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.
Comparison of Ham’s F10 with CO2 or Hepes buffer for storage of equine embryos at 5 C for 24 H. Forty equine embryos collected 7 d post-ovulation were stored at 5 C for 24 h in one of two culture media (n = 20/group): 1) Ham's F10 + 10% heat-treated fetal calf serum (FCS) buffered by gassing with 5% CO2, 5% O2 and 90% N2 and 2) Ham's F10 + 10% FCS with Hepes buffer (25 mM). Embryos cultured in Ham's F10 + CO2 maintained a better quality score and had a larger average increase in diameter (+34.8 micron) than embryos stored in Hepes buffered Ham's F10 (-10.2 micron). Embryos were transferred surgically into recipient mares that ovulated -3 to +1 d in relation to the donor mare. Twenty embr...
The major protamine from stallion sperm. Isolation and amino-acid sequence. The major stallion protamine was isolated from sperm cell nuclei by extraction with 6M guanidine/5% mercaptoethanol, alkylation with 4-vinylpyridine and subsequent reversed-phase high-performance liquid chromatography. The primary structure of stallion protamine was determined by N-terminal sequencing of the intact protein and of the fragments obtained from thermolysin cleavage of the S-pyridylethylated and from endoproteinase Lys-C cleavage of the S-aminoethylated protein. Stallion protamine consists of 49 amino-acid residues and shows 49% identity with all other sequenced mammalian type 1 pr...
Alpha- and beta-adrenergic receptors in the horse ureter. The presence of both alpha and beta adrenergic receptors in the caudal third ureter of the horse were studied in vitro under isometric conditions using adrenergic agonist and antagonist drugs. Isoprenaline and the beta 2- stimulating agent, salbutamol, elicited relaxation of the ureter smooth muscle strips. The responses were not affected by the beta 1- blocking agent, practolol, but were totally abolished by propranolol and the beta 2- blocking agent, butoxamine. The stimulation of alpha-adrenergic receptors with noradrenaline and phenylephrine evoked a contractile effect which was totally in...
The use of a passive hemolysis system to evaluate the complement activities of six mammalian species. A passive hemolysis assay system was developed which permitted comparisons of the hemolytic activities of complement (C) from six species. This system employs a single antigen and an antiserum raised in one species. Thus, variations resulting from different target antigens and those inherent in using antibodies (of different affinities and isotypes) raised in a variety of species were minimized. Of the erythrocytes (E) examined, those from horses and guinea pigs were most susceptible to lysis, and either would be suitable, as a tentative choice, for measuring C activity of a previously unstudi...
Assessment of neutrophil migration, phagocytosis and bactericidal capacity in neonatal foals. Comparison of neutrophil function was made between 8 clinically normal pony foals (3 to 7 days of age), and their dams. Random migration, stimulated migration to zymosan-activated serum, bacterial phagocytosis and bactericidal capacity of neutrophils were determined in vitro. Random migration was greater (P less than 0.01) and stimulated migration was less (P less than 0.01) in foals than in their dams. Bacterial phagocytosis and bactericidal capacity of neutrophils were not different (P greater than 0.05) between foals and mares. Results of this study suggested that neonatal foals have altere...
[Isolation and purification of proteolytic enzymes on organo-silica sorbents with immobilized gramicidin S]. Biospecific sorbents for affinity chromatography of proteolytic enzymes have been synthesized by attaching cyclopeptide antibiotic gramicidin S to organo-silica supports. It is shown possible to attach gramicidin S to the organo-silica supports using glutaric aldehyde, p-benzoquinone, soluble and insoluble carbodiimides. The sorbents prepared by these methods were successfully applied for the purification of the crude pepsin from horse gastric juice and proteolytic complex produced by Acremonium chrysogenum.
Nucleotide (cDNA) sequence encoding the horse gonadotrophin alpha-subunit. Several cDNA clones corresponding to mRNA for the alpha-subunit of the horse (Equus caballus) pituitary and placental (chorionic) gonadotrophic hormones have been isolated and sequenced. Polyadenylated mRNA was purified from horse pituitary glands (the source of FSH and LH) and horse placental tissues (the source of chorionic gonadotrophin; CG). The mRNA preparations were characterized by in-vitro translation and Northern hybridization techniques using human and ovine gonadotrophin cDNA clones as probes. Complementary DNA libraries were created from the pituitary and placental mRNAs and a huma...
