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Topic:In Vitro Research
In vitro research involving horses refers to the study of equine cells, tissues, or biological molecules outside their normal biological context, typically in controlled laboratory environments. This research approach allows scientists to investigate cellular processes, molecular interactions, and the effects of various treatments without the ethical and logistical complexities of in vivo studies. In vitro studies contribute to understanding equine physiology, pathology, and pharmacology by providing insights into cellular responses to pathogens, drugs, and other stimuli. This page compiles peer-reviewed research studies and scholarly articles that explore various in vitro methodologies and their applications in equine science, including cell culture techniques, molecular assays, and drug efficacy testing.
Effect of povidone-iodine on in vitro locomotion of equine neutrophils. Incubation of equine neutrophils with povidone-iodine solutions of greater than or equal to 0.2 per cent resulted in total inhibition of migration under agarose. This was caused by the cytotoxic effects of the solutions as shown by pyknosis and cell lysis. Lower concentrations of povidone-iodine, however, did not adversely affect neutrophil viability or locomotion.
Phagocytic function of equine neutrophils exposed to Mycoplasma felis in vitro and in vivo. Neutrophils were isolated from the peripheral blood of adult equids (group 1) and were purified on a density gradient of polyvinylpyrrolidone-coated silica gel. A bactericidal assay was developed, using an equine skin isolate of Staphylococcus epidermidis as target bacterium in medium containing pooled fresh equine serum for opsonization. Significant (P less than 0.05) killing was observed after 60 or 120 minutes' incubation. Reduction in bactericidal function of blood neutrophils was not found after incubation with a virulent strain of Mycoplasma felis for 30 or 60 minutes. Similarly, the fun...
Synthesis of 2-methoxy and 4-methoxy equine estrogens. 4-Methoxyequilin and 2-methoxyequilin were synthesized from the corresponding 4-bromoequilin and 2-iodoequilin derivatives, respectively, by nucleophilic displacement of halogen with methoxide ion in the presence of copper (II) chloride and 15-crown-5-ether. 4-Bromoequilin was prepared by reacting equilin with one equivalent of N-bromoacetamide. 2-Iodoequilin was prepared by reductive dehalogenation of 2,4-diiodoequilin, which in turn was obtained by treatment of equilin with two equivalents of iodine in methanolic ammonium hydroxide solution. 4-Methoxy-equilenin and 2-methoxyequilenin were pr...
An equine rotavirus (FI-14 strain) which bears both subgroup I and subgroup II specificities on its VP6. An equinine rotavirus FI-14 strain, originally isolated from a diarrheic foal in New York state, was shown to belong to serotype 3 by neutralization assay. In addition, it was found to react with both subgroup I and subgroup II monoclonal antibodies by enzyme-linked immunosorbent assay (ELISA), thus representing the first rotavirus strain to exhibit both subgroup specificities. By using hybridoma technology, we successfully produced monoclonal antibodies directed against the major inner capsid protein VP6 (the sixth gene product) of FI-14 virus. Such monoclonal antibodies reacted specifically ...
Further observations on the keratinolytic activity of strains of the genus Epidermophyton. The ability of 17 strains of Epidermophyton to perforate hair in vitro using the Ajello & Georg's test procedure and a modification of Lu's method has been studied. Following the Ajello & Georg's test procedure only E. stockdaleae perforated hair. Sporadically some strains of E. floccosum perforated horse hair. We noted as well unusual perforations originated from inside to outside of the hair. By the other technique, all strains, excepting E. floccosum var. nigricans in child hair, perforated hair. E. floccosum showed these perforations later than E. stockdaleae.
Comparison of naturally occurring poliovirus-reactive immunoglobulins in bovine and equine sera. Bovine and equine sera were screened for poliovirus-reactive immunoglobulins (PRIgs) by means of neutralization and precipitation reactions with type 1 poliovirus. Bovine serum B1826 and B36 were found to contain such PRIgs from their reactivity to various PRIgs-resistant mutants of type 1 poliovirus origin. Neutralization and precipitation reactions with six mono-specific antibodies obtained by absorbing antiserum with each of the six different PRIgs-resistant virus mutants revealed that three antibodies were active in precipitation reaction while the others were substantially ineffective. On...
