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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 postnatal development of serum zinc, copper and ceruloplasmin in the horse. 1. Serum samples were collected from ten foals at predetermined times during the first 12 months following birth and zinc and copper concentrations and ceruloplasmin activity were evaluated. 2. Serum zinc concentrations were found to be quite variable with respect to age (range = 67-95 micrograms/dl). 3. Serum copper concentrations increased in a linear fashion from day 0 to day 28 before levelling off at 190-247 micrograms/dl. 4. Ceruloplasmin activity was found to correlate with the concentration of serum copper (r = 0.92) and reached a plateau at an activity of 30-38 IU by day 28.
[Diagnosis of liver diseases in dogs, cats and horses]. Clinical symptoms of hepatopathies are not specific and must be verified by further investigation. Laboratory diagnosis is a very useful method to decide if liver disease is present or not. In individual cases laboratory methods can give hints as to the aetiology of the illness. If necessary, biopsy, angiography or/and cholecystography can be carried out for further clarification of the diagnosis.
Lipids in the laminated layer of liver, lung and daughter hydatid cysts of equine Echinococcus granulosus (Cestoda). Lipids extracted from the laminated layers of horse liver and lung hydatids, including a daughter liver cyst, were analysed using TLC. No differences in lipid composition was detected in 11 liver cysts, whether from the same or different livers, and di- and triacylglycerols, cholesterol, wax and steryl esters, oleic acid, sphingomyelin, phosphatidyl choline, phosphatidyl inositol and ceramide hexosides were detected. The daughter cyst differed from its "parent" cyst in lacking diacylglycerols and wax and steryl esters. The lung cyst differed from the liver cysts in that cholesterol, wax and st...
[Detection of dermatomycoses in horses with the dermatophyte test medium Fungassay]. For the inoculation of the dermatophyte-test-medium Fungassay, 200 skin scrapings from horses, 13 from cattle and 13 from artificially infected guinea pigs were used. As control methods, the alkali method, the fluorescent microscope technique and the usual mycological culture were available. For the analysis of skin scrapings, the Fungassay culture mediums are clearly inferior to the usual mycological culture. Fewer dermatophytes were isolated and false positive as well as false negative results occurred. The cultivation of Trichophyton verrucosum failed on the dermatophyte-test-medium.
Differentiation of meat from horse, donkey and their hybrids (mule/hinny) by electrophoretic separation of albumin. Meat from the species horse, donkey and their hybrids, mule/hinny, can be reliably identified by determination of genetic variants of serum albumin by starch gel electrophoresis of meat extracts. Staining of the starch gel for carboxylesterase activity permits differentiation of most horses from donkeys while mules/hinnies cannot be distinguished from horses by their esterase activity alone.
Characterization of equine plasma lipoproteins after separation by density gradient. 1. Plasma lipoproteins from six thoroughbred horses were separated by density gradient ultracentrifugation. For each sample, lipoprotein bands were visualized by means of a prestained plasma control and characterized by electrophoretic, chemical and morphological analysis. 2. Very low density lipoproteins (VLDL) were isolated at d less than 1.018 g/ml. 3. Two clearly resolved bands were detected in the low density lipoprotein fraction (LDL). The density limits were evaluated as follows: LDL1(1.028 less than d less than 1.045 g/ml) and LDL2(1.045 less than d less than 1.070 g/ml). Marked differ...
Expression of major histocompatibility complex (MHC) antigens on horse trophoblast. Antibodies to fetal major histocompatibility complex (MHC) antigens are routinely detected in the serum of pregnant mares some 2-4 weeks after formation of the endometrial cups at Day 36-38 after ovulation. Several experimental approaches were taken to determine whether paternal MHC antigens are expressed on horse placental tissues. First, absorption of anti-paternal MHC antisera with a large volume of endometrial cup cells removed antibody activity in only 2 of 4 experiments. Second, repeated immunization of horses with endometrial cup tissue recovered from a mare on Day 47 of pregnancy faile...
The proteins of equid herpesvirus 1 (EHV 1) recognised by equine antisera and their ability to promote antibody-dependent cell-mediated cytotoxicity. Equine sera were used to immunoprecipitate radiolabelled virus-infected cell proteins; subsequent resolution with polyacrylamide gel electrophoresis identified the EHV-1 polypeptides VP 2, 10a, 11, 13, 14, 15, 16, 20, 21 and 23a. The humoral support of ADCC by these sera was examined in vitro. Cytotoxicity could be demonstrated against both subtypes irrespective of the immunising isolate. The implications of these results are discussed.