Effect of estradiol and progesterone on antistaphylococcal activity of neutrophils from ovariectomized mares. Neutrophils isolated from jugular blood of ovariectomized mares were studied for the effect of estradiol and progesterone on bactericidal activity against Staphylococcus aureus. In experiment 1, neutrophils obtained from 4 mares were tested for bactericidal activity by adding estradiol (43 pg/ml) or progesterone (6.4 ng/ml) to the bactericidal assay. In experiment 2, 3 of the 4 ovariectomized mares were given 2 mg of estradiol, IM, daily for 3 days. Eighteen days after the initial estradiol injection, mares were given 300 mg of progesterone, IM, for 6 days. Neutrophils from these mares were te...
In vitro febantel transformation by sheep and cattle ruminal fluids and metabolism by hepatic subcellular fractions from different animal species. Febantel and one of its main metabolites, febantel sulphoxide, are chemically modified to only a slight extent when incubated in vitro with sheep and cattle ruminal fluids; other major metabolites, fenbendazole and oxfendazole, are respectively, oxidized to oxfendazole and reduced to fenbendazole. Febantel is negligibly metabolized by hepatic cytosol fractions but microsome preparations effect more extensive metabolic transformations. Important differences in this respect were found between microsome preparations from rat, horse, pig, cattle, sheep, chicken and trout livers.
Induction and characterization of acrosome reaction in equine spermatozoa. Equine spermatozoa were incubated in a chemically defined medium for 8 hours. The medium preserved spermatozoal viability, as assessed by total spermatozoal motility, progressive spermatozoal motility, and spermatozoal exclusion of eosin stain. Effects of time and divalent cation ionophore, A23187, on the occurrence and character of the spermatozoal acrosome reaction were determined. Two light microscopic assays, a triple-stain technique and a chlortetracycline fluorescence assay, were calibrated with transmission electron microscopy for detection of the acrosome reaction. Incubation time and ...
Preservation of skin by refrigeration for autogenous grafting in the horse. Eighteen stored split thickness meshed skin grafts were applied to surgically created lesions on the metacarpal and metatarsal regions of six horses. Donor skin was harvested from the sternal region, meshed and stored at 4 degrees C in a cell culture medium containing 10% serum. Stored grafts were applied to the wounds at 1, 2, and 3 week intervals. Acceptance of the grafts stored for 1 week was generally poor (1 of 6 grafts), whereas that of the 2 and 3 week old grafts was generally excellent (10 of 12 grafts). Poor acceptance of the 1 week old grafts was attributed to streptococcal infection...
Ultrastructure of equine endothelial cells exposed to endotoxin and flunixin meglumine and equine neutrophils. An in vitro system of cultured equine endothelial cells was evaluated as a model for endotoxin (ET) exposure in the horse. Primary cell lines from pulmonary vessels and aortas were cultured from tissues of 6 horses. Effects of ET alone with and without serum and in combination with the cyclo-oxygenase inhibitor flunixin meglumine and isolated equine neutrophils were evaluated by transmission electron microscopy. Cells plus serum were incubated with 10, 25, 50, or 100 micrograms of ET/ml of incubation medium for 1, 3, 8, or 24 hours. Cells without serum were cultured for 1 and 3 hours. Flunixin...
Ethoxyformylation of histidine residues in equine growth hormone. Reactivity of histidine residues in equine growth hormone to ethoxyformic anhydride was studied. The existence of two kinetically different sets was demonstrated: one of them including only the slow reacting histidine 169 (k = 0.164 min-1) and the other containing fast reacting histidines 19 and 21 (k = 0.892 min-1). A correlation between the decrease in the capacity to compete with 125I-labeled hormone for rat liver binding sites and the degree of ethoxyformylation of the fast group was found. Circular dichroism studies indicated no significant conformational changes in the protein with all t...
Influence of arachidonic acid metabolites in vitro and in uterine washings on migration of equine neutrophils under agarose. The influence of arachidonic acid metabolites on migration of equine neutrophils under agarose was investigated. Leukotriene B4 (LTB4) was chemotactic at concentrations between 0.1 and 1000 ng ml-1 and prostaglandin E2 (PGE2) at 1 and 10 ng ml-1 but not at higher or lower concentrations. Prostaglandin F2 alpha (PGF2 alpha) was not chemotactic for equine neutrophils at any concentration. Random migration was significantly inhibited (P less than 0.05) by suspension of neutrophils in LTB4 (0.1 to 1000 ng ml-1) and PGF2 alpha (0.1 ng ml-1) but not at high concentrations. There was a significant po...
