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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.
Equilibrium and kinetics of the folding of equine lysozyme studied by circular dichroism spectroscopy. The equilibrium unfolding and the kinetics of unfolding and refolding of equine lysozyme, a Ca2+-binding protein, were studied by means of circular dichroism spectra in the far and near-ultraviolet regions. The transition curves of the guanidine hydrochloride-induced unfolding measured at 230 nm and 292.5 nm, and for the apo and holo forms of the protein have shown that the unfolding is well represented by a three-state mechanism in which the molten globule state is populated as a stable intermediate. The molten globule state of this protein is more stable and more native-like than that of alp...
Evidence for different 5-HT1B/1D receptors mediating vasoconstriction of equine digital arteries and veins. 5-hydroxytryptamine (5-HT) is a potent vasoconstrictor of equine digital arteries and veins which may play a role in the ischaemic disease, laminitis. The present investigation compared the properties of 5-HT1B/1D receptors in arteries with those in veins using isolated rings of equine digital blood vessels. The 5-HT1B/1D receptor-selective agonists, anpirtoline and sumatriptan were 17.9 and 10 times more potent and produced 4.1 and 5.6 times greater maximum contractions, respectively, in veins when compared to arteries. Other agonists tested were of equal potency and produced the same maximum...
Equine TIMP-1 and TIMP-2: identification, activity and cellular sources. Matrix metalloproteinases (MMPs) are the main enzymes involved in connective tissue turnover. Regulation of MMPs is achieved by controlling production, activation of the pro-enzymes together with the presence of inhibitors, such as, tissue inhibitors of metalloproteinases (TIMPS). The presence of TIMPs in equine synovial fluid was assessed by the ability of the fluid to inhibit equine MMP-9 activity using a gelatin degradation ELISA. The cellular source of the TIMPs was determined using culture supernatants of resident articular cells (chondrocytes and synovial fibroblasts) and invading inflam...
Dextran-70 inhibits equine platelet aggregation induced by PAF but not by other agonists. Dextrans of mean molecular weight 70 kDa (dextran-70) have had clinical use as anti-thrombotics in man. A major part of the anti-thrombotic action is mediated via inhibition of platelet function. Greatorex (1975, 1977) treated thromboembolic colic in horses with infusions of dextran-70 and reported a 90% recovery rate, but this treatment is nonetheless rarely used. We have used an in vitro method to examine the effect of dextran-70 on equine platelet suspensions, in the hope that understanding the mechanism of action of dextran-70 might lead to the development of alternative therapeutic agents...
Equine CRISP-3: primary structure and expression in the male genital tract. Although originally described in the male rodent genital tract, cysteine-rich secretory proteins (CRISPs) are expressed in a variety of mammalian tissue and cell types. The proteins of the male genital tract have been observed associated to spermatozoa and are believed to play a role in mammalian fertilization. Here we describe the identification and primary structure of the first equine member of the CRISP family. Equine CRISP-3 is transcribed and expressed in the stallion salivary gland, in the ampulla and the seminal vesicle. It displays all 16 conserved cysteine residues and shows 82% homo...
Histamine-induced adherence and migration of equine eosinophils. To examine effects of histamine on equine eosinophil adherence in vitro and to determine the histamine receptor subtype(s) and cell surface adhesion molecules that mediate this response. In addition, to determine the receptor subtypes involved in histamine-induced eosinophil migration. Methods: 8 healthy ponies. Methods: Effects of histamine on equine eosinophil adherence to serum- or fibronectin-coated plastic, and migration in a microchemotaxis assay were examined. In some experiments, eosinophils were pretreated with histamine receptor antagonists or monoclonal antibodies raised against cel...
Isolation and chondrocytic differentiation of equine bone marrow-derived mesenchymal stem cells. To isolate mesenchymal stem cells from adult horses and determine specific monolayer culture conditions required to enhance biochemically and phenotypically defined chondrocytic differentiation. Methods: 2 adult horse bone marrow donors without skeletal or hematologic abnormalities. Methods: Bone marrow was aspirated from the sternebra, and mesenchymal stem cells were isolated by centrifugation and cultured in monolayers. Subcultures were established in 24-well plates on day 13. Culture medium was harvested every 2 days, and culture of 12 of the 24 wells was terminated on day 6 and of the rema...
Effect of three types of half-limb casts on in vitro bone strain recorded from the third metacarpal bone and proximal phalanx in equine cadaver limbs. To determine effect of 3 half-limb casts on bone strains recorded from the proximal phalanx (P-1) and third metacarpal bone (MCIII) of equine cadaver limbs, using a mechanical testing machine. Methods: 12 equine cadaver limbs and 4 live horses. Methods: Bone strains were recorded at middorsal P-1 and the dorsal cortical aspect of the distal third of MCIII while limbs were variably loaded with 100 to 1,000 lb of force. To determine ability of the cast to protect the distal portion of the limb from weight-bearing loads, strains were recorded with the limb in 1 of the 3 casts and with it unsuppor...