Secretion of luteinizing hormone and follicle stimulating hormone in intact and ovariectomized mares in summer and winter. Sequential samples of blood were drawn via jugular catheters every 15 min for 24 h from four mares in each of five reproductive states: intact anestrous mares in winter, intact diestrous mares in summer, intact estrous mares in summer, ovariectomized mares in winter and ovariectomized mares in summer. Estrous mares were sampled on d 4 or 5 of estrus and diestrous mares on d 10 or 11 of diestrus. Each sample of plasma was assessed for concentrations of luteinizing hormone (LH) and follicle stimulating hormone (FSH) in two independent radioimmunoassays. A computer program was developed that dete...
Development and survival of free-living stages of equine strongyles under laboratory conditions. In a series of laboratory studies the optimum conditions for the development and survival of the free-living stages of strongyle parasites occurring in horses in tropical north Queensland were determined. No differences in behaviour were noted between the strongyle species. Development to the infective stage occurred only between 10 and 35 degrees C. The rate was affected by temperature, taking 15-24 days and 3 days, respectively, at the lowest and highest temperatures for the developing stages to reach the infective third stage. Yields of infective larvae were very low outside the range 20-33...
Analysis of the equine lymphocyte antigen system by Southern blot hybridization. Fourteen Standardbred horses homozygous for one of six equine lymphocyte antigen (ELA) specificities (A1, A3, A4, A5, A6, or A10) were analyzed by Southern blot hybridization using DNA probes derived from the mouse major histocompatibility complex (MHC). Total genomic DNA from peripheral lymphocytes was digested with the restriction enzymes Hind III, Pvu II, or Eco RI. Twenty-three to thirty-three bands were generated for individual horses with the class I cDNA probe. The resulting band patterns revealed 12-14 nonpolymorphic fragments, which is consistent with the highly conserved Qa/Tla genes...
Detection of serum antibodies against Ehrlichia risticii in Potomac horse fever by enzyme-linked immunosorbent assay. An indirect enzyme-linked immunosorbent assay (ELISA) was developed which was specific and sensitive in detecting antibodies to Ehrlichia risticii in Potomac horse fever (PHF). The ELISA antibody titers were correlated with the indirect fluorescent antibody (IFA) titers. E. risticii propagated in human histiocyte culture was purified on renografin gradient and the band of the organisms at a density of 1.182 g/ml was used as antigen. ELISA antibody titers were determined through computer assisted analysis, the observed antibody titers were derived by serial serum dilutions and using a resultant...
Molecular genetic analysis of the major histocompatibility complex in an ELA typed horse family. Restriction fragment length polymorphism was studied in an ELA typed horse family which included a stallion, a mare with two full-sibs, another mare with three full-sibs and, in addition, three paternal half-sibs. DNA samples from all individuals were investigated by Southern blot analysis using three restriction enzymes (EcoRI, HindIII or TaqI) and human cDNA class I, class II (DR beta) and class III (C4) probes. In addition, a genomic class II DQ alpha probe was used. Fragments hybridized with the various probes revealed the existence of DNA sequences homologous to HLA class I, DR beta, DQ a...
ISO-DALT characterization of 12 ‘new’ equine plasma protease inhibitor (Pi) alleles. Twelve equine protease inhibitory alleles, PiE, H, J, K, L2, O, P, Q, R, V, X, Z, have been characterized in terms of isoelectric point, molecular mass and inhibitory activity to bovine trypsin and chymotrypsin by ISO-DALT electrophoresis. Protein maps for 20 Pi alleles including those of the eight 'Thoroughbred' alleles (PiF, G, I, L, N, S1, S2, U) have now been determined. Five pairs of alleles, S1/S2, G/K, L/L2, P/R and U/Z, possessed varying numbers of common proteins ranging from one protein in the case of G/K and L/L2 to six in the case of U/Z. Based on these results and studies of the a...
The effects of ammonium sulfate and acid on horse and human serum butyrylcholinesterase (EC 3.1.1.8). 1. Results of laboratory experiments which compared horse and human serum butyrylcholinesterase (EC 3.1.1.8) with respect to their acid inactivation and ammonium sulfate protection show: 2. Horse serum butyrylcholinesterase is more resistant to inactivation at pH 3.0 than human serum butyrylcholinesterase. 3. The loss of activity at pH 3.0 for both horse and human butyrylcholinesterase does not follow first order kinetics. 4. Both human and horse serum butyrylcholinesterase are protected from pH 3.0 inactivation by ammonium sulfate concentrations up to 33% saturation (1.37 M).