Immunoassay detection of drugs in horses. I. Particle concentration fluoroimmunoassay detection of fentanyl and its congeners. We investigated the use of particle concentration fluorescence immunoassay (PCFIA) as a technique for drug detection in racing horses. The test was constructed from an antiserum to a carboxyfentanyl-BSA conjugate and carboxyfentanyl linked to b-Phycoerythrin. Using these reagents and a PCFIA apparatus levels of fentanyl as low as 0.1 ng/ml could be detected by the assay. In addition, cross-reactivity studies on this assay showed that the anti-serum cross-reacted well with carfentanil, sufentanil and the methylated analogs of fentanyl. We therefore evaluated the ability of these agents to produ...
Site-directed chemical modification of horse cytochrome c results in changes in antigenicity due to local and long-range conformational perturbations. Comparative binding studies with peptide fragments of the whole antigen, or with evolutionarily related intact proteins with varying degrees of sequence homology, have been used extensively to map antigenic sites on proteins to the resolution of single amino acid residues. These methods are limited, however, since high affinity antibodies will often not react with peptides and evolutionarily related proteins are available for only a few antigens. In this study we use site-directed chemical modification of horse cytochrome c to identify residues involved in the binding sites of four monoclonal ...
Viability of stored equine embryos. Equine embryos were recovered nonsurgically 6.5 d after ovulation (Exp. 1) and those greater than 200 microns were stored in one of three media: 1) Ham's F10 + 10% fetal calf serum (FCS) under 5% CO2, 5% O2 and 90% N2 at 24 C (Ham's F10); 2) Minimal Essential Medium with Hank's balanced salts + 10% FCS in air (MEM) at 24 C or 3) MEM at 5 C n = 10/treatment). Embryos less than or equal to 200 micron (n = 10) were bisected microsurgically; one-half of each embryo was stored in Ham's F10 and the other half in either Dulbecco's phosphate-buffered saline + 10% FCS in air at 24 C (DPBS), or MEM in a...
Interaction of Rhodococcus equi with phagocytic cells from R. equi-exposed and non-exposed foals. The interaction of Rhodococcus equi with alveolar macrophages from adult horses, foals experimentally exposed to R. equi (sensitized foals) and non-exposed foals was studied using in vitro bactericidal assays, cytochemical staining and transmission electron microscopy. It was demonstrated that R. equi is a facultative intracellular parasite, able to survive and multiply within the alveolar macrophages of the host by interfering with phagosome-lysosome fusion. Opsonization of R. equi with antibody against capsular components was associated with increased phagosome-lysosome fusion and significan...
The interaction of Rhodococcus equi and foal neutrophils in vitro. Polymorphonuclear neutrophil leukocytes (PMNL) from 8 healthy foals (2-14 weeks of age) and 2 foals with bacterial pneumonia were separated from whole blood using a 2 step Percoll gradient. Purified PMNL were tested for bactericidal function against Rhodococcus equi and Staphylococcus aureus in the presence of normal horse serum. The percentage uptake after a 15-min pre-incubation of PMNL and bacteria was also calculated. Ultrastructural examination of the interaction of R. equi and normal foal PMNL was performed after 15 min incubation. Results indicated that foal PMNL effectively phagocytose...
The role of aromatic side chain residues in micelle binding by pancreatic colipase. Fluorescence studies of the porcine and equine proteins. Fluorescence techniques have been employed to study the interaction of porcine and equine colipase with pure taurodeoxycholate and mixed micelles. Nitrotyrosine-55 of porcine colipase is obtained by modification with tetranitromethane (low excess, in the presence of taurodeoxycholate) of the protein followed by gel filtration and ion-exchange chromatography. Verification of the residue modified was obtained by h.p.l.c. peptide purification and sequence analysis. Reduction and quantitative reaction with dansyl chloride yields a fluorescent derivative that is twice as active in conjunction with ...
The binding domain on horse cytochrome c and Rhodobacter sphaeroides cytochrome c2 for the Rhodobacter sphaeroides cytochrome bc1 complex. The interaction of the Rhodobacter sphaeroides cytochrome bc1 complex with Rb. sphaeroides cytochrome c2 and horse cytochrome c was studied by using specific lysine modification and ionic strength dependence methods. The rate of the reactions with both cytochrome c and cytochrome c2 decreased rapidly with increasing ionic strength above 0.2 M NaCl. The ionic strength dependence suggested that electrostatic interactions were equally important to the reactions of the two cytochromes, even though they have opposite net charges at pH 7.0. In order to define the interaction domain on horse cytochro...