The S2 gene of equine infectious anemia virus is dispensable for viral replication in vitro. Equine infectious anemia virus (EIAV) contains the simplest genome among lentiviruses in that it encodes only three putative regulatory genes (S1, S2, S3) in addition to the canonical gag, pol, and env genes, presumably reflecting its limited tropism to cells of monocyte/macrophage lineage. Tat and Rev functions have been assigned to S1 and S3, respectively, but the specific function for the S2 gene has yet to be determined. Thus, the function of S2 in virus replication in vitro was investigated by using an infectious molecular viral clone, EIAVUK. Various EIAVUK mutants lacking S2 were constr...
Development of an ELISA to assess the potency of horse therapeutic polyvalent antibothropic antivenom. The objective of this study was the search for a suitable venom antigen to be used in an in vitro alternative immunoassay, to the standard antivenom neutralization assay using mice. Bothrops jararaca venom was fractionated in DEAE-Sephacel columns and the fractions were tested for a correlation between antibody capture enzyme linked immunosorbent assay (ELISA) absorbance values and the 'in vivo' antivenom potency. Individual antivenoms from 14 horses and 15 separate FUNED polyspecific Bothrops ampouled antivenoms (final product) were used. Fractions showing the higher correlations were further...
Neutralizing potency of horse antibothropic antivenom. Correlation between in vivo and in vitro methods. The correlation coefficients between in vivo neutralization of lethal toxicity (ED50), neutralization of the hemolytic activity (PLA2) and levels of antibodies measured by ELISA, was investigated to test the potency of horse anti-bothropic antivenom. Twenty six horses were hyperimmunized with Bothrops venoms (B. alternatus, B. jararaca, B. jararacussu, B. neuwiedii and B. moojeni). To set up an indirect ELISA, for neutralization of PLA2 activity and for determination of ED50 in Swiss mice, the whole Bothrops jararaca venom (reference venom for assessing the bothropic antivenom potency in Brazi...
The potential of collagenase as a new therapy for separation of human retained placenta: hydrolytic potency on human, equine and bovine placentae. The purpose of this study was to determine to what degree bacterial collagenase may digest human placentae compared to equine and bovine placentae. Placenta samples from human, equine and bovine were incubated with bacterial collagenase solution at various concentrations. The degree of hydrolysis and collagen breakdown was measured by the release of total proteins and hydroxyproline into the incubation media. Also, whole placentae were injected via umbilical cord arteries with collagenase solution (200 U/ml, 200 ml total volume in human and 1000 ml in equine) and hydrolysis determined chemical...
Regional differences in endothelial function in horse lungs: possible role in blood flow distribution? We investigated regional differences of in vitro responses of pulmonary arteries (6-mm OD) from the dorsocaudal (top) and cranioventral (bottom) lung regions to endothelium-dependent vasodilators (methacholine, bradykinin, and calcium ionophore A-23187). Methacholine relaxed endothelium-intact top vessels; however, in bottom vessels, a small relaxation preceded a profound contraction. In top vessels, removal of endothelial cells converted relaxation to contraction, and in bottom vessels it abolished relaxation and enhanced contraction. Bradykinin and A-23187 were more potent and caused greater...
Fusion pore expansion in horse eosinophils is modulated by Ca2+ and protein kinase C via distinct mechanisms. Using the patch-clamp technique, we studied the role of protein phosphorylation and dephosphorylation on the exocytotic fusion of secretory granules with the plasma membrane in horse eosinophils. Phorbol 12-myristate 13-acetate (PMA) had no effect on the amplitude and dynamics of degranulation, indicating that the formation of fusion pores is insensitive to activation of protein kinase C (PKC). Fusion pore expansion, however, was accelerated approximately 2-fold by PMA, and this effect was abolished by staurosporine. Elevating intracellular Ca2+ to 1.5 microM also resulted in a 2-fold accelera...
Oocyte competence for in vitro maturation is associated with histone H1 kinase activity and is influenced by estrous cycle stage in the mare. The in vitro maturation rate of equine oocytes remains low, regardless of culture conditions. Our objective was to determine the reasons for failure of equine oocytes to resume meiosis during in vitro maturation and to ascertain the influence of the estrous cycle stage on meiotic competence. In 10 cyclic mares, 7 ultrasound-guided follicular punctures were performed alternately during the follicular phase (group DF; n = 3 punctures), at the end of the follicular phase (group EF; n = 2), and during the luteal phase (group DL; n = 2). We evaluated the competence of the oocytes for in vitro matur...