Acid-stable protease inhibiting polypeptides formed from denatured horse plasma by proteolysis. 1. Trypsin digestion of perchloric acid precipitated horse plasma yielded polypeptides with inhibitory properties for trypsin, chymotrypsin and, to a small extent, kallikrein. 2. The Mr of the inhibitory polypeptides were 73,000 and 24,000. 3. The number, enzyme specificity and Mr of the inhibitory polypeptides differed from the values known for the human being. 4. The inhibitory polypeptides were purified by affinity chromatography on Sepharose-trypsin and by gel filtration through Sephadex G-75. 5. Protease inhibitory polypeptides were generated in the same manner by chymotrypsin, elastase, ...
Serum immunoreactive gastrin activity in horses: basal and postprandial values. Using commercially available diagnostic reagents, serum immunoreactive gastrin activity was measured in five normal horses that were starved of food and water for 24 hours. Blood samples were taken every 15 minutes for two hours. The horses were then fed a pelleted diet for 15 minutes and samples were taken every 15 minutes for a further two hours. Three further samples were taken at hourly intervals. The total sampling period was seven hours. Basal immunoreactive gastrin activity was lower than that reported in other mammals, ranging from a mean of 7.0 pg/ml to 13.8 pg/ml. At 30, 60 and 75 mi...
Serological and virological investigations of an equid herpesvirus 1 (EHV-1) abortion storm on a stud farm in 1985. An extensive outbreak of EHV-1 abortions occurred on a stud farm in England in 1985. Of the 67 pregnant mares present on the stud farm, 31 were challenged with EHV-1, resulting in the loss of 22 fetuses or foals. Laboratory investigations revealed that the spread of the virus closely followed movement of apparently healthy mares (during the incubation period of the infection). During the outbreak mares were challenged 1-4 months before the expected foaling date. There was no relationship between the gestational age at the time of challenge and the subsequent outcome of infection in terms of ab...
Molecular pathogenesis of equine coital exanthema (ECE): temperature sensitivity (TS) and restriction endonuclease (RE) fragment profiles of several field isolates. Examination of six field isolates of equine herpesvirus 3, the causative agent of equine coital exanthema, indicates that all were temperature sensitive (ts) at the body temperature, 39 degrees C, of their host (Equine asinus and callabus) when grown in cell culture. The isolates were characterized by fingerprint analysis with the restriction endonucleases XbaI, EcoRI, BamHI and Hind III to establish possible epidemiologic relatedness. Three of the six isolates may be considered related. Variation in the mobility of the BamHI-A and Hind III-K fragments indicates that a small plaque isolate may...
Purification of horse (Equus caballus) serum lecithin:cholesterol acyltransferase. 1. A method for the purification of horse serum lecithin:cholesterol acyltransferase has been established. 2. The method involves the adsorption of the enzyme from diluted horse serum on DEAE-Sephadex A-50, (NH4)2SO4 fractionation, 1-butanol treatment, and chromatographic techniques of DEAE-Sepharose CL-6B, DEAE-Sephadex A-50, Affi-Gel blue and hydroxylapatite. 3. The resultant enzyme preparation essentially formed a single main band when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. 4. The final purification of the enzyme was 20,000-fold with 7% yi...
Comparison of two reference preparations for horse chorionic gonadotrophin in four in-vivo and in-vitro assays. A number of horse chorionic gonadotrophin (CG) preparations of different purities and from diverse sources have been compared in radioimmuno-, radioreceptor, in-vitro cell culture, and in-vivo assays. The relative activities of the great majority of the preparations tested were consistent in the 4 assay systems. Moreover, their relative activities in the 4 assays were consistent with those found for unfractionated plasmas. These preparations were therefore considered to represent the native form of hormone. The second International Reference Preparation (IRP2) was among the few preparations ex...
Light and electron microscopy of Ag-NORs in domestic horse chromosomes identified after R-banding. Silver staining shows the presence in the domestic horse of six NORs located on chromosomes 1, 26 and 31 as identified after R-banding. Following electron microscopy, the argyrophilic material was observed outside the terminal secondary constrictions (satellite stalks) on the terminal portion of the short arm of chromosome 1, outside the secondary constrictions on the proximal region of the long arms of chromosome 31, and beside the proximal region of the long arms of chromosome 26. Satellite staining applied to these chromosomes appears to reveal only the active NORs.