In vitro calibration and surgical implantation of electromagnetic blood flow transducers for measurement of left coronary blood flow and cardiac output in the pony. Electromagnetic flow transducers were implanted via left thoracotomy in 8 ponies (122.7 to 263.6 kg) around the main pulmonary and left main coronary arteries for continuous measurement of mean and pulsatile blood flow. Flow transducers were calibrated in vitro with a gravity flow system. The mean +/- SE pulmonary flow was 73.1 +/- 5.1 ml/kg of body weight/min. Left coronary flow was 0.95 +/- 0.07 ml/kg/min (1.3% of cardiac output) and was not believed to be an accurate measurement. This was caused by the inability to implant a zero-flow occluder, requiring the use of minimum flow during systo...
Large granular lymphocytes from SCID horses develop potent cytotoxic activity after treatment with human recombinant interleukin 2. Peripheral blood mononuclear cells from foals with hereditary severe combined immunodeficiency (SCID) have morphologic characteristics of large granular lymphocytes (LGL). Attempts to demonstrate cytotoxic activity were without success unless the LGL were incubated with 100 U of human recombinant interleukin 2 (rIL 2)/ml for 24 hr. With rIL 2 incubation, low effector to target ratios (10:1) consistently yielded high levels of cytotoxic activity (30 to 50%) in a standard 4-hr 51Cr-release assay using YAC-1 lymphoma or K562 erythroleukemia cell lines as targets. Monoclonal antibody EqT12 reacted...
Influence of chemotactic agents on the locomotion of equine polymorphonuclear and mononuclear leucocytes. Subpopulations of equine leucocytes, polymorphonuclear and mononuclear cells, were separated from whole blood on a discontinuous Percoll gradient and used in studies of chemokinesis and chemotaxis. Polymorphonuclear cells responded to the chemo-attractant properties of zymosan-activated plasma in Boyden chamber and agarose microdroplet assays but they responded only slightly (Boyden chamber) or not at all (agarose microdroplet) to the peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). Equine mononuclear cell movement was increased by FMLP in both assay systems and these cells also respond...
Calmodulin-mediated adenylate cyclase from mammalian sperm. Calmodulin (CaM), the calcium binding protein that modulates the activity of a number of key regulatory enzymes, is present at high levels in sperm. To determine whether CaM regulates adenylate cyclase in mammalian sperm, the actions of EGTA and selected CaM antagonists on a solubilized adenylate cyclase from mature equine sperm were examined. The activity of equine sperm adenylate cyclase was inhibited by EGTA in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 2 mM. Equine sperm adenylate cyclase was also inhibited in a concentration-dependent manner by...
Isolation and characterization of two protamines St1 and St2 from stallion spermatozoa, and amino-acid sequence of the major protamine St1. Two protamines, St1 and St2, were isolated from stallion sperm nuclei, where they represent about 75 and 25%, respectively, of the total basic protein complement. The primary structure of protamine St1 (49 residues; Mr approximately equal to 6600) has been determined. The structure of this protamine is compared to the amino-acid sequence of other mammalian protamines already known.
Actions of non-steroidal anti-inflammatory drugs on equine leucocyte movement in vitro. The direct effects of four non-steroidal anti-inflammatory drugs (NSAIDs) on equine polymorphonuclear (PMN) and mononuclear (MN) leucocyte movement were investigated using two in vitro assay systems. The Boyden chamber microfilter technique measures both chemokinetic and chemotactic locomotion, and the agarose microdroplet assay measures solely chemokinesis. Zymosan-activated plasma (ZAP) and the synthetic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) were used as standard chemoattractants for PMN and MN leucocytes, respectively. The actions of six concentrations of each NSAID, indome...
Cellular aspects of inflammation. The Ciba-Geigy Prize for Research in Animal Health. The migration of leucocytes to sites of acute and chronic inflammation is an event of central importance to the maintenance of inflammatory processes; extravascular leucocytes are responsible for generating chemical mediators of inflammation and the phagocytosis of particulate matter. They may also be involved in the conversion of acute to chronic inflammatory lesions. Leucocytes are attracted to sites of tissue injury by a range of chemoattractants. This paper describes the development of a method for separating on Percoll gradients purified populations of equine polymorphonuclear and mononuc...