Progesterone in mare follicular fluid induces the acrosome reaction in stallion spermatozoa and enhances in vitro binding to the zona pellucida. The aim of this study was to investigate whether mare follicular fluid (FF) induces the acrosome reaction (AR) in stallion spermatozoa and, if so, to identify the component in FF responsible for it. Furthermore, the effect of this component on sperm-zona binding and the subsequent AR was studied. Pooled FF, aspirated from the preovulatory follicles of mares in oestrous, was used and aliquots of the fluid were treated with charcoal to remove steroids (CFF). Charcoal treatment reduced the progesterone concentration in FF from 153 to < 2 ng/mL. Spermatozoa from fertile stallions collected by a...
Diversity among isolates of Actinobacillus equuli and related organisms as revealed by ribotyping. The objective of this work was to examine the diversity within Australian isolates of Actinobacillus equuli and related organisms by the genotypic method of ribotyping. Methods: Ribotyping, performed using the enzyme HaeIII, was used to examine the diversity in 12 field isolates of A equuli (five being capable of fermenting L-arabinose), one field isolate of Pasteurella caballi and two unclassifiable field isolates. Isolates were obtained from Australian horses, except for three isolates of A equuli (one L-arabinose positive and two L-arabinose negative) which were obtained from horses and a p...
Inhibition of glutathione S-transferase activity by the quinoid metabolites of equine estrogens. The risk factors for women developing breast and endometrium cancers are all associated with a lifetime of estrogen exposure. Estrogen replacement therapy (ERT) in particular has been correlated with a slight increased cancer risk, although the numerous benefits of ERT may negate this harmful side effect. Equilenin and equilin are equine estrogens which make up between 30% and 45% of the most widely prescribed estrogen replacement formulation, Premarin (Wyeth-Ayerst). In this study we have synthesized the catechol metabolites of equilenin [4-hydroxyequilenin (4-OHEN)] and equilin [4-hydroxyequ...
General method for the detection and in vitro expansion of equine cytolytic T lymphocytes. Equine immunological research is hindered by the lack of a simple yet reliable general protocol by which to assay CTL activity specific for viral or parasitic antigens. We present here the first comprehensive analysis of the parameters necessary to reliably culture equine T cells and to analyze the antigen specific cytolytic activity of T lymphocytes utilizing the equine infectious anemia virus (EIAV) infection of outbred ponies as a source for in vivo primed T lymphocytes. Effective long-term in vitro culture of equine T cells was determined to require minimally 200 U/ml of recombinant human ...
Endometrial connexin expression in the mare and pig: evidence for the suppression of cell-cell communication in uterine luminal epithelium. This investigation examines the relationship between implantation strategy and gap junction protein expression in uterine endometrium. The pattern of gap junction and connexin protein expression was analyzed in porcine and equine endometrium from cycling and pregnant animals using electron microscopy and immunocytochemistry. Functional analysis of cell-cell communication was also monitored by laser cytometry in primary cultures of endometrial epithelial cells. Gap junctions were detected in endometrial stroma of cycling and pregnant animals, which was correlated with immunoreactive Cx43 within...
Maturation-promoting factor (MPF) and mitogen activated protein kinase (MAPK) expression in relation to oocyte competence for in-vitro maturation in the mare. In the equine species, a large proportion of oocytes fail to complete meiosis during in-vitro culture. The biochemical and molecular basis of this failure is unknown. The meiotic cell cycle is controlled in part by the maturation-promoting factor (MPF) and the mitogen-activated protein kinase (MAPK). In this study, we evaluated the oocyte competence for in-vitro maturation and the expression of MPF components (p34cdc2 and cyclin B) and MAPK after in-vitro culture. The maturation rate was influenced by the culture medium and the physiological stage of the mare at the time of oocyte recovery. We...
Loading-induced changes in synovial fluid affect cartilage metabolism. The purpose of this study was to determine whether changes in the synovial fluid (SF) induced by in vivo loading can induce an alteration in the metabolic activity of chondrocytes in vitro. Therefore, SF was collected from ponies after a period of box rest and after they had exercise for a week. Normal, unloaded articular cartilage explants were cultured in 20% solutions of these SFs for 4 days and chondrocyte activity was determined by glycosaminoglycan (GAG) turnover. In explants cultured in post-exercise SF, GAG synthesis was enhanced and GAG release was diminished when compared to cultures...