A proton NMR study of the non-covalent complex of horse cytochrome c and yeast cytochrome-c peroxidase and its comparison with other interacting protein complexes. Cytochrome-c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) forms a noncovalent 1:1 complex with horse cytochrome c in low ionic strength solution that is detectable by proton NMR spectroscopy. When the entire proton hyperfine-shifted spectrum is considered only five hyperfine resonances exhibit unambiguously detectable shifts: the heme 8-CH3 and 3-CH3 resonances, single proton resonances near 19 ppm and -4 ppm and the methionine-80 methyl group. These shifts are very similar to those observed for the covalently crosslinked complex of cytochrome-c peroxidase and h...
A rapid microtitration serum agglutination test for the detection of contagious equine metritis antibodies. A microtitration serum agglutination test, based on that used for brucellosis, has been developed to detect antibodies in the sera of horses exposed to the contagious equine metritis (CEM) organism. Two known positive sera were tested 100 times in 15 separate tests. The results were reproducible to within a twofold range. The test is capable of being carried out within 100 min.
Characterization of equine infectious anemia virus long terminal repeat. The long terminal repeats (LTRs) of equine infectious anemia virus (EIAV) were examined with respect to their ability to function as transcriptional promoters in various cellular environments. Nucleotide sequence analyses of the LTRs derived from two unique proviral clones revealed the requisite consensus transcription and processing signals. One of the proviruses possessed a duplication of a 16-base-pair sequence in the CCAAT box region of the LTR which was absent in the other provirus. To assess its functional activity, each LTR was coupled to the bacterial chloramphenicol acetyltransferase ...
Influence of administration of ovarian steroids on the function of neutrophils isolated from the blood and uterus of ovariectomized mares. The function of blood and uterine luminal neutrophils from ovariectomized mares treated with ovarian steroids was investigated 18 h after intrauterine infusion of 1 X 10(9) Streptococcus zooepidemicus. Random migration of blood neutrophils under agarose was reduced by treatment with progesterone compared with that of neutrophils from oestradiol-treated and control mares. In-vitro addition of progesterone to blood neutrophils from acyclic ponies also reduced migration. Uterine neutrophils did not migrate under agarose which was probably an effect of bacterial phagocytosis. Hormone treatment had...
Infection of a poikilothermic cell line (XL-2) with eastern equine encephalitis and western equine encephalitis viruses. Eastern Equine Encephalitis (EEE) was in Cuba before the 1940s; the virus has been isolated from horses, birds, and rodents during epizootic as well as interepizootic periods. The only isolation of Western Equine Encephalitis (WEE) virus was from a sick pigeon found in the vicinity of Havana University. Both viruses can cause human disease; the isolation of WEE virus from the centre of an urban area emphasises the need for the prompt isolation and rapid identification of these agents. The object of this work was to compare the sensitivity of a continuous cell line (XL-2) from the toad, Xenopus...
In vitro strength of the suspensory apparatus in training and resting horses. Forty-eight limbs of 12 freshly euthanized horses were used to generate data on the strength of the equine suspensory apparatus. The point of failure of the suspensory apparatus of each limb was determined. Immediately before euthanasia, 6 of the 12 horses (thoroughbreds and standardbreds) had been engaged in active training or racing, and six horses in stall and/or pasture activity. In the actively training or racing horses, the point of acute failure of the suspensory apparatus was within the proximal sesamoid bones in 20 (83%) limbs (resulting in 17 apical fractures, 2 basilar fractures, an...
Quantitative determination of acylphosphatase levels in horse tissues by enzyme-linked immunosorbent assay. A non competitive enzyme-linked immunosorbent assay (ELISA) specific for horse muscle acylphosphatase (E.C. 3.6.1.7.) has been developed. The purified anti-acylphosphatase antibodies were immobilized by passive absorption to a solid-phase support and incubated with known and unknown amounts of antigen. The antibody-acylphosphatase complex was quantified using the same antibody conjugated to horseradish peroxidase. The assay yields positive reactions with as little as 0.05 ng of antigen, with intra- and interassay coefficients of variation of 5% and 7%, respectively. On the basis of this assay ...