Motility and ATP content of extended equine spermatozoa in different storage conditions. The role of various environmental conditions on sperm motility and ATP content was investigated by incubating raw and washed spermatozoa collected with an open-ended artificial vagina from 10 stallions in various biological and artificial media under different atmospheric conditions. Spermatozoa did not survive for more than 12 h when kept unextended in the original seminal fluid in any circumstances. The most favourable media tested for long-term sperm survival were Kenney's medium or Kenney's medium supplemented with 10 mM-theophylline and 10 mM-Hepes, pH 7.2. Centrifugation and slow cooling...
Steroid secretion by different cell types of the horse conceptus. Horse conceptuses were recovered non-surgically at Day 12-Day 15 and were dissociated with collagenase. Separation of the cells on a 31.8% Percoll gradient gave two bands of cells and indirect evidence suggests that the low density cells (LDC) are endoderm and the higher density cells (HDC) are trophectoderm. Each band was incubated for 24 h in Minimum Essential Medium and concentrations of oestradiol and progesterone in the medium were measured by radioimmunoassay (RIA). The LDC secreted predominately progesterone (log oestradiol/progesterone = -0.994 +/- 0.141; N = 15) whereas the HDC secret...
Evaluation of cellulose acetate/nitrate filters for the study of stallion sperm motility. Stallion semen was diluted in a Hepes-supplemented buffer (CM) (10(6) spermatozoa/ml) and placed in the upper well of a Sykes-Moore chemotaxis chamber. Chambers were incubated in a humidified atmosphere (5% CO2 in air) at 37 degrees C for 1 and 2 h and spermatozoa were allowed to swim through filters with a mean pore size of 3,5 or 8 micron. Spermatozoa entered filters of all three pore sizes. Distance travelled was greater for each increase in pore size (P less than 0.01) but did not differ (P greater than 0.05) between 1 and 2h of incubation. Extended semen from stallions of different fertil...
Horse plasma ceruloplasmin molecular weight and subunit analysis. Ceruloplasmin is a blue copper-containing serum glycoprotein with oxidase activity. It as been proposed that the physiological function of ceruloplasmin involves the oxidation of ferrous iron and its incorporation into apotransferrin. There are several reports demonstrating that ceruloplasmin is made up of multiple chains. Ryden has questioned the multichain structure of ceruloplasmin from human, pig, horse and rabbit sera, arguing that the dissociation observed by previous workers could be attributed to cleavage of labile bands in the protein by enzymatic contaminants present in commercial pr...
Stromal cells from human long-term marrow cultures, but not cultured marrow fibroblasts, phagocytose horse serum constituents: studies with a monoclonal antibody that reacts with a species-specific epitope common to multiple horse serum proteins. This report describes an IgG1 mouse monoclonal antibody derived after immunization of mice with washed stromal cells from human, long-term bone marrow cultures. The antigen recognized by the antibody (BMS-1) is a carbohydrate-containing prosthetic group that is common to and specific for multiple horse serum proteins. These proteins are avidly ingested by stromal cells and concentrated in endocytic vesicles. Cultured smooth muscle cells took up the horse proteins in a similar manner to marrow stromal cells while cultured marrow fibroblasts, endothelial cells, and hepatoma cells did not. These ...
Endotoxin-induced production of thromboxane and prostacyclin by equine peritoneal macrophages. Equine peritoneal macrophages were isolated and cultured in vitro to assess their ability to produce thromboxane (TxA2) and prostacyclin (PGI2) in response to endotoxin. Peritoneal macrophages (2.5 x 10(6)/ml) were incubated in tissue culture media, containing 1) no additive (nonstimulated control), 2) endotoxin (0.5 to 100 ng/ml) or 3) the calcium ionophore, A23187 (0.95 microM) for two and six h. Concentrations of the stable metabolites of TxA2 and PGI2 thromboxane B2 (TxB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), in the incubation media were determined by radioimmunoassay. Th...
[Differentiation of equine influenza viruses subtype 2 with monoclonal antibodies]. Infections and clinical diseases caused by equine 2 influenza A viruses are observed worldwide. The frequency of these outbreaks supports the hypothesis that antigenic variation of the surface proteins may play an important role. For the demonstration of these variations, monoclonal antibodies (Mabs) were prepared. They are directed against the hemagglutinin or the neuraminidase of the prototype strain a/eq/Miami/1/63. In hemagglutination-inhibition assays with Mabs two reaction patterns were observed: four Mabs inhibited 14 out of 17 strains tested. Another Mab recognized the hemagglutinin of...