Influence of exogenous hyaluronan on synthesis of hyaluronan and collagenase by equine synoviocytes. To evaluate the influence of exogenous hyaluronan (HA) on in vitro synthesis of HA and collagenase by equine synoviocytes from normal and inflamed joints. Methods: 9 adult horses. Methods: Synoviocytes for culture were taken from the middle carpal joint of 3 horses with normal joints (control) and 6 horses with osteochondral fractures (principal). Synoviocytes were propagated in monolayer cultures and were incubated with 3 commercial HA products at concentrations of 0, 200, 400, and 1,500 micrograms/ml. Newly synthesized HA was radiolabeled with [3H]glucosamine and quantified by cetylpyridiniu...
Monoclonal antibody against Babesia equi: characterization and potential application of antigen for serodiagnosis. Monoclonal antibody (MAb) BEG3 was produced against Babesia equi parasites to define a species-specific antigen for diagnostic use. The MAb reacted with single, paired, and Maltese cross forms of B. equi, and no reaction was observed with this MAb on acetone-fixed Babesia caballi, Babesia ovata, or Babesia microti parasites in the indirect immunofluorescent antibody test. Confocal laser and immunoelectron microscopic studies showed that the antigen which was recognized by this MAb was located on the surface of B. equi parasites. This MAb recognized a 19-kDa protein of B. equi antigen and did n...
Determination of the volume changes for pressure-induced transitions of apomyoglobin between the native, molten globule, and unfolded states. The volume change for the transition from the native state of horse heart apomyoglobin to a pressure-induced intermediate with fluorescence properties similar to those of the well-established molten globule or I form was measured to be -70 ml/mol. Complete unfolding of the protein by pressure at pH 4.2 revealed an upper limit for the unfolding of the intermediate of -61 ml/mol. At 0.3 M guanidine hydrochloride, the entire transition from native to molten globule to unfolded state was observed in the available pressure range below 2.5 kbar. The volume change for the N-->I transition is relat...
Direct sequencing of the HA gene of clinical equine H3N8 influenza virus and comparison with laboratory derived viruses. Equine influenza viruses propagated in the laboratory in alternate hosts such as embryonated hens' eggs or mammalian cell culture have been analysed by HA sequencing and antigenically and their sequence compared to the original virus present in clinical material. In contrast to clinically derived human influenza virus which generally grows in MDCK cells without change, the data for equine influenza virus were less clear in that variants of equine virus were derived in both eggs and cells. The study indicated that the current use of eggs for equine influenza virus surveillance and vaccine produ...
Dung-derived biological agents associated with reduced numbers of infective larvae of equine strongyles in faecal cultures. Two sets of dung-derived organisms from soil routinely fertilized with manure (MA) and soil chemically fertilized (CH) were cultured separately in the laboratory. Baermannized organisms from these cultures were added to 20 g of faeces from strongyle-infected horses to form three treatment groups: (i) no soil organisms; (ii) low inoculum of soil organisms containing all organisms present in a suspension of approximately 100 adult female free-living nematodes; and (iii) high inoculum containing those soil organisms present with approximately 1000 adult female free-living nematodes. Three studies...
Monoclonal antibodies to subclass-specific antigenic determinants on equine immunoglobulin gamma chains and their characterization. This paper describes the production of a panel of monoclonal antibodies (mAbs) identifying the four recognised equine IgG subisotypes IgG, IgGa, IgGb, IgGc and IgG(T). Pure preparations of the subisotypes for use in immunisations and testing were produced using a combination of gel filtration, salt precipitation, ion exchange chromatography and protein A and Protein G affinity chromatography. The specificity of mAbs for the IgG subisotypes was confirmed using ELISA assays, by characterisation of affinity purified proteins recognised by the mAbs, and by Western blotting of equine serum proteins...
Multiple kinetic intermediates accumulate during the unfolding of horse cytochrome c in the oxidized state. The unfolding kinetics of horse cytochrome c in the oxidized state has been studied at 10, 22, and 34 degreesC as a function of guanidine hydrochloride (GdnHCl) concentration. Rapid (millisecond) measurements of far-UV circular dichroism (CD) as well as fluorescence quenching due to tryptophan to heme excitation energy transfer have been used to monitor the unfolding process. At 10 degreesC, the decrease in far-UV CD signal that accompanies unfolding occurs in two phases. The unobservable burst phase is complete within 4 ms, while the slower phase occurs over tens to hundreds of milliseconds. ...
Equine monocyte-derived macrophage cultures and their applications for infectivity and neutralization studies of equine infectious anemia virus. Equine infectious anemia virus (EIAV) has been shown to infect cells of monocyte/macrophage lineage. These primary cells are intrinsically difficult to obtain, to purify and to culture in vitro for extended periods of time. As a result, most in vitro studies concerning this lentivirus make use of primary equine fibroblasts or transformed canine or feline cell lines. We describe methods that yield reproducibly pure cultures of equine blood monocytes from peripheral blood mononuclear cells. The in vitro differentiation of these cells into mature equine macrophage was verified using various cytoc...