Radiolabeling of equine platelets in plasma with 111In-(2-mercaptopyridine-N-oxide) and their in vivo survival. A method is presented for the in vitro isolation and radiolabeling of equine platelets with the isotope indium 111 (111In: half-life = 2.8 days, gamma = 173 keV, 89%; 247 keV, 94%). The technique described involves complexing 111In with the lipid-soluble chelating agent, 2-mercaptopyridine-N-oxide (merc), in an aqueous medium. 111In-merc platelet-labeling efficiencies in autologous plasma pretreated with or without ferric citrate reagent were 82 +/- 7% and 24 +/- 12%, respectively. Mean intravascular survivals of 111In-merc-radiolabeled platelets in 8 healthy horses according to simple linear,...
Neutrophil phagocytic and serum opsonic response of the foal to Corynebacterium equi. This study was undertaken to examine the neutrophil response to Corynebacterium (Rhodococcus) equi, and to assess the possibility of neutrophil immaturity or malfunction in predisposition to C. equi pneumonia in foals. Neutrophil phagocytosis of Corynebacterium (Rhodococcus) equi was studied in foals from birth to 6 months of age. Chemiluminescence (CL) and bactericidal assays were used to assay the phagocytic response of peripheral blood neutrophils to C. equi in vitro. Results of in vitro bactericidal and CL assays indicate that foal neutrophils are able to ingest and kill C. equi, however a...
Inactivation of horse liver mitochondrial aldehyde dehydrogenase by disulfiram. Evidence that disulfiram is not an active-site-directed reagent. The inhibition of mitochondrial (pI 5) horse liver aldehyde dehydrogenase by disulfiram (tetraethylthiuram disulphide) was investigated to determine if the drug was an active-site-directed inhibitor. Stoichiometry of inhibition was determined by using an analogue, [35S]tetramethylthiuram disulphide. A 50% loss of the dehydrogenase activity was observed when only one site per tetrameric enzyme was modified, and complete inactivation was not obtained even after seven sites per tetramer were modified. Modification of only two sites accounted for a loss of 75% of the initial catalytic activity. Th...
Inhibition of equine neutrophil chemotaxis and chemokinesis by a Taenia taeniaeformis proteinase inhibitor, taeniaestatin. Taeniaestatin, a recently isolated Taenia taeniaeformis proteinase inhibitor, was used to inhibit equine neutrophil migration. Taeniaestatin itself was not chemotactic when used as a chemotactic factor but taeniaestatin did inhibit neutrophil chemokinesis when tested in a Zigmond-Hirsch checkerboard assay. A dose-dependent inhibition of both chemokinesis and chemotaxis was observed when zymosan activated bovine sera (ZABS) was used as the chemotactic factor. This inhibition was greater than 95% when 5 mu of taeniaestatin was present on both the cell and chemotactic factor side of the chambers....
Equine zona pellucida and capsule: some physicochemical and antigenic properties. The capsule which surrounds the pre-attachment equine embryo has been compared with the zona pellucida (zp) that it replaces, as well as with the rabbit blastocyst coverings, by means of physicochemical and immunological methods. Trypsin solution at pH varying between 7.5 and 9.0 completely solubilized the capsule, as did Na borohydride. However, solutions of pH 2.0 or 12.0, urea, high temperature (65 degrees C, 60 min or 80 degrees C, 30 min), mercaptoethanol and dithiothreitol were able to solubilize the zp but not the capsule at the concentrations used. Indirect immunofluorescence on cryost...
Effects of stallion seminal plasma on hydrogen peroxide release by leukocytes exposed to spermatozoa and bacteria. The ability of stallion seminal plasma to modify phagocytosis of spermatozoa and Streptococcus zooepidemicus was examined. Phagocytosis was monitored indirectly as the H2O2 produced by peripheral blood leukocytes after addition of spermatozoa or bacteria. Hydrogen peroxide production after addition of ejaculated spermatozoa was greater (P less than 0.01) than after addition of epididymal sperm. Furthermore, pre-incubation of epididymal sperm with 6.25-50% seminal plasma caused a dose-dependent increase in subsequent H2O2 production by leukocytes (P less than 0.05). In addition, equine serum wa...
