamine oxidase (DAO) and caeruloplasmin oxidase in the blood of thoroughbred horses. These enzymes were measured in 178 normal thoroughbreds stabled throughout the United Kingdom. The relationships between the activities of SOD, DAO and caeruloplasmin oxidase and the blood concentrations of their associated trace metals (copper, zinc and manganese) were studied in 52 of the thoroughbreds. Trace metals were measured by electrothermal atomic absorption spectrophotometry. No relationships were found between the activities ...
Sodium and potassium ion-dependent change in oligomerization of Na,K-ATPase in C12E8 detected by low-angle laser light scattering technique in combination with high performance porous silica-gel chromatography. Approximate molecular weights and the subunit structures of Na,K-ATPase from horse kidney were estimated by means of the combination of porous silica gel chromatography, laser light scattering (LS) and refractive index (RI) measurements in C12E8. When the enzymes were eluted with NaCl- or KCl-containing solution, 3 or 4 protein peaks, respectively were detected except that of low molecular weight range. These peaks were tentatively named Na-1, Na-2, Na-2', Na-3 (NaCl-containing eluents), K-1, K-2, K-3 (KCl-containing eluents), respectively. Na,K-ATPase and K-p-nitrophenylphosphatase activities...
The in vitro effects of EDTA-tris, EDTA-tris-lysozyme, and antimicrobial agents on equine genital isolants of Pseudomonas aeruginosa. Five isolants of Pseudomonas aeruginosa collected from clinical cases of equine genital infection and one standard strain of P. aeruginosa were exposed to various concentrations of ethylene-diaminetetraacetic acid (EDTA) and tris (hydroxymethyl) aminomethane (tris buffer pH 8) and EDTA-tris lysozyme. Colony forming units of the isolants and minimal inhibitory concentrations for 11 antimicrobial agents were determined with each isolant before and after exposure to the EDTA solutions. Decreased cellular viability was found with all six isolants after exposure to the EDTA-tris solutions. Reversal...
Unfolding pathway of myoglobin. Evidence for a multistate process. The free energy of unfolding of horse myoglobin has been calculated from the denaturation pattern induced by guanidine hydrochloride as well as by acid. The delta GH2O, i.e., the value in the absence of denaturant obtained by using the two-state transition model, was found to be 25% lower than that determined from the acid denaturation pattern, i.e., 12.0 kcal/mol, although the extent of protein denaturation produced by acid was much lower. The amount of helical structure surviving the acid-induced conformational change was estimated to be 50% of that present in the native protein, and it coul...
Interspecies activation of lecithin-cholesterol acyltransferase by apolipoprotein A-I isolated from the plasma of humans, horses, sheep, goats and rabbits. The abilities of apolipoprotein A-I species isolated from humans, horses, sheep, goats and rabbits to activate purified human lecithin-cholesterol acyltransferase and the enzyme from homologous plasmas and plasma of other mammalian species were compared. Each purified apolipoprotein A-I species was individually incorporated into phosphatidylcholine/cholesterol vesicles by the cholate dialysis method to form proteoliposome common substrates (apolipoprotein A-I/phosphatidylcholine/cholesterol molar ratio of 1:250:12.5) for the enzyme activity assay. All apolipoprotein A-I species tested had the ...
Concentration of nucleotides and deoxynucleotides in peripheral and phytohemagglutinin-stimulated mammalian lymphocytes. Effects of adenosine and deoxyadenosine. Concentrations of purine and pyrimidine ribonucleotides were measured with HPLC in lymphocytes of man, horse, pig and sheep and in rat thymocytes. The ATP concentration was highest in lymphocytes of all species and about 850 pmol/10(6) cells in human and equine lymphocytes, higher in porcine and lower in ovine lymphocytes and rat thymocytes. The GTP concentration was comparable in human, equine and porcine lymphocytes, but lower in ovine lymphocytes. ATP concentration was also measured in lymphocytes of man, horse and pig with a luciferin-luciferase assay. During culturing with or without phyt...
Serodiagnosis of western equine encephalitis virus infections: relationships of antibody titer and test to observed onset of clinical illness. Sera from horses and human beings with clinically diagnosed western equine encephalitis (WEE) virus infections were tested for hemagglutination-inhibition (HI), complement-fixation (CF), and neutralizing (N) antibody to WEE virus. These tests confirmed infection in 43.8% (HI), 56.3% (CF), and 80.4% (N) of horses and 54.5% (HI), 59.1% (CF), and 77.3% (N) of human beings. Use of the N test as an adjunct to the HI and CF tests increased the likelihood of serologic confirmation to 91.7%. In both horses and human beings, N antibody increased steeply at the end of the 1st week after onset. The resul...
Simplified technique for histochemical determination of three fiber types in equine skeletal muscle. For determination of 3 muscle fiber types in equine skeletal muscle, a comparison of 2 preincubation buffers, each followed by myosin adenosine triphosphatase staining, was made. Serial sections of the muscle samples (n = 75) were preincubated in an acid buffer (pH 4.6) or a formaldehyde-glycine buffer (pH 7.25) and then were stained for myosin adenosine triphosphatase. Differentiation of muscle fibers into type I, IIA, and IIB was identical with both techniques; however, in the samples prepared at pH 4.6, type I fibers were black; type IIA, light gray; and type IIB, dark gray. In the samples ...