In-vitro biosynthesis of C18 neutral steroids in horse testes. Deuterium, 14C- and 3H-labelled steroid substrates were incubated with minced testicular tissue from stallions of different ages. After extraction and separation of the neutral and phenolic fractions the metabolites were identified by gas chromatography-mass spectrometry. The presence of the expected C19 neutral and C18 phenolic steroids was confirmed. An isomer of 5(10)-oestrene-3,17-diol was also identified.
Susceptibility of various cell culture systems to pseudorabies virus. A comparative study was carried out to determine the susceptibility of five different cell lines to pseudorabies virus (PRV), a herpes virus of pigs. The cell systems tested were swine testicle (ST), mink lung (ML), equine dermal (ED), porcine kidney (PK15), and bovine turbinate (BT) cells. Virus titers obtained were 10(4.88), 10(4.38), 10(3.75), 10(2.63), and 10(0.25) for ML, ST, PK15, BT and ED cells, respectively indicating that ML, ST, and PK15 are optimal cell lines for the growth of PRV whereas BT and ED are not very sensitive.
[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.
Analysis of X-chromosome inactivation in horse embryos. To define the time of X-chromosome inactivation in the horse, 122 conceptuses were collected transcervically between Days 6 and 28 (ovulation = Day 0) and subjected to cytogenetic analysis: 59 of the embryos were divided and in 41 of these separate cytogenetic analysis of the embryonic disc and remaining tissues was possible. Conceptuses were measured and photographed before capsule removal, culture in the presence of 5-bromodeoxyuridine and subsequent fixation for cytogenetic analysis. On average, 15 slides were prepared per conceptus. C-banding was used to determine the sex of each conceptus...
Radionuclide, radiographic, and histomorphometric evaluation of healing of surgically created subchondral defects in equine bone. In the present study, radionuclide scintigraphy and radiography were used to evaluate the rate and degree of healing that occurred in surgically created subchondral bone defects in horses. Following radionuclide scintigraphy and radiography the horses were killed, and histomorphometric analysis was performed on the defect sites. The histomorphometric results were compared to the radionuclide scintigraphic and radiographic results to determine which noninvasive technique provided the most accurate information concerning healing of the bone defects. It was concluded that radionuclide scintigraph...
Evaluation of mare oocyte collection methods and stallion sperm penetration of zona-free hamster ova. Comparisons were made between 2 methods of oocyte recovery from the ovarian follicles of slaughtered mares: 500 oocytes (3 per mare) were obtained by aspiration of follicular fluid from ovaries of 162 mares, and 120 oocytes (8 per mare) by isolation and rupture of follicles from ovaries of 14 mares. In the oocytes recovered after rupture of follicles, 89.2% were morphologically unchanged, in comparison to 29.3% obtained by aspiration of follicular fluid. Stallion spermatozoa capacitated in vitro were tested on zona-free hamster oocytes. The stallion spermatozoa were washed in TCM-199 and prein...
Application of recombinant DNA techniques to structure-function studies of equine protein hormones. Complementary (c)DNA libraries have been made from horse pituitary gland and endometrial cup tissues with the aim of isolating the genes for the horse gonadotrophins (FSH, LH and CG) and growth hormone (GH). Southern (DNA) and Northern (RNA) blotting techniques were used to demonstrate that several heterologous (human and ovine) cDNA probes would be adequate for isolating the horse genes. A human cDNA probe was then used to isolate the horse gonadotrophin alpha-subunit cDNA from the pituitary and endometrial cup libraries. The nucleotide sequences from both tissue sources were identical, there...
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.
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...
Proteins in stallion seminal plasma. Motility and fertility of frozen-thawed semen differs greatly amongst stallions. Differences in seminal plasma might be one cause of this variation. For 8 ejaculates from each of 17 stallions, seminal plasma was saved at -20 degrees C and spermatozoa were cryopreserved. Based on post-thaw sperm motility, seminal plasma samples from 7 stallions (2 good, 3 variable, 2 poor sperm motility) were selected for measurement of electrolytes, protein content and analysis by sodium dodecylsulphate gel electrophoresis (10% gel, Coomassie blue stain). Variation in seminal plasma was significant (P less tha...
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...
